Edward Ziff, Ph.D.

Exposure of neurons to excitotoxic levels of glutamate induces cleavage of the RNA editing enzyme, adenosine deaminase acting on RNA 2, and loss of GLUR2 editing
Mahajan, S S; Thai, K H; Chen, K; Ziff, E
2011 Aug 25;189:305-315, Neuroscience
AMPA receptors are glutamate receptors that are tetramers of various combinations of GluR1-4 subunits. AMPA receptors containing GluR1, 3 and 4 are Ca(2+) permeable, however, AMPA receptors containing even a single subunit of GluR2 are Ca(2+) impermeable. Most AMPA receptors are Ca(2+) impermeable due to the presence of GluR2. GluR2 confers special properties on AMPA receptors through the presence of arginine at the pore apex; other subunits (GluR1, 3, 4) contain glutamine at the pore apex and allow Ca(2+) influx. Normally, an RNA editing step changes DNA-encoded glutamine to arginine, introduces arginine in the GluR2 pore apex. GluR2 RNA editing is carried out by an RNA-dependent adenosine deaminase (ADAR2). Loss of GluR2 editing leads to the formation of highly excitotoxic AMPA channels [Mahajan and Ziff (2007) Mol Cell Neurosci 35:470-481] and is shown to contribute to loss of motor neurons in amyotrophic lateral sclerosis (ALS). Relatively higher levels of Ca(2+)-permeable AMPA receptors are found in motor neurons and this has been correlated with lower GluR2 mRNA levels. However, the reason for loss of GluR2 editing is not known. Here we show that exposure of neurons to excitotoxic levels of glutamate leads to specific cleavage of ADAR2 that leads to generation of unedited GluR2. We demonstrate that cleaved ADAR2 leads to a decrease or loss of GluR2 editing, which will further result in high Ca(2+) influx and excitotoxic neuronal death
— id: 135556, year: 2011, vol: 189, page: 305, stat: Journal Article,

Effects of food restriction and sucrose intake on synaptic delivery of AMPA receptors in nucleus accumbens
Peng XX; Ziff EB; Carr KD
2011 Oct;65(10):1024-31 L, Synapse
Insertion and removal of AMPA receptors from the synaptic membrane underlie dynamic tuning of synaptic transmission and enduring changes in synaptic strength. Preclinical addiction research suggests that AMPA receptor trafficking plays an important role in nucleus accumbens (NAc) neuroplasticity underlying the compulsive and persistent quality of drug-seeking. Considering the parallels between drug addiction and compulsive eating, plus the supranormal reward properties of sucrose, and the role of dieting as a risk factor in development of binge pathology, the present study used a biochemical subcellular fractionation approach to determine whether brief intake of a 10% sucrose solution increases synaptic delivery of AMPA receptors in NAc of chronically food-restricted (FR) relative to ad libitum fed (AL) rats. FR, alone, produced a small but significant increase in synaptic expression of AMPA receptors. This may contribute to NAc integrative mechanisms that mediate the enhanced behavioral responsiveness of FR subjects to phasic reward stimuli, including food and drugs. Brief intake of sucrose increased GluR1 in the PSD, regardless of dietary condition, though the net effect was greater in FR than AL subjects. A marked increase in GluR2 was also observed, but only in FR rats. This set of results suggests that in FR subjects, sucrose may have primarily increased delivery of GluR1/GluR2 heteromers to the PSD, while in AL subjects sucrose increased delivery of GluR2-lacking channels. The functional consequences of these possible differences in subunit composition of trafficked AMPA receptors between diet groups remain to be determined. Nevertheless, the present set of results suggest a promising new avenue to pursue in the effort to understand synaptic plasticity involved in adaptive and pathological food-directed behavior and the mechanistic basis of severe dieting as a risk factor for the latter. Synapse, 2011. (c) 2011 Wiley-Liss, Inc
— id: 134092, year: 2011, vol: 65, page: 1024, stat: Journal Article,

Synaptic Autoregulation by Metalloproteases and {gamma}-Secretase
Restituito, Sophie; Khatri, Latika; Ninan, Ipe; Mathews, Paul M; Liu, Xin; Weinberg, Richard J; Ziff, Edward B
2011 Aug 24;31(34):12083-12093, Journal of neuroscience
The proteolytic machinery comprising metalloproteases and gamma-secretase, an intramembrane aspartyl protease involved in Alzheimer's disease, cleaves several substrates in addition to the extensively studied amyloid precursor protein. Some of these substrates, such as N-cadherin, are synaptic proteins involved in synapse remodeling and maintenance. Here we show, in rats and mice, that metalloproteases and gamma-secretase are physiologic regulators of synapses. Both proteases are synaptic, with gamma-secretase tethered at the synapse by delta-catenin, a synaptic scaffolding protein that also binds to N-cadherin and, through scaffolds, to AMPA receptor and a metalloprotease. Activity-dependent proteolysis by metalloproteases and gamma-secretase takes place at both sides of the synapse, with the metalloprotease cleavage being NMDA receptor-dependent. This proteolysis decreases levels of synaptic proteins and diminishes synaptic transmission. Our results suggest that activity-dependent substrate cleavage by synaptic metalloproteases and gamma-secretase modifies synaptic transmission, providing a novel form of synaptic autoregulation
— id: 136951, year: 2011, vol: 31, page: 12083, stat: Journal Article,

A single subanesthetic dose of ketamine relieves depression-like behaviors induced by neuropathic pain in rats
Wang, Jing; Goffer, Yossef; Xu, Duo; Tukey, David S; Shamir, D B; Eberle, Sarah E; Zou, Anthony H; Blanck, Thomas J J; Ziff, Edward B
2011 Oct;115(4):812-821, Anesthesiology
BACKGROUND: Chronic pain is associated with depression. In rodents, pain is often assessed by sensory hypersensitivity, which does not sufficiently measure affective responses. Low-dose ketamine has been used to treat both pain and depression, but it is not clear whether ketamine can relieve depression associated with chronic pain and whether this antidepressant effect depends on its antinociceptive properties. METHODS: The authors examined whether the spared nerve injury model of neuropathic pain induces depressive behavior in rats, using sucrose preference test and forced swim test, and tested whether a subanesthetic dose of ketamine treats spared nerve injury-induced depression. RESULTS: Spared nerve injury-treated rats, compared with control rats, showed decreased sucrose preference (0.719 +/- 0.068 (mean +/- SEM) vs. 0.946 +/- 0.010) and enhanced immobility in the forced swim test (107.3 +/- 14.6s vs. 56.2 +/- 12.5s). Further, sham-operated rats demonstrated depressive behaviors in the acute postoperative period (0.790 +/- 0.062 on postoperative day 2). A single subanesthetic dose of ketamine (10 mg/kg) did not alter spared nerve injury-induced hypersensitivity; however, it treated spared nerve injury-associated depression-like behaviors (0.896 +/- 0.020 for ketamine vs. 0.663 +/- 0.080 for control rats 1 day after administration; 0.858 +/- 0.017 for ketamine vs. 0.683 +/- 0.077 for control rats 5 days after administration). CONCLUSIONS: Chronic neuropathic pain leads to depression-like behaviors. The postoperative period also confers vulnerability to depression, possibly due to acute pain. Sucrose preference test and forced swim test may be used to compliment sensory tests for assessment of pain in animal studies. Low-dose ketamine can treat depression-like behaviors induced by chronic neuropathic pain
— id: 139733, year: 2011, vol: 115, page: 812, stat: Journal Article,

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development. L
Akaneya, Yukio; Sohya, Kazuhiro; Kitamura, Akihiko; Kimura, Fumitaka; Washburn, Chris; Zhou, Renping; Ninan, Ipe; Tsumoto, Tadaharu; Ziff, Edward B
2010 ;5(8):e12486-e12486, PLoS ONE
BACKGROUND: Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca(2+) fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents. CONCLUSIONS/SIGNIFICANCE: These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation
— id: 112206, year: 2010, vol: 5, page: e12486, stat: Journal Article,

AMPA receptor subunit GluR1 downstream of D-1 dopamine receptor stimulation in nucleus accumbens shell mediates increased drug reward magnitude in food-restricted rats
Carr, K D; Chau, L S; Cabeza de Vaca, S; Gustafson, K; Stouffer, M; Tukey, D S; Restituito, S; Ziff, E B
2010 Feb 17;165(4):1074-1086, Neuroscience
Previous findings suggest that neuroadaptations downstream of D-1 dopamine (DA) receptor stimulation in nucleus accumbens (NAc) are involved in the enhancement of drug reward by chronic food restriction (FR). Given the high co-expression of D-1 and GluR1 AMPA receptors in NAc, and the regulation of GluR1 channel conductance and trafficking by D-1-linked intracellular signaling cascades, the present study examined effects of the D-1 agonist, SKF-82958, on NAc GluR1 phosphorylation, intracranial electrical self-stimulation reward (ICSS), and reversibility of reward effects by a polyamine GluR1 antagonist, 1-NA-spermine, in ad libitum fed (AL) and FR rats. Systemically administered SKF-82958, or brief ingestion of a 10% sucrose solution, increased NAc GluR1 phosphorylation on Ser845, but not Ser831, with a greater effect in FR than AL rats. Microinjection of SKF-82958 in NAc shell produced a reward-potentiating effect that was greater in FR than AL rats, and was reversed by co-injection of 1-NA-spermine. GluR1 abundance in whole cell and synaptosomal fractions of NAc did not differ between feeding groups, and microinjection of AMPA, while affecting ICSS, did not exert greater effects in FR than AL rats. These results suggest a role of NAc GluR1 in the reward-potentiating effect of D-1 DA receptor stimulation and its enhancement by FR. Moreover, GluR1 involvement appears to occur downstream of D-1 DA receptor stimulation rather than reflecting a basal increase in GluR1 expression or function. Based on evidence that phosphorylation of GluR1 on Ser845 primes synaptic strengthening, the present results may reflect a mechanism via which FR normally facilitates reward-related learning to re-align instrumental behavior with environmental contingencies under the pressure of negative energy balance
— id: 106493, year: 2010, vol: 165, page: 1074, stat: Journal Article,

Regulation of synaptic structure and function by palmitoylated AMPA receptor binding protein
Misra, Charu; Restituito, Sophie; Ferreira, Jainne; Rameau, Gerald A; Fu, Jie; Ziff, Edward B
2010 Apr;43(4):341-352, Molecular & cellular neurosciences
AMPA receptor binding protein (ABP) is a multi-PDZ domain scaffold that binds and stabilizes AMPA receptor (AMPAR) GluR2/3 subunits at synapses. A palmitoylated N-terminal splice variant (pABP-L) concentrates in spine heads, whereas a non-palmitoylated form (ABP-L) is intracellular. We show that postsynaptic Sindbis viral expression of pABP-L increased AMPAR mediated mEPSC amplitude and frequency and elevated surface levels of GluR1 and GluR2, suggesting an increase in AMPA receptors at individual synapses. Spines were enlarged and more numerous and nerve terminals contacting these cells displayed enlarged synaptophysin puncta. A non-palmitoylated pABP-L mutant (C11A) did not change spine density or size. Exogenous pABP-L and endogenous GRIP, a related scaffold, colocalized with NPRAP (delta-catenin), to which ABP and GRIP bind, and with cadherins, which bind NPRAP. Thus postsynaptic pABP-L induces pre and postsynaptic changes that are dependent on palmitoylation and likely achieved through ABP association with a multi-molecular cell surface signaling complex
— id: 108427, year: 2010, vol: 43, page: 341, stat: Journal Article,

How the mind works: Revelations (Jean-Pierre Changeux)
Rosenfield, I; Ziff, E
2008 JUN 26 ;55(11):62-65, New York Review of Books
— id: 86956, year: 2008, vol: 55, page: 62, stat: Journal Article,

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity
Serulle, Yafell; Arancio, Ottavio; Ziff, Edward B
2008 Jul-Aug;2(4):230-232, Channels (Austin, Tex.)
Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity
— id: 93359, year: 2008, vol: 2, page: 230, stat: Journal Article,

Stable synaptic retention of serine-880-phosphorylated GluR2 in hippocampal neurons
States, Bradley A; Khatri, Latika; Ziff, Edward B
2008 Jun;38(2):189-202, Molecular & cellular neurosciences
Phosphorylation of S880 within the GluR2 C-terminus has been reported to promote endocytosis of AMPA receptors (AMPARs) by preventing GluR2 interaction with the putative synaptic anchoring proteins GRIP and ABP. It is not yet established however, whether S880 phosphorylation induces removal of AMPARs from synaptic sites, and the trafficking of phosphorylated GluR2 subunits with surface and endocytosed GluR2 has not been directly compared within the same intact neurons. Here we show that phosphorylation of GluR2 subunits by PKC activated with phorbol esters is compartmentally restricted to receptors located at the cell surface. Endogenous AMPARs containing S880-phosphorylated GluR2 remained highly synaptic and colocalized with postsynaptic markers to the same extent as AMPARs which did not contain S880-phosphorylated GluR2. Moreover, following S880 phosphorylation, exogenous GluR2 homomers were found specifically at the cell surface and did not co-traffic with the internalized endosomal GluR2 population. We also show that GluR2 is endogenously phosphorylated by a constitutively active kinase pharmacologically related to PKC, and this phosphorylation is opposed by the protein phosphatase PP1. Our results demonstrate a population of hippocampal AMPARs which do not require interaction with GRIP/ABP for synaptic anchorage
— id: 83101, year: 2008, vol: 38, page: 189, stat: Journal Article,

Molecular determinants of AMPA receptor subunit assembly
Greger, Ingo H; Ziff, Edward B; Penn, Andrew C
2007 Aug;30(8):407-416, Trends in neurosciences
AMPA-type (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review
— id: 94126, year: 2007, vol: 30, page: 407, stat: Journal Article,

Activity-dependent AIDA-1 nuclear signaling regulates nucleolar numbers and protein synthesis in neurons
Jordan, Bryen A; Fernholz, Brian D; Khatri, Latika; Ziff, Edward B
2007 Apr;10(4):427-435, Nature neuroscience
Neuronal development, plasticity and survival require activity-dependent synapse-to-nucleus signaling. Most studies implicate an activity-dependent regulation of gene expression in this phenomenon. However, little is known about other nuclear functions that are regulated by synaptic activity. Here we show that a newly identified component of rat postsynaptic densities (PSDs), AIDA-1d, can regulate global protein synthesis by altering nucleolar numbers. AIDA-1d binds to the first two postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domains of the scaffolding protein PSD-95 via its C-terminal three amino acids. Stimulation of NMDA receptors (NMDARs), which are also bound to PSD-95, results in a Ca(2+)-independent translocation of AIDA-1d to the nucleus, where it couples to Cajal bodies and induces Cajal body-nucleolar association. Long-term neuronal stimulation results in an AIDA-1-dependent increase in nucleolar numbers and protein synthesis. We propose that AIDA-1d mediates a link between synaptic activity and control of protein biosynthetic capacity by regulating nucleolar assembly
— id: 71907, year: 2007, vol: 10, page: 427, stat: Journal Article,

Novel toxicity of the unedited GluR2 AMPA receptor subunit dependent on surface trafficking and increased Ca2+-permeability
Mahajan, S S; Ziff, E B
2007 Jul;35(3):470-481, Molecular & cellular neurosciences
RNA editing modifies the GluR2 AMPA receptor subunit pore loop at the Q/R site and limits receptor Ca(2+) permeability. Editing failure is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis. We show that channels with unedited GluR2 are highly toxic in cultured hippocampal neurons. Toxicity exceeds that of other Ca(2+)-permeable AMPA receptor types and is influenced by agonist binding site mutations, ability to desensitize, and extracellular Ca(2+) levels. Significantly, toxicity also depends on GluR2's constitutive surface trafficking, a function dependent on GluR2 C-terminal domain interaction with NSF, a specialized chaperone. We have exploited the interaction between unedited GluR2 and NSF to reduce GluR2 surface levels. We show that a peptide that blocks the GluR2-NSF interaction reduces unedited GluR2 toxicity by reducing receptor surface expression. Peptide block of trafficking provides a model for design of drugs to reduce unedited GluR2 excitotoxicity in neurodegenerative diseases that result from editing failure
— id: 73862, year: 2007, vol: 35, page: 470, stat: Journal Article,

Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death
Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B
2007 Mar 28;27(13):3445-3455, Journal of neuroscience
Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS
— id: 71906, year: 2007, vol: 27, page: 3445, stat: Journal Article,

A GluR1-cGKII interaction regulates AMPA receptor trafficking
Serulle, Yafell; Zhang, Shuang; Ninan, Ipe; Puzzo, Daniela; McCarthy, Maria; Khatri, Latika; Arancio, Ottavio; Ziff, Edward B
2007 Nov 21;56(4):670-688, Neuron
Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. We have found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity
— id: 75310, year: 2007, vol: 56, page: 670, stat: Journal Article,

Synaptic anchorage of AMPA receptors by cadherins through neural plakophilin-related arm protein AMPA receptor-binding protein complexes
Silverman, Joshua B; Restituito, Sophie; Lu, Wei; Lee-Edwards, Laveria; Khatri, Latika; Ziff, Edward B
2007 Aug 8;27(32):8505-8516, Journal of neuroscience
Cadherins function in the adhesion of presynaptic and postsynaptic membranes at excitatory synapses. Here we show that the cadherin-associated protein neural plakophilin-related arm protein (NPRAP; also called delta-catenin) binds via a postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 (PDZ) interaction to AMPA receptor (AMPAR)-binding protein (ABP) and the related glutamate receptor (GluR)-interacting protein (GRIP), two multi-PDZ proteins that bind the GluR2 and GluR3 AMPAR subunits. The resulting cadherin-NPRAP-ABP/GRIP complexes serve as anchorages for AMPARs. Exogenous NPRAP that was bound to cadherins at adherens junctions of Madin-Darby canine kidney cells recruited ABP from the cytosol to form cadherin-NPRAP-ABP complexes, dependent on NPRAP interaction with the ABP PDZ domain 2. The cadherin-NPRAP-ABP complexes also bound GluR2. In cultured hippocampal neurons, dominant-negative mutants of NPRAP designed to disrupt tethering of ABP to NPRAP-cadherin complexes reduced surface levels of endogenous GluR2, indicating that interaction with cadherin-NPRAP-ABP complexes stabilized GluR2 at the neuronal plasma membrane. Cadherins, NPRAP, GRIP, and GluR2 copurified in the fractionation of synaptosomes and the postsynaptic density, two fractions enriched in synaptic proteins. Furthermore, synaptosomes contain NPRAP-GRIP complexes, and NPRAP localizes with the postsynaptic marker PSD-95 and with AMPARs and GRIP at spines of hippocampal neurons. Thus, tethering is likely to take place at synaptic or perisynaptic sites. NPRAP also binds PSD-95, which is a scaffold for NMDA receptors, for AMPARs in complexes with auxiliary subunits, the TARPs (transmembrane AMPA receptor regulator proteins), and for adhesion molecules. Thus, the interaction of scaffolding proteins with cadherin-NPRAP complexes may anchor diverse signaling and adhesion molecules at cadherins
— id: 73706, year: 2007, vol: 27, page: 8505, stat: Journal Article,

TARPs and the AMPA receptor trafficking paradox
Ziff, Edward B
2007 Mar 1;53(5):627-633, Neuron
AMPA receptors (AMPARs) conduct fast, excitatory currents that depolarize neurons and trigger action potentials. AMPARs took on new importance when it was shown that AMPAR transport can increase or decrease the number of AMPARs at synapses and give rise to synapse plasticity, including long-term potentiation (LTP) and long-term depression (LTD). This review considers how transmembrane AMPAR regulatory proteins (TARPs), a novel family of AMPAR auxiliary subunits, have changed our view of AMPAR transport and raised some perplexing questions
— id: 71421, year: 2007, vol: 53, page: 627, stat: Journal Article,

Developmentally regulated, combinatorial RNA processing modulates AMPA receptor biogenesis
Greger, Ingo H; Akamine, Pearl; Khatri, Latika; Ziff, Edward B
2006 Jul 6;51(1):85-97, Neuron
The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner
— id: 71908, year: 2006, vol: 51, page: 85, stat: Journal Article,

Getting to synaptic complexes through systems biology
Jordan, Bryen A; Ziff, Edward B
2006 ;7(4):214-214, Genome biology
ABSTRACT : Large numbers of synaptic components have been identified, but the effect so far on our understanding of synaptic function is limited. Now, network maps and annotated functions of individual components have been used in a systems biology approach to analyzing the function of NMDA receptor complexes at synapses, identifying biologically relevant modular networks within the complex
— id: 65253, year: 2006, vol: 7, page: 214, stat: Journal Article,

Membrane localization of membrane type 5 matrix metalloproteinase by AMPA receptor binding protein and cleavage of cadherins
Monea, Sara; Jordan, Bryen A; Srivastava, Sapna; DeSouza, Sunita; Ziff, Edward B
2006 Feb 22;26(8):2300-2312, Journal of neuroscience
Matrix metalloproteinases (MMPs) have been proposed to remodel the extracellular environment of neurons. Here, we report that the metalloproteinase membrane-type 5 MMP (MT5-MMP) binds to AMPA receptor binding protein (ABP) and GRIP (glutamate receptor interaction protein), two related postsynaptic density (PSD) PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain proteins that target AMPA receptors to synapses. The MT5-MMP C terminus binds ABP PDZ5 and the two proteins coimmunoprecipitated and colocalized in heterologous cells and neurons. MT5-MMP localized in filopodia at the tips of growth cones in young [2-5 d in vitro (DIV)] cultured embryonic hippocampal neurons, and at synapses in mature (21 DIV) neurons. Its enrichment in synaptosomes also indicated a synaptic localization in the mature brain. Deletion of the PDZ binding site impaired membrane trafficking of MT5-MMP, whereas exogenous ABP splice forms that are associated either with the plasma membrane or with the cytosol, respectively, colocalized with MT5-MMP in synaptic spines or recruited MT5-MMP to intracellular compartments. We show that endogenous MT5-MMP is found in cultured neurons and brain lysates in a proenzyme form that is activated by furin and degraded by auto-proteolysis. We also identify cadherins as MT5-MMP substrates. These results suggest that ABP directs MT5-MMP proteolytic activity to growth cones and synaptic sites in neurons, where it may regulate axon pathfinding or synapse remodeling through proteolysis of cadherins or other ECM or cell adhesion molecules
— id: 63742, year: 2006, vol: 26, page: 2300, stat: Journal Article,

Identification of transcriptional regulators of neuropeptide FF gene expression
Nystedt, Johanna M; Brandt, Annika; Vilim, Ferdinand S; Ziff, Edward B; Panula, Pertti
2006 May;27(5):1020-1035, Peptides
Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain insights into the transcriptional mechanisms that regulate NPFF gene expression, we cloned and sequenced 9.8 and 1.5 kb of the mouse and rat NPFF 5'-flanking region, respectively. Regions with high sequence homology between mouse, rat and human were expected to have high probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with the homeobox proteins Oct-1, PDX1, Pit-1 and MEIS and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The distribution of NPFF gene expression was examined in the mouse using in situ hybridization and RT-PCR. NPFF expression was also evident during mouse embryogenesis. A fixed transcription initiation site for the mouse NPFF gene was found. A novel splice variant with a retained intron of the NPFF gene was characterized. Chimeric luciferase reporter gene constructs for the mouse NPFF gene revealed a minimal promoter region and a region with transcriptional suppressor features. An NGF responsive area was found using mouse NPFF reporter gene constructs. We postulate that Oct-1, PDX1, Pit-1, MEIS and DREAM are likely transcriptional regulators of NPFF gene expression
— id: 71909, year: 2006, vol: 27, page: 1020, stat: Journal Article,

PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking
Lu, Wei; Ziff, Edward B
2005 Aug 4;47(3):407-421, Neuron
PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking
— id: 71911, year: 2005, vol: 47, page: 407, stat: Journal Article,

EphB2 gets a GRIP on the dendritic arbor
Misra, Charu; Ziff, Edward B
2005 Jul;8(7):848-850, Nature neuroscience
— id: 71910, year: 2005, vol: 8, page: 848, stat: Journal Article,

Identification and verification of novel rodent postsynaptic density proteins
Jordan, Bryen A; Fernholz, Brian D; Boussac, Muriel; Xu, Chongfeng; Grigorean, Gabriela; Ziff, Edward B; Neubert, Thomas A
2004 Sep;3(9):857-871, Molecular & cellular proteomics
The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission
— id: 48196, year: 2004, vol: 3, page: 857, stat: Journal Article,

Bidirectional regulation of neuronal nitric-oxide synthase phosphorylation at serine 847 by the N-methyl-D-aspartate receptor
Rameau, Gerald A; Chiu, Ling-Yu; Ziff, Edward B
2004 Apr 2;279(14):14307-14314, Journal of biological chemistry
At glutamatergic synapses, the scaffolding protein PSD95 links the neuronal isoform of nitric-oxide synthase (nNOS) to the N-methyl-d-aspartate (NMDA) receptor. Phosphorylation of nNOS at serine 847 (Ser(847)) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca(2+)-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser(847) phosphorylation. nNOS phosphorylated at Ser(847) colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 microm glutamate stimulated CaMKII phosphorylation of nNOS at Ser(847), whereas excitotoxic concentrations of glutamate, 100 and 500 microm, induced Ser(847)-PO(4) dephosphorylation by protein phosphatase 1. Strong NMDA receptor stimulation was likely to activate nNOS under these conditions because protein nitration to form nitrotyrosine, a marker of nNOS activity, correlated in individual neurons with Ser(847)-PO(4) dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser(847) could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser(847) by different doses of glutamate suggests two mechanisms with opposite effects: 1). a time-dependent negative feedback induced by physiological concentrations of glutamate that limits nNOS activation and precludes the overproduction of NO; and 2). a pathological stimulation by high concentrations of glutamate that leads to unregulated nNOS activation and production of toxic levels of NO. These mechanisms may share pathways, respectively, with NMDA receptor-induced forms of synaptic plasticity and excitotoxicity
— id: 44853, year: 2004, vol: 279, page: 14307, stat: Journal Article,

Protein interactions and the control of AMPA receptor trafficking
Ziff, EB; Khatri, L; Kong, X; Greger, I
2004 FEB ;88(5):5-5, Journal of neurochemistry
— id: 42487, year: 2004, vol: 88, page: 5, stat: Journal Article,

Intracellular membrane targeting and suppression of Ser880 phosphorylation of glutamate receptor 2 by the linker I-set II domain of AMPA receptor-binding protein
Fu, Jie; deSouza, Sunita; Ziff, Edward B
2003 Aug 20;23(20):7592-7601, Journal of neuroscience
AMPA receptor-binding protein (ABP) is a multi-postsynaptic density-95/discs large/zona occludens (PDZ) protein that binds to the glutamate receptor 2/3 (GluR2/3) subunits of the AMPA receptor and is implicated in receptor membrane anchorage. A palmitoylated form of ABP localizes to spine heads, whereas a nonpalmitoylated form is found in intracellular clusters. Here, we investigate intracellular cluster formation by ABP and the ability of ABP to associate with GluR2 while in these clusters. We show that ABP interacts with intracellular membranes via the ABP linker I (LI)-set II (SII) subdomain, a region consisting of ABP linker 1 and PDZ4, -5, and -6. This suggests that cluster formation results from LI-SII ABP association with the membrane of a vesicular structure. We present evidence that ABP can self-associate at intracellular membrane surfaces via interactions involving SII. ABP in such membrane clusters can bind and retain GluR2 that has trafficked endocytotically from the plasma membrane. Phosphorylation of GluR2 at serine 880, proximal to the ABP binding site, has been implicated by others in the release of ABP from GluR2 and the mobilization of AMPA receptors for trafficking. We show that binding of GluR2 to ABP blocks phosphorylation of serine 880. This suggests that ABP can stabilize its own association with GluR2. We discuss a model in which ABP can form a protein scaffold at a vesicular membrane that is capable of binding GluR2, leading to formation of an intracellular AMPA receptor pool. Receptors in such a pool may contribute to receptor endocytotic and exocytotic trafficking and recycling
— id: 39103, year: 2003, vol: 23, page: 7592, stat: Journal Article,

AMPA receptor assembly determined by Q/R editing
Greger, I. H.; Khatri, L.; Kong, X.; Ziff, E. B.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
AMPA receptors are tetrameric cation channels that mediate the majority of fast excitatory transmission in the brain. Four subunits, GluR1-4 (or A-D) assemble in various stoichiometries, resulting in a spectrum of functionally distinct channels. Rules that govern assembly are largely unknown. The majority of AMPARs contain GluR2, which dominates transmission properties via Arg607, introduced into the pore loop by RNA editing (at the Q/R-site). Here we report that Arg607 also determines AMPAR assembly. Sedimentation analysis reveals that edited GluR2-R channels remain incompletely assembled and ER-retained, whereas unedited GluR2-Q channels readily tetramerize and exit from the ER, in neurons and HeLa cells. Mutagenesis reveals that the structure of the pore loop affects tetramerization. Therefore, by modulating a critical assembly surface, Q/R-editing determines AMPAR assembly and subunit stoichiometry
— id: 92627, year: 2003, vol: 2003, page: ?, stat: Journal Article,

AMPA receptor tetramerization is mediated by Q/R editing
Greger, Ingo H; Khatri, Latika; Kong, Xiangpeng; Ziff, Edward B
2003 Nov 13;40(4):763-774, Neuron
AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain
— id: 44855, year: 2003, vol: 40, page: 763, stat: Journal Article,

Dream is a potential transcriptional regulator of neuropeptide FF gene expression
Nystedt, J. M.; Vilim, F. S.; Ziff, E. B.; Panula, P.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain further insights into the transcriptional mechanisms that regulate NPFF gene expression, we performed extensive homology analyses on the NPFF 5'-flanking region cloned from mouse, rat and human. Regions with high sequence homology between mouse, rat and human were expected to have higher probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with homeobox proteins and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The region containing the DRE elements was found to repress reporter gene transcription as studied with chimeric luciferase reporter gene constructs. Co-transfections with DREAM expression vectors and chimeric NPFF promoter/luciferase gene constructs are in progress. We postulate that DREAM, a known transcriptional regulator of prodynorphin gene expression, is also a likely transcriptional regulator of NPFF gene expression
— id: 92628, year: 2003, vol: 2003, page: ?, stat: Journal Article,

NMDA receptor regulation of nNOS phosphorylation and induction of neuron death
Rameau, Gerald A; Chiu, Ling-Yu; Ziff, Edward B
2003 Dec;24(8):1123-1133, Neurobiology of aging
Stimulation of NMDA receptors activates neuronal nitric oxide synthase (nNOS) and the production of nitric oxide (NO). Dephosphorylation of nNOS increases nNOS enzymatic activity. We have examined the regulation of nNOS phosphorylation in rat cortical neurons following NMDA receptor activation. We show that nNOS is constitutively phosphorylated and that NMDA receptor activation decreases the level of nNOS phosphorylation by a mechanism that is blocked specifically by NMDA receptor antagonists and inhibitors of the Ca2+-regulated phosphatases calcineurin and PP1/PP2A. Using quantitative digital microscopy, we show that NMDA receptor activation induces the accumulation of nitrotyrosine, a measure of nNOS activity, and TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) positivity, a measure of cell death. A calcineurin inhibitor blocked the increase in both TUNEL and nitrotyrosine positivity. Notably, TUNEL was increased in those neurons that were most strongly positive for nitrotyrosine. We conclude that NMDA receptor activation induces death of neurons by a cell autonomous pathway involving nNOS dephosphorylation by a calcineurin-dependent mechanism
— id: 44854, year: 2003, vol: 24, page: 1123, stat: Journal Article,

Protein interactions and the trafficking of AMPA receptors
Ziff, EB; Greger, I; Fu, J; deSouza, S; States, B; Lu, W; Khatri, L; Lee-Edwards, L
2003 DEC ;87(1):35-35, Journal of neurochemistry
— id: 42522, year: 2003, vol: 87, page: 35, stat: Journal Article,

Receptor trafficking and the plasticity of excitatory synapses
Barry, Michael F; Ziff, Edward B
2002 Jun;12(3):279-286, Current opinion in neurobiology
Newly discovered features of the trafficking of AMPA receptors to and from the postsynaptic membrane of excitatory synapses are now bringing the mechanisms of synaptic plasticity into focus. Recent advances, including the existence of slots, anchors, transport factors and pathways for activity-dependent control, have elucidated the role of the individual AMPA receptor subunits and their binding partners. The latest views describe how subunit type dictates the assembly of heteromeric receptors, and how these heteromers interact with the receptor trafficking machinery and synaptic anchorage factors. Moreover, phosphorylation may play an important role in receptor transport and synaptic turnover
— id: 35235, year: 2002, vol: 12, page: 279, stat: Journal Article,

Ca2+-dependent formation of a dynamin-synaptophysin complex: potential role in synaptic vesicle endocytosis
Daly, Christopher; Ziff, Edward B
2002 Mar 15;277(11):9010-9015, Journal of biological chemistry
Synaptophysin is a synaptic vesicle (SV) protein of unknown function. Here we show that a repeated sequence in the cytoplasmic tail of synaptophysin mediates the formation of a protein complex containing the GTPase dynamin. The formation of this complex requires a high Ca(2+) concentration, suggesting that it occurs preferentially at the sites of SV exocytosis. Coimmunoprecipitation of a dynamin-synaptophysin complex from brain extracts is promoted by dissociation of vesicle-associated membrane protein 2 from synaptophysin. This finding suggests that dynamin only associates with synaptophysin in vivo after vesicle-associated membrane protein 2 (VAMP2) enters the SNARE complex. GTP binding releases dynamin from synaptophysin, possibly serving to regulate dynamin selfassembly during endocytosis. Our results suggest that synaptophysin plays a role in SV recycling by recruiting dynamin to the vesicle membrane
— id: 35238, year: 2002, vol: 277, page: 9010, stat: Journal Article,

Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters
DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B
2002 May 1;22(9):3493-3503, Journal of neuroscience
Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse
— id: 35236, year: 2002, vol: 22, page: 3493, stat: Journal Article,

AMPA receptors do the electric slide
deSouza, Sunita; Ziff, Edward B
2002 Oct 29;2002(156):PE45-PE45, Science's STKE
How the synapse is organized and how its organization changes during events that result in long-term changes in synaptic efficacy is the subject of intense study. Various anchoring proteins work in concert to organize the postsynaptic side of the membrane, and the interactions of these proteins can be altered by synaptic activity. DeSouza and Ziff discuss the evidence that the reversible palmitoylation of the postsynaptic density protein PSD-95 may result in the movement of AMPA-type glutamate receptors into and out of lipid raft domains, ultimately controlling AMPA receptor accumulation at the postsynaptic membrane
— id: 35231, year: 2002, vol: 2002, page: PE45, stat: Journal Article,

INTRACELLULAR MEMBRANE TARGETING AND SUPPRESSION OF SER880 PHOSPHORYLATION OF GluR2 BY THE Li - SII SUBDOMAIN OF ABP
Fu, J.; DeSouza, S.; Ma, X.; Ziff, E. B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Regulated trafficking of AMPA receptors (AMPAR) contributes to the plasticity of excitatory synapses. AMPAR consisting of GluR2/3 subunit heteromers can cycle constitutively between intracellular and synaptic membranes. The multi-PDZ protein AMPA receptor Binding Protein (ABP) binds to GluR2/3 and may function as an AMPAR membrane anchor. ABP synaptic membrane association depends upon palmitoylation of the N-terminal variable exon of the pABP-L isoform. Here we identify an ABP subdomain, LI-SII, consisting of PDZ4-6 plus the N-terminally neighboring linker region that targets non-palmitoylated ABP (ABP-L) to intracellular membranes, where it clusters GluR2. We show that following endocytosis from the plasma membrane, GluR2 trafficks to intracellular membranes where it localizes with ABP-L via LI-SII. Phosphorylation of GluR2 at serine 880, which releases AMPAR for traffficking, is blocked by association of GluR2 with LI-SII. These data indicate that the ABP region LI-SII functions as an intracellular GluR2 tether that can stabilize the receptor against transport by inhibiting S880 phosphorylation
— id: 92629, year: 2002, vol: 2002, page: ?, stat: Journal Article,

Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina
Gabriel, Robert; de Souza, Sunita; Ziff, Edward B; Witkovsky, Paul
2002 Jul 22;449(2):129-140, Journal of comparative neurology
We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain
— id: 35233, year: 2002, vol: 449, page: 129, stat: Journal Article,

RNA EDITING AT ARG607 CONTROLS AMPA RECEPTOR EXIT FROM THE ENDOPLASMIC RETICULUM
Greger, I. H.; Khatri, L.; Hong, K.; Ziff, E. B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C-terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER-retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER-exit, and may thereby ensure the availability of GluR2 for assembly into AMPARs
— id: 92632, year: 2002, vol: 2002, page: ?, stat: Journal Article,

RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum
Greger, Ingo H; Khatri, Latika; Ziff, Edward B
2002 May 30;34(5):759-772, Neuron
AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs
— id: 35234, year: 2002, vol: 34, page: 759, stat: Journal Article,

NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex
Hanley, Jonathan G; Khatri, Latika; Hanson, Phyllis I; Ziff, Edward B
2002 Mar 28;34(1):53-67, Neuron
AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity
— id: 35237, year: 2002, vol: 34, page: 53, stat: Journal Article,

Analysis of human neuropeptide FF gene expression
Nystedt, Johanna M; Brandt, Annika M; Mandelin, Jami; Vilim, Ferdinand S; Ziff, Edward B; Panula, Pertti
2002 Sep;82(6):1330-1342, Journal of neurochemistry
As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites
— id: 35232, year: 2002, vol: 82, page: 1330, stat: Journal Article,

NMDA RECEPTORS ACTIVATION REGULATE NOS PHOSPHORYLATION AND APOPTOTIC NEURON DEATH. G.A. RAMEAU*, P. WITKOVSKY, L. CHIU AND E. B. ZIFF. HHMI, DEPT. OF BIOCHEMISTRY, NYU SCH. OF MED., NEW YORK, NY 10016
Rameau, G. A.; Witkovsky, P.; Chiu, L.; Ziff, E. B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
We have studied the role and the control of neuronal nitric oxide synthase (nNOS) by NMDA receptors (NMDARs) in excitotoxicity. Phosphorylation of nNOS regulates its activity. NMDAR activation in neurons greatly reduces the constitutive phosphorylation of nNOS by a mechanism sensitive to the NMDAR antagonist, MK801. Okadaic acid, a general phosphatase inhibitor and cyclosporine, a specific inhibitor of the Ca2+-activated phosphatase calcineurin, reduce the NMDAR-induced dephosphorylation of nNOS. We demonstrated that in brain nNOS is phosphorylated at two different phosphorylation sites by Western blotting and immunocytochemistry in brain, retina, and cortical cultured neurons using phospho-specific antibodies. NMDAR-induced death in these experiments is apoptotic since cell death is blocked by the baculovirus-encoded caspase inhibitor protein, p35, and is accompanied by caspase-3 activation. FK506 was neuroprotective since it reduced NMDAR-dependent nitrotyrosine formation and TUNEL positivity. This suggests that NMDA-induced cell death of neurons involves nNOS activation via dephosphorylation leading to caspase activation and apoptosis
— id: 92630, year: 2002, vol: 2002, page: ?, stat: Journal Article,

DELETION OF THE JUXTAMEMBRANE 10 AMINO ACIDS OF THE GluR2 C - TERMINUS INCREASES INTERNALIZATION THE EXOGENOUS RECEPTOR IN CULTURED HIPPOCAMPAL NEURONS
States, B. A.; Khatri, L.; Ziff, E. B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Movement of AMPA receptors into and out of the postsynaptic membrane is correlated with LTP and LTD, respectively. The roles of specific AMPA receptor subunits, in particular their cytosolic C-termini, have been examined with respect to exocytosis of exogenous receptors in neurons. Contributions of AMPA receptor C-terminal sequences to endocytosis of exogenous receptor subunits, however, have not been examined in intact neurons.)We have employed mutagenesis to examine the role of protein binding to the GluR2 C-terminus in endocytic trafficking of exogenously expressed GluR2 subunits in cultured hippocampal neurons. Using immunocytochemistry we analyzed internalization of GluR2 mutants in which binding of proteins known to interact with the GluR2 C-terminus was abrogated. We also examined a mutation in which the juxtamembrane 10 amino acids of the GluR2 C-terminus were deleted. Mutation of the NSF binding site resulted in a modest but significant (26%) increase in receptor internalization. Deletion of the juxtamembrane region however resulted in a 96% increase in the rate of internalization of the receptor.)We show that internalization of exogenous GluR2 subunits takes place mainly in dendritic shafts and is largely absent in dendritic spines. Internalized GluR2 was highly colocalized with the early endosomal marker EEA1 when observed after 10 minutes of exposure to extracellular antibodies.)We will discuss models which could account for the contribution of the GluR2 juxtamembrane region in stabilizing AMPA receptors in the plasma membrane
— id: 92631, year: 2002, vol: 2002, page: ?, stat: Journal Article,

Characterization of the phenotype of neuropeptide FF knockout mice
Brandt, A. M.; Westerlund, J. M.; Pietila, P.; Vilim, F. S.; Ziff, E. B.; Panula, P.
2001 ;27(1):70-70, Abstracts (Society for Neuroscience)
Establishment of mice deficient in neuropeptide FF (NPFF) was achieved using homologous recombination of the entire NPFF precursor coding region in embryonic stem cells. The disruption of the NPFF locus was confirmed by Southern blotting and PCR. The absence of NPFF precursor mRNA expression in null mutant mice was confirmed using in situ hybridization and RT-PCR. The expression of related peptide precursor mRNAs such as prolactin releasing peptide (PrRP) mRNA and RFamide related peptide (RFRP) mRNA was not affected in the NPFF null mutant mice. Immunohistochemical analysis of NPFF in the CNS of null mutant mice revealed weakly NPFF-positive fibers in the nucleus tractus solitarius. This result may be due to antibody crossreactivity to closely related RFamide peptides such as RFRP-1 and RFRP-3. In heterozygous mice, variable levels of NPFF-immunoreactivity were observed in the CNS, suggesting a complex regulation of RFamide peptide expression. Interestingly, wild type mice were observed to contain an abundant variant form of NPFF mRNA that retains the first intron. Whether this NPFF splice variant excerts a regulatory role in the expression of the NPFF or generates additional peptides remains to be elucidated. Characterization of the behavioural phenotype of the NPFF null mutant mice is in progress
— id: 92634, year: 2001, vol: 27, page: 70, stat: Journal Article,

Differential Cellular and Subcellular Localization of AMPA Receptor-Binding Protein and Glutamate Receptor-Interacting Protein
Burette A; Khatri L; Wyszynski M; Sheng M; Ziff EB; Weinberg RJ
2001 Jan 15;21(2):495-503, Journal of neuroscience
Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP
— id: 17519, year: 2001, vol: 21, page: 495, stat: Journal Article,

Association of AMPA receptor sub-units GluR2/3 with two AMPA receptor-specific proteins, GRIP and ABP, in rat retinal neurons
Gabriel, R; Witkovsky, P; Ziff, E; deSouza, S
2001 MAR 15 ;42(4):S728-S728, Investigative ophthalmology & visual science. IOVS
— id: 54988, year: 2001, vol: 42, page: S728, stat: Journal Article,

The majority of the GluR2 AMPA-receptor subunit stably resides in the endoplasmic reticulum
Greger, I. H.; Ziff, E. B.
2001 ;27(1):77-77, Abstracts (Society for Neuroscience)
The regulation of AMPA-receptor (AMPAR) transport to synapses may be involved in the modulation of synaptic strength. We describe the existence of a prominent intracellular pool of the Ca2+-impermeable AMPAR subunit GluR2. The intracellular form of GluR2 is characterised by the presence of high-mannose type glycans, which are linked to proteins traversing the early part of the secretory pathway, i.e the ER, the intermediate compartment and the cis-Golgi. The existence of this GluR2 pool is based on subcellular fractionations, sedimentation on sucrose gradients, de-glycosylation and cell surface crosslinking experiments. An ER localisation is suggested by the co-fractionation and co-sedimentation with the ER marker calnexin. Interestingly, the subcellular localisation and ER export kinetics of GluR2 are strikingly different from GluR1, a receptor subunit that oligomerises with GluR2 to form a functional AMPAR. Since this form of GluR2 is unexpectedly stable (t1/2apprx24 hrs) it may represent an intracellular GluR2 reserve pool
— id: 92635, year: 2001, vol: 27, page: 77, stat: Journal Article,

Regulation of AMPA receptor GLuR2 subunit-PDZ protein interactions by NSF and alpha/beta-SNAPs
Hanley, J. G.; Khatri, L.; Hanson, P. I.; Ziff, E. B.
2001 ;27(1):403-403, Abstracts (Society for Neuroscience)
Recent studies have revealed that trafficking of AMPA receptors may be responsible for some forms of synaptic plasticity. For example, LTD is mediated at least in part by endocytosis of AMPA receptors from the synaptic plasma membrane, and LTP can involve insertion of AMPARs into the synaptic membrane by exocytosis. The PDZ proteins ABP/GRIP and PICK1 in addition to the ATPase NSF bind to the C-terminal tail of the GluR2 subunit and are thought to play roles in these processes. NSF is a well characterized molecular chaperone which disrupts SNARE complexes upon ATP hydrolysis as a crucial step in membrane fusion events. alpha-SNAP is a required co-factor for this reaction. PICK1 may function to traffic AMPA receptors to or from the plasma membrane. We studied the interactions between GluR2, PDZ proteins, NSF and alpha/beta-SNAPs in an isolated system of purified, bacterially expressed components. The GluR2-NSF interaction in the absence if other proteins is sensitive to ATP hydrolysis, and the GluR2-PICK1 complex has a much higher affinity for NSF than GluR2 alone. The GluR2-PICK1 interaction requires non-PDZ contacts within the NSF binding site. Both alpha- and beta-SNAP bind directly to PICK1. We demonstrate association of PICK1 with NSF and SNAPs in brain. ATP hydrolysis by NSF will disrupt the PICK1-GluR2 complex when alpha-SNAP is bound, and this is inhibited by the binding of beta-SNAP. We propose a model whereby NSF and SNAPs modulate levels of AMPA receptor at the synaptic membrane by regulating interaction of PICK1 with GluR2
— id: 92633, year: 2001, vol: 27, page: 403, stat: Journal Article,

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf
Hornyak TJ; Hayes DJ; Chiu L; Ziff EB
2001 Mar;101(1-2):47-59, Mechanisms of development
A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration
— id: 17518, year: 2001, vol: 101, page: 47, stat: Journal Article,

Transcriptional repression activity and induction of RPE neural differentiation revealed by expression of Mash-1 transcription factor in pigment cell precursors in transgenic mice
Hornyak, T; Gavin, J; Jiao, Z; Gawel, J; Ziff, E
2001 AUG ;117(2):508-508, Journal of investigative dermatology
— id: 54903, year: 2001, vol: 117, page: 508, stat: Journal Article,

PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2
Perez JL; Khatri L; Chang C; Srivastava S; Osten P; Ziff EB
2001 Aug 1;21(15):5417-5428, Journal of neuroscience
The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane
— id: 26724, year: 2001, vol: 21, page: 5417, stat: Journal Article,

Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles
Daly C; Sugimori M; Moreira JE; Ziff EB; Llinas R
2000 May 23;97(11):6120-6125, Proceedings of the National Academy of Sciences of the United States of America
The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca(2+)-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission
— id: 9869, year: 2000, vol: 97, page: 6120, stat: Journal Article,

ABP splice variants: differential co-localization with AMPA receptors in hippocampal neurons
de Souza, S.; Osten, P.; Khatri, L.; Ziff, E. B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
PDZ domains are structural motifs that interact with the C-termini of proteins. Several labs, including ours, have identified two homologous proteins that interact with the C-terminus of the GluR2 subunit of the glutamate receptor: AMPA receptor Binding Protein (ABP - containing 6 PDZ domains) and Glutamate Receptor Interacting Protein (GRIP - containing 7 PDZ domains). The role that ABP, GRIP and other molecules that interact with glutamate receptors, such as PICK1 and NSF, play in the precise synaptic localization, targeting, clustering and anchoring of the receptor is still not clear. Several splice variants of ABP were cloned from a rat brain cDNA library, including a form that contains 7 PDZ domains (ABP2) and two ABP forms with alternatively spliced N-termini. We have characterized the expression and localization of these different ABP forms both in Hela cells and hippocampal neurons. Currently, we are investigating the molecular basis underlying the differential localization of these ABP splice variants
— id: 92636, year: 2000, vol: 26, page: ?, stat: Journal Article,

Microphthalmia and loss of coat pigmentation from transgenic expression of a neurogenic factor in pigment cell precursors [In Process Citation]
Hornyak TJ; Gavin J; Jiao Z; Ziff EB
2000 Aug;115(2):332-332, Journal of investigative dermatology
— id: 11537, year: 2000, vol: 115, page: 332, stat: Journal Article,

Cell-density-dependent regulation of expression and glycosylation of dopachrome tautomerase/tyrosinase-related protein-2
Hornyak TJ; Hayes DJ; Ziff EB
2000 Jul;115(1):106-112, Journal of investigative dermatology
The expression of the dopachrome tautomerase gene (Dct) and its protein product, tyrosinase-related protein-2, was studied in the cultured, phorbol-ester-dependent murine melanocyte cell line melan-a. Increased cell density was found to stimulate Dct expression both in cells stably transfected with a Dct promoter-lacZ construct and endogenously in nontransfected cells. Increased Dct expression under these conditions corresponds to increased tyrosinase-related protein-2 production. Tyrosinase-related protein-2 was found to exist in two distinct glycoforms with different endoglycosidase sensitivities. Density-dependent expression of tyrosinase-related protein-2 was independent of time of cell growth, cell proliferation, and soluble factors, implying that cell-cell contact is the important determinant governing increased Dct expression under these conditions. Tyrp1 gene expression and tyrosinase-related protein-1 production were also induced under similar conditions. The results show that cell-cell contact between melanocytes induces a coordinated response at both transcriptional and nontranscriptional levels that induces production of the tyrosinase-related proteins that have a significant role in melanization
— id: 11610, year: 2000, vol: 115, page: 106, stat: Journal Article,

Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles
Llinas, R.; Daly, C.; Sugimori, M.; Moreira, J. E.; Ziff, E. B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca2+-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. Here we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is activated by the high Ca2+ concentration at SV release sites
— id: 92313, year: 2000, vol: 26, page: ?, stat: Journal Article,

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor
Osten P; Khatri L; Perez JL; Kohr G; Giese G; Daly C; Schulz TW; Wensky A; Lee LM; Ziff EB
2000 Aug;27(2):313-325, Neuron
We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate
— id: 17521, year: 2000, vol: 27, page: 313, stat: Journal Article,

Role of NMDA receptor functional domains in excitatory cell death
Rameau GA; Akaneya Y; Chiu L; Ziff EB
2000 Sep;39(12):2255-2266, Neuropharmacology
The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death
— id: 17522, year: 2000, vol: 39, page: 2255, stat: Journal Article,

NMDA-induced apoptotic cell death mediated by activated caspase-3
Rameau, G. A.; Chiu, L.; Ziff, E. B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
NMDA-induced apoptosis in cultured primary neurons was characterized by analysis of DNA laddering and formation of TUNEL positive cells. Western blot analyses showed the appearance of activated caspase-3 immediately following NMDA stimulation of apoptotic cell death of cultured cortical neurons. The activation of caspase-3 was correlated with increased NO., levels detected in real time using the cell permeable DAF2-DA fluorophore. To determine the role of activated caspaases in NMDA receptor mediated apoptotic death we used the luciferase cell assay system (Rameau et al., (2000) Neuropharmacology, in press) to investigate the ability of cell survival factors and a broad inhibitor of caspases to reduce NMDA induced cell death. Cortical neurons were sensitive to both the baculovirus p35 caspase inhibitor expressed by transfection, and the Z-DEVD-FMK peptide, another inhibitor of caspases. Although the inhibitory effects of p35 and Z-DEVD-FMK are not specific to caspase-3 alone, these results indicate a primary role for caspases and in particular the action of activated caspase-3 in the mechanism of NMDA-induced excitotoxicity. Furthermore, the overexpression of Bcl-2, a cell survival factor also reduced NMDA induced cell death. These data add further support to a role for an apoptotic mechanism of NMDA receptor induced cell death and identify physiological effects of NMDA induced activation of caspases in the death of cultured primary cortical neurons
— id: 92638, year: 2000, vol: 26, page: ?, stat: Journal Article,

Novel interactions with AMPA receptor binding protein (ABP)
Silverman, J. B.; Bloomfield, S.; Ziff, E. B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
AMPA receptors mediate the majority of fast synaptic neurotransmission at excitatory synapses within the CNS. One approach to studying these receptors is to construct a blueprint of the proteins located within the PSD which interact with AMPA receptor subunits. Towards this goal, a number of PDZ-containing proteins have been shown to interact with the cytoplasmic C-terminus of GluR2, the dominant AMPA receptor subunit. ABP is one such protein that contains six or seven PDZ domains, depending on its form. PDZ domain 5 of ABP shows the highest binding affinity for GluR2, and therefore it is likely that this domain mediates the interaction with AMPA receptors in vivo. Presumably, the rest of the PDZ domains of ABP are free to bind other proteins. These interactions may be responsible for anchoring AMPA receptors at the synapse, or alternatively, may be involved with the transport of AMPA receptors into and out of the postsynaptic membrane. Therefore, in order to understand the processing of AMPA receptors it is necessary to decipher the identity of additional proteins that interact with ABP. Using the Yeast Two-Hybrid system, a rat brain cDNA library was screened for potential ABP binding partners. Specifically, the first three PDZ domains of ABP were used as bait to discover novel interactions. Additional biochemical methods have been used to prove the specificity of binding and the function of these novel interacting proteins
— id: 92637, year: 2000, vol: 26, page: ?, stat: Journal Article,

Consistent and selective expression of the discoidin domain receptor-1 tyrosine kinase in human brain tumors
Weiner HL; Huang H; Zagzag D; Boyce H; Lichtenbaum R; Ziff EB
2000 Dec;47(6):1400-1409, Neurosurgery
OBJECTIVE: Few molecular targets are both consistently and selectively expressed in a majority of central nervous system (CNS) neoplasms. Receptor tyrosine kinases have been implicated in brain tumor oncogenesis. We previously isolated one such receptor, discoidin domain receptor-1 (DDR1), from high-grade pediatric brain tumors. Here, we analyze the cellular origin and distribution of DDR1 expression in human brain tumors and its expression in tumor cells relative to surrounding brain. METHODS: By use of a digoxigenin-labeled DDR1 riboprobe, we investigated the expression of DDR1 messenger ribonucleic acid in a prospective series of 30 resected human primary and metastatic brain neoplasms, nonneoplastic human brain, and mouse embryonic brain, as well as a mouse glioblastoma model, by in situ hybridization. RESULTS: All the high-grade primary brain and metastatic brain tumors showed unequivocal, intense DDR1 expression within the majority of tumor cells, whereas expression was not observed in hyperplastic tumor blood vessels, normal brain blood vessels, inflammatory cells, or in the normal brain tissue that surrounded the tumor. Receptor expression was limited to tumor cells located within solid tumor tissue. Overall, 27 of 29 resected CNS tumors exhibited tumor cell-specific DDR1 expression, whereas one specimen composed of isolated glioblastoma cells within invaded brain parenchyma showed no detectable staining for this receptor. DDR1 was also expressed preferentially in the ventricular zone (a region of highly proliferating precursor cells) of mice at embryonic Day 15.5. CONCLUSION: We found that DDR1 is consistently expressed in all high-grade brain neoplasms studied and is selective for tumor cells in the specimens analyzed. The expression of DDR1 by tumor cells of CNS neoplasms and by primitive cells of the embryonic ventricular zone suggests that DDR1 is a potentially useful marker of tumor cells within the CNS
— id: 17520, year: 2000, vol: 47, page: 1400, stat: Journal Article,

KCl induces an activity-dependent expression of NMDA receptors in early development of rat hippocampal neurons in culture
Akaneya, Y.; Ziff, E. B.
1999 ;25(1-2):203-203, Abstracts (Society for Neuroscience)
— id: 92643, year: 1999, vol: 25, page: 203, stat: Journal Article,

Activity-dependent regulation of AMPA receptors and AMPA receptor binding proteins in the adult mouse olfactory bulb
Baker, H.; Liu, N.; Berlin, R. A.; DeSouza, S.; Ziff, E. B.
1999 ;25(1-2):128-128, Abstracts (Society for Neuroscience)
— id: 92641, year: 1999, vol: 25, page: 128, stat: Journal Article,

FGF-2 potentiates Ca(2+)-dependent inactivation of NMDA receptor currents in hippocampal neurons
Boxer AL; Moreno H; Rudy B; Ziff EB
1999 Dec;82(6):3367-3377, Journal of neurophysiology
Peptide growth factors such as the neurotrophins and fibroblast growth factors have potent effects on synaptic transmission, development, and cell survival. We report that chronic (hours) treatment with basic fibroblast growth factor (FGF-2) potentiates Ca(2+)-dependent N-methyl-D-aspartate (NMDA) receptor inactivation in cultured hippocampal neurons. This effect is specific for the NMDA-subtype of ionotropic glutamate receptor and FGF-2. The potentiated inactivation requires ongoing protein synthesis during growth factor treatment and the activity of protein phosphatase 2B (PP2B or calcineurin) during agonist application. These results suggest a mechanism by which FGF-2 receptor signaling may regulate neuronal survival and synaptic plasticity
— id: 11900, year: 1999, vol: 82, page: 3367, stat: Journal Article,

Cloning and functional characterization of the 5' flanking region of neuropeptide FF promoter
Brandt, A.; Westerlund, J.; Vikstrom, S.; Vilim, F. S.; States, B.; Ziff, E.; Panula, P.
1999 ;25(1-2):2225-2225, Abstracts (Society for Neuroscience)
— id: 92640, year: 1999, vol: 25, page: 2225, stat: Journal Article,

ABP splice variants: Differential co-localization with AMPA receptors in hippocampal neurons
deSouza, S.; Osten, P.; Khatri, L.; Rameau, G.; Ziff, E. B.
1999 ;25(1-2):732-732, Abstracts (Society for Neuroscience)
— id: 92639, year: 1999, vol: 25, page: 732, stat: Journal Article,

Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF
Lee TC; Ziff EB
1999 Jan 8;274(2):595-606, Journal of biological chemistry
The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes
— id: 7384, year: 1999, vol: 274, page: 595, stat: Journal Article,

AMPA receptor forms a biochemically functional complex with NSF and alpha- and beta-SNAPs
Osten P; Ziff EB
1999 Apr 30;868:558-560, Annals of the New York Academy of Sciences
— id: 56457, year: 1999, vol: 868, page: 558, stat: Journal Article,

The role of the GluR2 C-terminus in trafficking of the AMPA receptor
Osten, P.; Khatri, L.; Perez, J. L.; Ziff, E. B.
1999 ;25(1-2):25-25, Abstracts (Society for Neuroscience)
— id: 92642, year: 1999, vol: 25, page: 25, stat: Journal Article,

ABP: a novel AMPA receptor binding protein
Srivastava S; Ziff EB
1999 Apr 30;868:561-564, Annals of the New York Academy of Sciences
We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor
— id: 56458, year: 1999, vol: 868, page: 561, stat: Journal Article,

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli
Vilim FS; Aarnisalo AA; Nieminen ML; Lintunen M; Karlstedt K; Kontinen VK; Kalso E; States B; Panula P; Ziff E
1999 May;55(5):804-811, Molecular pharmacology
Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord
— id: 56427, year: 1999, vol: 55, page: 804, stat: Journal Article,

Consistent and selective expression of the DDR1 tyrosine kinase in high grade human brain tumors
Weiner HL; Huang HY; Zagzag D; Ziff EB
1999 ;1:345-345, Neuro-oncology
— id: 34023, year: 1999, vol: 1, page: 345, stat: Journal Article,

Recent excitement in the ionotropic glutamate receptor field
Ziff EB
1999 Apr 30;868:465-473, Annals of the New York Academy of Sciences
The synapse is a specialized cellular junction with an elaborate and highly evolved capacity for signal transduction. At excitatory synapses, the neurotransmitter glutamate is released from the presynaptic nerve terminal and stimulates several types of glutamate receptors in the postsynaptic membrane. These include the ionotropic receptors, which are glutamate-gated cation channels, and the metabotropic receptors, which are G protein-coupled seven-transmembrane receptors. The ionotropic glutamate receptors have received special attention because of growing evidence that changes in their synaptic abundance, posttranslational modification, or molecular interactions can provide long-term changes in synaptic strength. This review summarizes new information about the ionotropic glutamate receptors and relates receptor function to the organization of the postsynaptic membrane and the regulation of electrophysiologic and biochemical signaling at the synapse
— id: 56456, year: 1999, vol: 868, page: 465, stat: Journal Article,

Expression of glutamate receptor-binding PDZ proteins from adenovirus vectors in the CNS in vitro and in vivo
Akaneya, Y.; States, B.; Khatri, L.; Schneider, R.; Ziff, E. B.
1998 ;24(1-2):571-571, Abstracts (Society for Neuroscience)
— id: 92645, year: 1998, vol: 24, page: 571, stat: Journal Article,

AP-1, CREB and CBP transcription factors differentially regulate the tyrosine hydroxylase gene
Ghee M; Baker H; Miller JC; Ziff EB
1998 Mar 30;55(1):101-114, Brain research. Molecular brain research
The tyrosine hydroxylase (TH) gene encodes the rate-limiting enzyme in the biosynthesis of catecholamines. We have investigated the roles of two elements of the TH promoter, the TH-'Fat Specific Element' (TH-FSE) which binds the Fos-Jun complex, and the cAMP Response Element (CRE), which binds CREB and the co-activator protein, CREB Binding Protein (CBP) in regulating TH gene transcription. In PC12 cells, the TH-FSE was required for induction by NGF while the CRE was required for induction by cAMP. We show that both elements can function independently and contribute strongly to TH promoter basal activity in PC12 cells. We employed transient expression in the F9 teratocarcinoma cell line to vary experimentally the levels of the nuclear regulators implicated in TH control by the PC12 studies. In F9 cells, the TH promoter was strongly activated by Fos and Jun, and by PKA-stimulated CREB protein. In F9 and NIH3T3 cells, CBP, a co-activator which targets Fos-Jun and PKA-stimulated CREB, also induced the TH promoter. Immunohistochemical studies in rat brain regions enriched in dopaminergic neurons, including the midbrain and olfactory bulb (OB), suggest that Fos-Jun and CREB make differential contributions to TH gene activity in different tissues. Whereas changes in Fos protein levels parallel decreases in TH protein upon olfactory deprivation, CBP levels remain unchanged. This suggests that CRE-associated factors, including CBP, are not major regulators in the OB. In contrast, the presence of CREB and the absence of Fos immunoreactivity in midbrain dopaminergic cells suggests that the CRE is the primary regulator in this region
— id: 57153, year: 1998, vol: 55, page: 101, stat: Journal Article,

Role of MITF in melanocyte development: A transgenic analysis
Hornyak, TJ; Harada, FT; Ziff, EB
1998 APR ;110(4):477-477, Journal of investigative dermatology
— id: 53520, year: 1998, vol: 110, page: 477, stat: Journal Article,

Altered expression of helix-loop-helix transcriptional regulators and cyclin D1 in Wnt-1-transformed PC12 cells
Issack PS; Ziff EB
1998 Oct;9(10):837-845, Cell growth & differentiation
Nerve growth factor induces PC12 cells to differentiate from a chromaffin cell to a sympathetic neuronal phenotype. In contrast, PC12 cells, which stably express Wnt-1, a secreted signaling factor required for development of mammalian midbrain and cerebellum, fail to express differentiation-specific genes in response to nerve growth factor. Analysis of factors binding to E box-containing regulatory elements of the terminal differentiation gene encoding peripherin suggested a differentiation-specific control of expression of helix-loop-helix transcriptional regulators. Specifically, the MASH-1 (mammalian achaete-scute homologue) helix-loop-helix transcription factor, which plays a positive role in neuronal differentiation, is reduced in Wnt-1/PC12 cells, and HES-1, a negative regulator of MASH-1, is increased. These data suggest that the differentiation block may result from induction of HES-1. Wnt-1/PC12 cells also proliferate more rapidly and express increased levels of cyclin D1. Thus, Wnt-1 may block the differentiation and enhance the proliferation of PC12 cells by activating HES-1 and cyclin D1 and repressing MASH-1
— id: 57040, year: 1998, vol: 9, page: 837, stat: Journal Article,

Genetic elements regulating HES-1 induction in Wnt-1-transformed PC12 cells
Issack PS; Ziff EB
1998 Oct;9(10):827-836, Cell growth & differentiation
PC12 cells differentiate in response to nerve growth factor from a chromaffin cell to a sympathetic neuronal phenotype. Wnt-1 is a secreted signaling factor required for development of mammalian midbrain and cerebellum. PC12 cells transformed by Wnt-1 fail to express several differentiation-specific genes in response to nerve growth factor. We have previously shown that HES-1, a negative regulator of neuronal differentiation, is increased in Wnt-1/PC12 cells (P. S. Issack and E. B. Ziff. Altered expression of helix-loop-helix transcriptional regulators and cyclin D1 in Wnt-1-transformed PC12 cells. Cell Growth & Differ., 9: 837-845). Here, we show that the HES-1 promoter is more active in Wnt-1/PC12 cells relative to PC12 and that the binding sites for the transcription factor RBP-J kappa contribute to this induction. We also identify two additional promoter elements required for elevated HES-1 expression. One element binds Wnt-1-induced protein complexes in a sequence-specific manner. Identification of Wnt-1 responsive elements in potential target genes may provide clues to nuclear pathways regulated by Wnt-1
— id: 57172, year: 1998, vol: 9, page: 827, stat: Journal Article,

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs
Osten P; Srivastava S; Inman GJ; Vilim FS; Khatri L; Lee LM; States BA; Einheber S; Milner TA; Hanson PI; Ziff EB
1998 Jul;21(1):99-110, Neuron
In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor
— id: 7726, year: 1998, vol: 21, page: 99, stat: Journal Article,

Functional characterization of the AMPA receptor-NSF-SNAP complex
Osten, P.; Khatri, L.; States, B. A.; Barry, M. F.; Ziff, E. B.
1998 ;24(1-2):842-842, Abstracts (Society for Neuroscience)
— id: 92644, year: 1998, vol: 24, page: 842, stat: Journal Article,

Identification of NMDA receptor dominant mutations and study of receptor apoptotic function
Rameau, G.; Akaneya, Y.; Ziff, E. B.
1998 ;24(1-2):279-279, Abstracts (Society for Neuroscience)
— id: 92646, year: 1998, vol: 24, page: 279, stat: Journal Article,

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP
Srivastava S; Osten P; Vilim FS; Khatri L; Inman G; States B; Daly C; DeSouza S; Abagyan R; Valtschanoff JG; Weinberg RJ; Ziff EB
1998 Sep;21(3):581-591, Neuron
We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors
— id: 7975, year: 1998, vol: 21, page: 581, stat: Journal Article,

Expression of NPFF mRNA in carrageenan inflammation in the spinal cord of rat
Aarnisalo, A. A.; Nieminen, M.; Kontinen, V. K.; Vilim, S.; Kalso, E.; Ziff, E.; Panula, P.
1997 ;23(1-2):1806-1806, Abstracts (Society for Neuroscience)
— id: 92648, year: 1997, vol: 23, page: 1806, stat: Journal Article,

Post-transcriptional regulation of synaptic vesicle protein expression and the developmental control of synaptic vesicle formation
Daly C; Ziff EB
1997 Apr 1;17(7):2365-2375, Journal of neuroscience
The regulated expression of synaptic vesicle (SV) proteins during development and the assembly of these proteins into functional SVs are critical aspects of nervous system maturation. We have examined the expression patterns of four SV proteins in embryonic hippocampal neurons developing in culture and have found that increases in the levels of these proteins result primarily from post-transcriptional regulation. Synaptotagmin I, vamp 2, and synapsin I proteins are synthesized at nearly constant rates as the neurons develop. However, these proteins are relatively unstable at early times in culture and undergo a progressive increase in half-life with time, possibly as a result of an increase in the efficiency with which they are incorporated into SVs. In contrast, synaptophysin is synthesized at a very low rate at early times in culture, and its rate of synthesis increases dramatically with time. The increase in synaptophysin synthesis is not simply the result of an increase in mRNA level, but is largely attributable to an increase in the rate of translational initiation. Despite the nearly constant rates of synthesis of synaptotagmin I, vamp 2, and synapsin I, we show that the number of SVs in these developing neurons increases, and that SV proteins are more efficiently targeted to SVs at later times in culture. Our results suggest that SV production during development is not limited by the rates of transcription of genes encoding the component proteins, thus allowing control of this process by cytoplasmic mechanisms, without signaling to the nucleus
— id: 7318, year: 1997, vol: 17, page: 2365, stat: Journal Article,

Density-dependent activation of TRP-2 in mouse melanocytes
Hornyak, TJ; Ziff, EB; Perelman, RO
1997 APR ;108(4):215-215, Journal of investigative dermatology
— id: 53216, year: 1997, vol: 108, page: 215, stat: Journal Article,

Myc represses transcription of the growth arrest gene gas1
Lee TC; Li L; Philipson L; Ziff EB
1997 Nov 25;94(24):12886-12891, Proceedings of the National Academy of Sciences of the United States of America
Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. We show that c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen was sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in promotion of cell growth by Myc
— id: 11472, year: 1997, vol: 94, page: 12886, stat: Journal Article,

Identification of AMPA interacting proteins by the yeast two hybrid system
Osten, P.; Srivastava, S.; Inman, G. J.; Villim, S.; Lee, L.; Khatri, L.; Ziff, E. B.
1997 ;23(1-2):923-923, Abstracts (Society for Neuroscience)
— id: 92650, year: 1997, vol: 23, page: 923, stat: Journal Article,

Identification of NMDA receptor dominant negative mutations and apoptotic function of the NMDA receptors in CHO cells
Rameau, G. A.; Ziff, E. B.
1997 ;23(1-2):2304-2304, Abstracts (Society for Neuroscience)
— id: 92647, year: 1997, vol: 23, page: 2304, stat: Journal Article,

AMPA receptor interacting protein: PSD 96
Srivastava, Sapna; Villim, Sven; Osten, Pavel; Khatri, Latika; States, Bradley; Inman, Gareth J.; Daly, Chris; Ziff, Edward B.
1997 ;23(1-2):1392-1392, Abstracts (Society for Neuroscience)
— id: 92649, year: 1997, vol: 23, page: 1392, stat: Journal Article,

Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor
Yan GZ; Ziff EB
1997 Aug 15;17(16):6122-6132, Journal of neuroscience
The PC12 pheochromocytoma cell line responds to nerve growth factor (NGF) by gradually exiting from the cell cycle and differentiating to a sympathetic neuronal phenotype. We have shown previously () that NGF induces the expression of the p21 WAF1/CIP1/Sdi1 (p21) cyclin-dependent kinase (Cdk) inhibitor protein and the G1 phase cyclin, cyclin D1. In this report, we show that induction is at the level of transcription and that the DNA elements in both promoters that are required for NGF-specific induction are clusters of binding sites for the Sp1 transcription factor. NGF also induced a synthetic promoter with repeated Sp1 sites linked to a core promoter, and a plasmid regulated by a chimeric transactivator in which the Gal4 DNA binding domain is fused to the Sp1 transactivation domain, indicating that this transactivation domain is regulated by NGF. Epidermal growth factor, which is a weak mitogen for PC12, failed to induce any of these promoter constructs. We consider a model in which the PC12 cell cycle is arrested as p21 accumulates and attains inhibitory levels relative to Cdk/cyclin complexes. Sustained activation of p21 expression is proposed to be a distinguishing feature of the activity of NGF that contributes to PC12 growth arrest during differentiation
— id: 7285, year: 1997, vol: 17, page: 6122, stat: Journal Article,

Enlightening the postsynaptic density
Ziff EB
1997 Dec;19(6):1163-1174, Neuron
— id: 7871, year: 1997, vol: 19, page: 1163, stat: Journal Article,

Chronic FGF-2 treatment increases NMDA receptor desensitization in cultured hippocampal neurons by a cycloheximide-sensitive mechanism that requires calcineurin activity
Boxer, Adam L.; Ziff, Edward B.
1996 ;22(1-3):1959-1959, Abstracts (Society for Neuroscience)
— id: 92652, year: 1996, vol: 22, page: 1959, stat: Journal Article,

Regulated expression of synaptic vesicle proteins in cultured hippocampal neurons
Daly, C.; Ziff, E. B.
1996 ;22(1-3):1949-1949, Abstracts (Society for Neuroscience)
— id: 92653, year: 1996, vol: 22, page: 1949, stat: Journal Article,

Dominant negative mutants of Myc inhibit cooperation of both Myc and adenovirus serotype-5 E1a with Ras
MacGregor D; Li LH; Ziff EB
1996 Apr;167(1):95-105, Journal of cellular physiology
We have used dominant negative Myc mutants to analyze the Myc and E1a mechanisms of cooperation with Ras. We show that mutants of Myc with an altered basic region (BR; RR366, 367EE) or deletion of the leucine zipper (LZ; delta aa 414-439), changes which modify the DNA binding domain, or with deletions in the Myc amino terminal conserved regions box 1 (dlMB1; delta aa 46-55) and box 2 (dlMB2; delta aa 132-140) inhibit cooperation of wt Myc and activated Ras to transform rat embryo fibroblasts (REF). Expression of the amino terminal 104 aa had no effect whereas wt Myc stimulated focus formation. Mutant dlMB1 cooperated with Ras with one half wt efficiency while dlMB2 was inactive. No mutant tested was toxic during neomycin cotransformation of REF to G418 resistance. Interestingly, these Myc mutants exerted a parallel inhibition of E1a-Ras cooperation to transform REF. This suggests that the Myc-Ras and E1a-Ras cooperation pathways intersect and require common protein factors. A Myc box 2 deletion mutant which is a wt transactivator of the Myc responsive ornithine decarboxylase promoter, but unlike the wt does not repress the adenovirus-2 core promoter (Li et al., 1994, EMBO J., 13:4070-4079), inhibits Myc-Ras and E1a-Ras cooperation. This suggests that a box 2-dependent step, potentially gene repression, is required for both the E1a- and Myc-Ras cooperation mechanisms
— id: 8005, year: 1996, vol: 167, page: 95, stat: Journal Article,

Expression of neuropeptide FF precursor in rat CNS
Panula, P.; Nieminen, M.; Aarnisalo, A. A.; Lintunen, M.; Karhunen, T.; Vilim, F. S.; Ziff, E.; Karlstedt, K.
1996 ;22(1-3):1557-1557, Abstracts (Society for Neuroscience)
— id: 92651, year: 1996, vol: 22, page: 1557, stat: Journal Article,

Analysis of the bovine and rat neuropeptide FF and AF gene
Vilim, F. S.; Ziff, E.
1996 ;22(1-3):1925-1925, Abstracts (Society for Neuroscience)
— id: 92654, year: 1996, vol: 22, page: 1925, stat: Journal Article,

Pediatric brain tumors express multiple receptor tyrosine kinases including novel cell adhesion kinases
Weiner HL; Rothman M; Miller DC; Ziff EB
1996 Aug;25(2):64-71, Pediatric neurosurgery
We have used the polymerase chain reaction to clone and characterize growth factor receptor tyrosine kinases (RTKs) expressed in 3 pathologically distinct pediatric brain tumors, an anaplastic ependymoma, a glioblastoma multiforme and a primitive neuroectodermal tumor (PNET). These neoplasms are presumed to be derived from embryonic neuroepithelial precursor cells of the central nervous system. This cloning demonstrated expression of 24 distinct kinase genes: 16 receptor type kinases and 8 nonreceptor type kinases. The expression of 6 receptors, including Hek2, IRR, Ryk, FGFR3, and 2 members of the newly identified cell adhesion kinase receptor family, DDR and TKT, in such tumors has not been reported previously. Northern analysis of mRNA levels revealed DDR expression in 6 of 7 pediatric brain tumors including an ependymoma, PNET, glioblastoma and astrocytoma, and also in an adult pheochromocytoma. Thus, the DDR cell adhesion kinase may be widely expressed in pediatric brain tumors. Also, PCR cloning may be an effective procedure for characterizing RTKs in clinical tissue samples and revealing the expression of novel RTK species
— id: 7276, year: 1996, vol: 25, page: 64, stat: Journal Article,

The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity
Wong HK; Ziff EB
1996 Jan;70(1):332-340, Journal of virology
We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level
— id: 57385, year: 1996, vol: 70, page: 332, stat: Journal Article,

Basic fibroblast growth factor selectively increases desensitization of NMDA receptor currents in cultured rat hippocampal neurons
Boxer, A. L.; Ziff, E. B.
1995 ;21(1-3):1547-1547, Abstracts (Society for Neuroscience)
— id: 92655, year: 1995, vol: 21, page: 1547, stat: Journal Article,

Regulation of the tyrosine hydroxylase gene requires cooperation between the TH-FSE and CRE
Ghee, M.; Miller, J. C.; Ziff, E. B.
1995 ;21(1-3):92-92, Abstracts (Society for Neuroscience)
— id: 92658, year: 1995, vol: 21, page: 92, stat: Journal Article,

Regulation of cell proliferation and differentiation by Myc
Hopewell R; Li L; MacGregor D; Nerlov C; Ziff EB
1995 ;19:85-89, Journal of cell science. Supplement
Myc is a nuclear phosphoprotein which controls cellular proliferation, most likely by regulating gene activity. The finding that the neuronal model cell line PC12 lacks the Myc DNA binding partner, the Max protein, and the demonstration that Myc is a repressor of gene activity as well as a transactivator, lead to models for Myc action in regulating cell growth
— id: 6917, year: 1995, vol: 19, page: 85, stat: Journal Article,

The nerve growth factor-responsive PC12 cell line does not express the Myc dimerization partner Max
Hopewell R; Ziff EB
1995 Jul;15(7):3470-3478, Molecular & cellular biology
Heterodimerization of Max with the nuclear oncoprotein Myc and the differentiation-associated proteins Mad and Mxi1 enables these factors to bind E-box sites in DNA and control genes implicated in cell proliferation and differentiation. We show that in the PC12 pheochromocytoma tumor cell line, functional Max protein is not expressed because of the synthesis of a mutant max transcript. This transcript encodes a protein incapable of homo- or heterodimerization. Furthermore, the mutant Max protein, unlike wild-type Max, is incapable of repressing transcription from an E-box element. Synthesis of mutant max transcripts appears to be due to a homozygous chromosomal alteration within the max gene. Reintroduction of max into PC12 cells results in repression of E-box-dependent transcription and a reduction in growth rate, which may explain the loss of Max expression either during the growth of the pheochromocytoma or in subsequent passage of the PC12 cell line in vitro. Finally, the ability of these cells to divide, differentiate, and apoptose in the absence of Max demonstrates for the first time that these processes can occur via Max- and possibly Myc-independent mechanisms
— id: 12751, year: 1995, vol: 15, page: 3470, stat: Journal Article,

Raf phosphorylates p53 in vitro and potentiates p53-dependent transcriptional transactivation in vivo
Jamal S; Ziff EB
1995 Jun 1;10(11):2095-2101, Oncogene
Using recombinant baculovirus expressed p53 and Raf proteins, we show that activated Raf-1 kinase can phosphorylate mouse p53 in vitro. We also show that co-expression of vRaf and p53 in NIH3T3 fibroblasts, potentiates the ability of p53 to transactivate a minimal promoter with a p53 cognate DNA binding site. A dominant negative mutant of Raf inhibits the transactivation function of p53 in NIH3T3 fibroblasts. Incubation of Raf-1 kinase with a series of p53 derived synthetic peptides maps the Raf-1 phosphorylation sites to the 27 amino terminal residue region of p53 which coincides with the transactivation domain. Phosphorylation occurs on serines which are phosphorylated in vivo. Our results suggest that the transactivation function of p53 can be regulated by a signaling cascade involving Raf
— id: 12766, year: 1995, vol: 10, page: 2095, stat: Journal Article,

Cyclin D2 and Ha-Ras transformed rat embryo fibroblasts exhibit a novel deregulation of cell size control and early S phase arrest in low serum
Kerkhoff E; Ziff EB
1995 May 1;14(9):1892-1903, EMBO journal
The D-type cyclins are growth factor-regulated delayed early functions which peak at the G1/S transition, are thought to regulate entry into S phase and have been implicated in tumorigenesis. Here, we show that cyclin D2 can co-operate with Ha-Ras to impose a novel transformed state on rat embryo fibroblasts (REF). While clonal cyclin D2/Ha-Ras REF transformants exhibit a characteristic transformed phenotype in high serum, in low serum they arrest cell proliferation and display profound morphological and cytological changes indicating loss of control of cell mass and deregulation of the G1/S transition. Notably, in low serum, despite re-establishment of actin cables and arrest of proliferation, cell mass continues to increase, creating giant cells up to 10 x normal size. Also, during low-serum culture the cells make a very gradual but progressive entry into S phase, reaching a 2.4N DNA content after 6 days. PCNA is expressed and 2N and 4N cells are largely absent, and thus the cells undergo a novel S phase arrest. While transfer to low serum induced the retinoblastoma protein to enter its dephosphorylated state, and cyclin A, cyclin B and cdc2 levels to decrease, all as normal, cyclin E, cdk4, cdk2 and the exogenous cyclin D2 persisted at high levels. These results indicate that cyclin D2 and Ha-Ras can transform cells when mitogenic signals from growth factors are provided. However, in low serum, co-operation of cyclin D2 and Ha-Ras provides only a subset of the progression signals and these are sufficient for G1-related cell mass increase and S phase entry, but are insufficient for full cell cycling
— id: 6658, year: 1995, vol: 14, page: 1892, stat: Journal Article,

Deregulated messenger RNA expression during T cell apoptosis
Kerkhoff E; Ziff EB
1995 Dec 11;23(23):4857-4863, Nucleic acids research
The IL-2 dependent murine cytotoxic T cell line CTLL-2 undergoes programmed cell death when deprived of its specific cytokine. We analyzed the expression of cell cycle related genes after IL-2 deprivation. Here we show that a generalized decrease and re elevation of the levels of mRNA takes place as part of the apoptotic program. The levels of several mRNAs encoding cell cycle functions, including cyclin D2, cyclin D3, cyclin B1, c-myc and max all declined at 1.5-3 h following IL-2 deprivation. Notably, the maxmRNA, which was shown to be expressed in proliferating, growth arrested and differentiated cells, is down regulated with the same kinetics as the other mRNAs. Surprisingly, the mRNAs whose levels declined at 1.5-3 h rose again at 10-14 h, a time which closely followed the time of the first detection of apoptotic DNA degradation, at 8 h, but which precedes actual loss of viability, at 14 h, as measured by trypan blue exclusion. Of all analyzed genes only the expression of the S-phase specific histone H4 gene resists the initial decrease and declines gradually over the course of cell death. Measurement of c-Myc protein synthesis at a late stage of the apoptotic program revealed that the accumulated reinduced mRNA is not translated into protein. Because transcriptional regulation has been shown to be dependent on the chromatin structure, the reinduction may be triggered by relaxation of the chromatin caused by alterations in the chromatin structure of apoptotic cells
— id: 8001, year: 1995, vol: 23, page: 4857, stat: Journal Article,

CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells
Nerlov C; Ziff EB
1995 Sep 1;14(17):4318-4328, EMBO journal
We have analysed the molecular basis for the function of the C/EBP alpha transactivation domain. We have previously found that the three C/EBP alpha transactivation elements (TEs) synergistically activate transcription in mammalian cells. We now report that two of these elements, TE-I and -II, co-operatively mediate in vitro binding of C/EBP alpha to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP alpha coincide, and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and -II in both yeast and mammalian cells. Our data demonstrate a biochemical basis for the modular buildup of transactivation domains, and indicate that this modularity is conserved in eukaryote evolution. We also show that the same amino acid motifs in a cellular activator can co-operate to mediate contacts between the activator and two distinct basal transcription factors. These results suggest that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related, and support the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process
— id: 17523, year: 1995, vol: 14, page: 4318, stat: Journal Article,

Cloning of the neuropeptide NPFF and NPAF precursor from bovine, rat, mouse and human
Vilim, F. S.; Ziff, E.
1995 ;21(1-3):760-760, Abstracts (Society for Neuroscience)
— id: 92657, year: 1995, vol: 21, page: 760, stat: Journal Article,

NGF regulates the PC12 cell cycle machinery through specific inhibition of the Cdk kinases and induction of cyclin D1
Yan GZ; Ziff EB
1995 Sep;15(9):6200-6212, Journal of neuroscience
We have examined the effects of NGF on components of the PC12 cell cycle machinery. We show that NGF represses over 6-8 d the levels of specific cdk kinase proteins and the G2-M phase specific cyclin B1 and the S phase marker PCNA as well as the level of phosphorylation of the retinoblastoma (Rb) protein. All of these changes may provide a basis for a NGF block to cell cycling. Unexpectedly, the G1 phase-specific cyclin D1 was dramatically increased by inducers of differentiation (NGF and FGF), but not by inducers of proliferation (EGF and insulin). Although the levels of cyclin D1/cdk2 and cyclin D1/cdk4 complexes increased following NGF treatment, as did cyclin D1/Rb complexes, the associated kinase activities declined, indicating that NGF also induces an inhibitor of cdk kinase activity. In agreement, NGF induced the cdk inhibitory protein, p21, which was found in cyclin D1/cdk kinase complexes after NGF treatment. We show that vector over expression of cyclin D1 in PC12 is sufficient on its own to arrest the cells in G1 phase and inhibit expression of PCNA. These results indicate that NGF induction of cyclin D1 and inactivation of cdk kinases, the latter possibly by increase of p21, play a central role in the NGF block of PC12 cell cycling
— id: 8053, year: 1995, vol: 15, page: 6200, stat: Journal Article,

NGF inducible regulatory elements in the rat cyclin D1 promoter
Yan, G.-Z.; Ziff, E. B.
1995 ;21(1-3):1049-1049, Abstracts (Society for Neuroscience)
— id: 92656, year: 1995, vol: 21, page: 1049, stat: Journal Article,

Selective down regulation of NMDA receptor currents and NMDAR2B messenger RNA levels by basic fibroblast growth factor in cultured rat hippocampal neurons
Boxer, A. L.; Moreno, H.; Ruby, B.; Ziff, E. B.
1994 ;20(1-2):673-673, Abstracts (Society for Neuroscience)
— id: 92664, year: 1994, vol: 20, page: 673, stat: Journal Article,

Nerve growth factor regulates the expression and activity of p33cdk2 and p34cdc2 kinases in PC12 pheochromocytoma cells
Buchkovich KJ; Ziff EB
1994 Nov;5(11):1225-1241, Molecular biology of the cell
In the absence of serum, nerve growth factor (NGF) promotes the survival and differentiation of the PC12 pheochromocytoma cell line. In the presence of serum, NGF acts primarily as a differentiation factor and negative regulator of cell cycling. To investigate NGF control of cell cycling, we have analyzed the regulation of cyclin dependent kinases during PC12 cell differentiation. NGF treatment leads to a reduction in the steady-state protein levels of p33cdk2 and p34cdc2, two key regulators of cell cycle progression. The decrease in p33cdk2 and p34cdc2 coincides with a decrease in the enzymatic activity of cyclinA-p34cdc2, cyclinB-p34cdc2, cyclinE-p33cdk2, and cyclinA-p33cdk2 kinases. The decline in p33cdk2 and p34cdc2 kinase activity in response to NGF is accelerated in cells that over-express the p140trk NGF receptor, suggesting that the timing of the down- regulation is dependent on the level of p140trk and the strength of the NGF signal. The level of cyclin A, a regulatory subunit of p33cdk2 and p34cdc2, is relatively constant during PC12 differentiation. Nevertheless, the DNA binding activity of the cyclinA-associated transcription factor E2F/DP decreases. Thus, NGF down-regulates the activity of cyclin dependent kinases and cyclin-transcription factor complexes during PC12 differentiation
— id: 56616, year: 1994, vol: 5, page: 1225, stat: Journal Article,

Nerve growth factor regulates the expression and kinase activity of p33-cdk2 and p34-cdc2
Buchkovich, K. J.; Ziff, E. B.
1994 ;20(1-2):1481-1481, Abstracts (Society for Neuroscience)
— id: 92659, year: 1994, vol: 20, page: 1481, stat: Journal Article,

Regulation of the tyrosine hydroxylase promoter
Ghee, M.; Miller, J. C.; Ziff, E. B.
1994 ;20(1-2):280-280, Abstracts (Society for Neuroscience)
— id: 92666, year: 1994, vol: 20, page: 280, stat: Journal Article,

Fos family members successively occupy the tyrosine hydroxylase gene AP-1 site after nerve growth factor or epidermal growth factor stimulation and can repress transcription
Gizang-Ginsberg E; Ziff EB
1994 Feb;8(2):249-262, Molecular endocrinology
Nerve growth factor induces the neuronal-like differentiation of PC12 cells, and epidermal growth factor promotes PC12 viability and is weakly mitogenic. Despite these differences, both growth factors induce indistinguishable patterns of transient delayed transcription of the tyrosine hydroxylase (TH) gene and the expression of proteins encoded by Fos gene family members. Thus, TH expression is sensitive to signaling pathways common to these two growth factors. We show that c-fos and fosB successively occupy an AP-1 site-like element of the TH promoter after nerve growth factor treatment. Furthermore, under conditions of transient transfection, Fos family proteins may synergize with c-jun to transrepress TH gene transcription through the TH-fat-specific element. We show that the target of repression is the AP-1 site-like element that lies within the TH-fat-specific element. We demonstrate that this site is also a major positive acting site for TH control. These results suggest a model in which the long term effect of c-fos family protein expression is to limit the expression of the TH gene. We consider the novel properties of this element in providing temporal and cell type-specific regulation of TH transcription
— id: 56532, year: 1994, vol: 8, page: 249, stat: Journal Article,

Origin of PC12 pheochromocytoma cells: Evidence that loss of the MYC dimerization partner Max was a transforming event
Hopewell, R.; Ziff, E. B.
1994 ;20(1-2):1050-1050, Abstracts (Society for Neuroscience)
— id: 92662, year: 1994, vol: 20, page: 1050, stat: Journal Article,

Regulation of neuronal determination genes in PC12 and P19 cells
Issack, P. S.; Ziff, E. B.
1994 ;20(1-2):1077-1077, Abstracts (Society for Neuroscience)
— id: 92660, year: 1994, vol: 20, page: 1077, stat: Journal Article,

c-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II
Li LH; Nerlov C; Prendergast G; MacGregor D; Ziff EB
1994 Sep 1;13(17):4070-4079, EMBO journal
We show that c-Myc, in addition to activating transcription through E-box Myc binding sites (Ems), also represses transcription by a mechanism dependent on initiator (Inr) elements of the basal promoters of susceptible genes. Repression was first observed as a component of c-Myc biphasic regulation of the adenovirus-2 major late promoter (MLP), which contains both Inr and Ems sequences. Two differentiation-specific genes containing Inr, the C/EBP alpha and albumin genes, are repressed through their basal promoters by c-Myc, but are activated by the related B-HLH-LZ factor, USF. Repression requires both the B-HLH-LZ and Myc box II (MBII) domains. Significantly, a MBII deletion mutant which is deficient in repression, but transactivates normally, fails to cooperate with an activated ras gene to transform primary fibroblasts. Thus Myc-dependent transactivation is insufficient for Ras cooperation and the novel transcription repression function is implicated in Ras cooperation as well as the suppression of Inr-dependent genes
— id: 6680, year: 1994, vol: 13, page: 4070, stat: Journal Article,

Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-alpha on the serum albumin promoter
Nerlov C; Ziff EB
1994 Feb 1;8(3):350-362, Genes & development
We have studied the activation of the serum albumin promoter by transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) in the HepG2 hepatoma cell line. We find that three distinct mechanisms determine the ability of C/EBP alpha to activate this promoter in a cell-type-specific and cooperative manner. First, the trans-activating function of C/EBP alpha is generated through cooperation between three separate domains of the protein that we have named trans-activation elements (TE-I through TE-III). The TEs have little or no ability to activate transcription by themselves, but any two can cooperate to do so, both in the C/EBP alpha protein and when linked to the GAL4 DNA-binding domain. Second, TE-III was found to contain a negative regulatory subdomain, the function of which was alleviated when C/EBP alpha was bound in the environment of the albumin promoter. This formed the basis for cooperative activation of this promoter by C/EBP alpha. Finally, we demonstrate that the leucine zipper of C/EBP alpha participates in determining the cell type specificity of albumin promoter activation, as it exerts a strong negative effect on albumin promoter activation in the nonhepatic HeLa cell line but not in HepG2 cells. These findings shed new light on the mode of action of C/EBP alpha and show a novel function for leucine zipper in cell-type-specific gene expression
— id: 56569, year: 1994, vol: 8, page: 350, stat: Journal Article,

Defective processing of human adenovirus 2 late transcription unit mRNAs during abortive infections in monkey cells
Ross D; Ziff E
1994 Jul;202(1):107-115, Virology
Growth of human adenoviruses is severely restricted in monkey cells. We examined the synthesis of mRNAs from the Ad2 late transcription unit (LTU) in abortively infected monkey cells at late times in infection. All L2, L3, and L5 mRNAs were absent or drastically reduced in abortive infections. Most L1 and L4 mRNAs were also greatly decreased in abortive infections; however, a single large messenger was produced from each of the L1 and L4 families, at levels approaching those found in productive infections. The pattern of i-leader containing mRNAs was also changed in abortive infections. These defects could be corrected in monkey cells by the presence of SV40 T antigen or an altered adenoviral DNA binding protein. These defects in late gene expression in abortive infections could not be attributed to differences in transcription along the LTU or levels of DNA replication. In abortively infected cells, nuclear levels of L5 mRNA were decreased 2 to 6 fold, while cytoplasmic levels were decreased over 200-fold. These findings imply a general defect in processing of viral mRNAs, most likely due to defective splicing and/or transport, during abortive infections
— id: 6502, year: 1994, vol: 202, page: 107, stat: Journal Article,

Antibodies to the Aplysia neuropeptide myomodulin stain varicose processes in rat brain
Vilim, F. S.; Ziff, E.
1994 ;20(1-2):515-515, Abstracts (Society for Neuroscience)
— id: 92665, year: 1994, vol: 20, page: 515, stat: Journal Article,

Complementary functions of E1a conserved region 1 cooperate with conserved region 3 to activate adenovirus serotype 5 early promoters
Wong HK; Ziff EB
1994 Aug;68(8):4910-4920, Journal of virology
The amino-terminal region of the adenovirus type 5 E1a protein including conserved regions (CRs) 1 and 2 binds the 105-kDa retinoblastoma protein and a second, 300-kDa, cellular protein. We show that mutant viruses with deletions of CR1 which release the binding of either p105 or p300 still activate early promoters and infect cells productively. However, mutations which disrupt binding of both proteins disrupt early promoter activity and block the viral life cycle. Ela CR3, which has an established role in early promoter activation, can act in trans to the amino-terminal functions. This suggests that the amino terminus provides distinct, redundant functions related to p300 and Rb binding that synergize with CR3 to transactivate early genes
— id: 56604, year: 1994, vol: 68, page: 4910, stat: Journal Article,

A PC12 cells differentiation induced by NGF is associated with induction of cyclin D1 and its complexes
Yan, G-Z.; Ziff, E.
1994 ;20(1-2):675-675, Abstracts (Society for Neuroscience)
— id: 92663, year: 1994, vol: 20, page: 675, stat: Journal Article,

The cloning of multiple protein tyrosine kinase genes expressed in human pediatric brain tumors and developing brain
Ziff, Edward B.; Rothman, Marc; Weiner, Howard L.; McCarthy, Maria
1994 ;20(1-2):1050-1050, Abstracts (Society for Neuroscience)
— id: 92661, year: 1994, vol: 20, page: 1050, stat: Journal Article,

Adenovirus 5 E1A proteins disrupt the neuronal phenotype and growth factor responsiveness of PC12 cells by a conserved region 1-dependent mechanism
Boulukos KE; Ziff EB
1993 Feb;8(2):237-248, Oncogene
Expression in PC12 cells of adenovirus 5 E1a proteins dramatically changes cell morphology and disrupts neuronal differentiation. We demonstrate that the nerve growth factor (NGF) receptors, p140trk and p75NGFR, as well as the epidermal growth factor receptor are undetectable in E1a-expressing PC12 cells. This correlates with a repression of mRNAs for the chromaffin- and neuronal-specific proteins, tyrosine hydroxylase and peripherin, while more ubiquitously expressed genes remain unaffected. One possible mechanism of E1a action could thus be the repression of a coordinately regulated group of chromaffin- and/or neuronal-specific genes. Furthermore, we show taht E1a conserved region 1, which binds p105Rb and p300, is necessary for this E1a-dependent effect. This indicates that cellular proteins interacting with E1a conserved region 1 may be implicated in growth arrest, expression of neuron-specific functions and orderly differentiation of PC12 cells in response to NGF
— id: 13280, year: 1993, vol: 8, page: 237, stat: Journal Article,

Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain
Ferre-D'Amare AR; Prendergast GC; Ziff EB; Burley SK
1993 May 6;363(6424):38-45, Nature
The three-dimensional structure of the basic/helix-loop-helix/leucine zipper domain of the transcription factor Max complexed with DNA has been determined by X-ray crystallography at 2.9 A resolution. Max binds as a dimer to its recognition sequence CACGTG by direct contacts between the alpha-helical basic region and the major groove. This symmetric homodimer, a new protein fold, is a parallel, left-handed, four-helix bundle, with each monomer containing two alpha-helical segments separated by a loop. The two alpha-helical segments are composed of the basic region plus helix 1 and helix 2 plus the leucine repeat, respectively. As in GCN4, the leucine repeat forms a parallel coiled coil
— id: 17524, year: 1993, vol: 363, page: 38, stat: Journal Article,

DOMINANT NEGATIVE MYC MUTANTS SHED NEW LIGHT ON MYC FUNCTION
MACGREGOR, DN; ZIFF, EB
1993 APR ;41(2):A266-A266, Clinical research
— id: 54272, year: 1993, vol: 41, page: A266, stat: Journal Article,

Biphasic effect of Max on Myc cotransformation activity and dependence on amino- and carboxy-terminal Max functions
Prendergast GC; Hopewell R; Gorham BJ; Ziff EB
1992 Dec;6(12A):2429-2439, Genes & development
In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino- and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino- or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo
— id: 56575, year: 1992, vol: 6, page: 2429, stat: Journal Article,

A new bind for Myc
Prendergast GC; Ziff EB
1992 Mar;8(3):91-96, Trends in genetics
Recent studies centered on the c-Myc basic/helix-loop-helix/leucine zipper (B/HLH/LZ) motifs have led to the identification of a DNA recognition sequence for c-Myc and the isolation of a novel protein that forms a DNA-binding complex with c-Myc in vitro. These advances may make it possible to address directly the long-standing question of c-Myc function in vivo
— id: 17525, year: 1992, vol: 8, page: 91, stat: Journal Article,

Defective synthesis of early region 4 mRNAs during abortive adenovirus infections in monkey cells
Ross D; Ziff E
1992 May;66(5):3110-3117, Journal of virology
Human adenovirus 2 grows poorly in monkey cells, partly because of defects in late gene expression. Since deletions in early region 4 (E4) cause similar defects in late gene expression, we examined E4 mRNA expression in abortive infections. Processing of E4 mRNAs was defective during abortive infections, most likely at the level of splicing. At early times in productive infections in HeLa cells, the major E4 species produced is a 2-kb mRNA; at late times, a shift occurs so that smaller spliced E4 mRNAs are also produced. In CV-1 cells, a nonpermissive monkey cell line, this shift did not take place and only the 2-kb species was produced at late times, suggesting a defect in E4 mRNA splicing during abortive infections. The adenovirus DNA-binding protein (DBP) was required for normal processing of E4 mRNAs, since a host range mutant (hr602) containing an altered DBP gene showed a normal late E4 mRNA pattern in CV-1 cells; in addition, DBP was required during infections in HeLa cells for late E4 mRNA expression. DBP was not required for production of the late E4 pattern in transient expression assays in HeLa or 293 cells, suggesting that a second factor in addition to the DBP, present during infection but not transfection, modulates E4 mRNA processing
— id: 13625, year: 1992, vol: 66, page: 3110, stat: Journal Article,

Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a negative regulatory element
Thompson MA; Lee E; Lawe D; Gizang-Ginsberg E; Ziff EB
1992 Jun;12(6):2501-2513, Molecular & cellular biology
The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor (NGF) stimulates PC12 cells to differentiate into neurons. We have studied its transcriptional regulation in order to better understand the neuronal-specific end steps of the signal transduction pathway of NGF. By 5' deletion mapping of the peripherin promoter, we have localized two positive regulatory elements necessary for full induction by NGF: a distal positive element and a proximal constitutive element within 111 bp of the transcriptional start site. In addition, there is a negative regulatory element (NRE; -179 to -111), the deletion of which results in elevated basal expression of the gene. Methylation interference footprinting of the NRE defined a unique sequence, GGCAGGGCGCC, as the binding site for proteins present in nuclear extracts from both undifferentiated and differentiated PC12 cells. However, DNA mobility shift assays using an oligonucleotide probe containing the footprinted sequence demonstrate a prominent retarded complex in extracts from undifferentiated PC12 cells which migrates with slower mobility than do the complexes produced by using differentiated PC12 cell extract. Transfection experiments using peripherin-chloramphenicol acetyltransferase constructs in which the footprinted sequence has been mutated confirm that the NRE has a functional, though not exclusive, role in repressing peripherin expression in undifferentiated and nonneuronal cells. We propose a two-step model of activation of peripherin by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in depression of the gene
— id: 13578, year: 1992, vol: 12, page: 2501, stat: Journal Article,

PC12 CELL DIFFERENTIATION IS ALTERED BY ADENOVIRUS-5 E1A PRODUCTS
BOULUKOS K E; ZIFF E B
1991 ;17(1-2):37-37, Abstracts (Society for Neuroscience)
— id: 92667, year: 1991, vol: 17, page: 37, stat: Journal Article,

Differential spatial and temporal expression of two type III intermediate filament proteins in olfactory receptor neurons
Gorham JD; Ziff EB; Baker H
1991 Sep;7(3):485-497, Neuron
Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons
— id: 13913, year: 1991, vol: 7, page: 485, stat: Journal Article,

cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to trans-locate to the nucleus and induce c-fos transcription
Metz R; Ziff E
1991 Oct;5(10):1754-1766, Genes & development
The c-fos serum response element (SRE) is a multifunctional regulatory region of the c-fos promoter that responds to a variety of inducers. Recently, we have demonstrated that the SRE binds the C/EBP-related transcription factor rat NFIL-6 (rNFIL-6). In this study we show that rNFIL-6 is regulated by the cAMP second messenger pathway in the rat pheochromocytoma PC12 cell line. Following forskolin treatment, rNFIL-6 binding to the SRE is increased, and the factor becomes phosphorylated and undergoes a trans-location to the nucleus. In transient cotransfection assays, rNFIL-6 is capable of trans-activating the c-fos promoter in a manner dependent on the SRE. These data show that rNFIL-6 undergoes a novel activation in which cAMP-induced nuclear trans-location allows rNFIL-6 to bind to the SRE and contribute to c-fos activation. We propose that rNFIL-6 is an additional regulatory component of the c-fos gene, which provides cAMP responsiveness to the multifunctional SRE
— id: 13895, year: 1991, vol: 5, page: 1754, stat: Journal Article,

THE HELIX LOOP HELIX PROTEIN-RE12 AND THE C/EBP-RELATED FACTOR-RNFIL-6 BIND TO NEIGHBORING SITES WITHIN THE C-FOS SERUM RESPONSE ELEMENT
METZ, R; ZIFF, E
1991 DEC ;6(12):2165-2178, Oncogene
We show that members of two major families of transcription factors, the helix-loop-helix and C/EBP families, interact with the c-fos serum response element (SRE). Two cDNA clones encoding SRE binding factors (clones 9 and 21) were isolated by the direct screening of a PC12 lambda-gt11 cDNA library using SRE oligonucleotide sequences as probes. Clone 9 encodes the rat homolog of the human HLH transcription factor, E12 (called here rE12). Clone 21 encodes a b-zip domain polypeptide that is related to the liver transcription factor C/EBP, and is homologous to the human NFIL-6 transcription factor (called here rNFIL-6). Using in vitro-translated products we show that rNFIL-6 recognizes a 'CCAATT' motif which overlaps the c-fos dyad symmetry element (DSE), the binding site for serum regulatory factor (SRF). Factor rE12 binds to an E-box enhancer sequence, 'CATCTG', immediately adjacent to the rNFIL-6 site, within the SRE. Antibodies specific to rE12 and rNFIL-6 disrupt nucleoprotein complexes with these DNA-binding sites, confirming the interaction of native in vivo factors. We present evidence that rNFIL-6 and SRF binding are mutually exclusive, consistent with the overlap of their binding sites. The demonstration that rE12 and rNFIL-6 bind to the SRE at sites adjacent to the major c-fos regulatory element, the DSE, raises the possibility that helix-loop-helix and C/EBP families regulate the SRE and provide a new basis for the multifunctional properties of the SRE, including possible tissue specificity of expression
— id: 52123, year: 1991, vol: 6, page: 2165, stat: Journal Article,

Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation
Prendergast GC; Lawe D; Ziff EB
1991 May 3;65(3):395-407, Cell
Myn, a novel murine approximately 18 kd basic/helix-loop-helix/'leucine zipper' (B/HLH/LZ) protein, forms a specific DNA-binding complex with the c-Myc oncoprotein through the HLH/LZ motif in both proteins. c-Myc/Myn recognizes a c-Myc-binding site (GACCACGTGGTC) with higher affinity than either protein by itself. CpG methylation of the recognition site greatly inhibits DNA binding, suggesting that DNA methylation may regulate the c-Myc/Myn complex in vivo. In 3T3 fibroblasts, Myn mRNA levels are induced several-fold by serum with delayed early kinetics, suggesting regulation by immediate-early gene products. Coexpression of Myn in a myc/ras rat embryo fibroblast focus formation assay specifically augmented c-myc transforming activity. We suggest that interaction of Myn with c-Myc stabilizes sequence-specific DNA binding in vivo
— id: 14025, year: 1991, vol: 65, page: 395, stat: Journal Article,

Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo
Prendergast GC; Ziff EB
1991 Apr;10(4):757-766, EMBO journal
We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin
— id: 14087, year: 1991, vol: 10, page: 757, stat: Journal Article,

Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region
Prendergast GC; Ziff EB
1991 Jan 11;251(4990):186-189, Science
The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo
— id: 17526, year: 1991, vol: 251, page: 186, stat: Journal Article,

Nerve growth factor regulates tyrosine hydroxylase gene transcription through a nucleoprotein complex that contains c-Fos
Gizang-Ginsberg E; Ziff EB
1990 Apr;4(4):477-491, Genes & development
We have studied nerve growth factor (NGF) regulation of the expression of the tyrosine hydroxylase (TH) gene in PC12 cells. The TH gene encodes the initial and rate-limiting enzyme of the catecholamine biosynthetic pathway. We show that the TH gene is transiently transcriptionally induced by a mechanism reliant on new protein synthesis during 1-2 hr of NGF stimulation, a time following the induction of the c-fos gene at 15 min post-NGF treatment. A potential regulatory sequence located within the TH gene promoter, the TH-FSE, shares homology to a known regulatory element, the fat-specific element (FSE), which is found upstream from genes activated during adipocyte differentiation and binds the Fos-Jun transcription factor complex. We show that the TH-FSE DNA sequence elevates the basal level of transcription from the rat TH promoter and is required for NGF inducibility. This DNA element binds authentic Fos-Jun products produced abundance during NGF stimulation and by in vitro translation. We demonstrate further that the TH-FSE can bind proteins present in PC12 nuclear extracts in a sequence-specific manner. The DNA/nucleoprotein complex that forms increases in abundance during NGF stimulation and reaches a maximum level at 4 hr of treatment. Antibody inhibition studies utilizing an anti-Fos antibody indicate that Fos and/or Fos-related antigen(s) associate with the TH-FSE and suggest that the Fos protein family contributes to the regulation of TH in vivo. These results support a model in which NGF-induced immediate early genes, including c-Fos, contribute to the regulation of delayed early genes such as TH and thereby control neuronal differentiation
— id: 17528, year: 1990, vol: 4, page: 477, stat: Journal Article,

EXPRESSION OF THE NEURONAL INTERMEDIATE FILAMENT PROTEIN IFP PERIPHERIN IN THE RAT CNS AND OLFACTORY NEURONS
GORHAM J D; BAKER H; ZIFF E B
1990 ;16(1):51-51, Abstracts (Society for Neuroscience)
— id: 92668, year: 1990, vol: 16, page: 51, stat: Journal Article,

The expression of the neuronal intermediate filament protein peripherin in the rat embryo
Gorham JD; Baker H; Kegler D; Ziff EB
1990 Dec 15;57(2):235-248, Brain research. Developmental brain research
The expression of the neuronal type III intermediate filament protein peripherin was examined in the rat embryo during and following neuronogenesis in the spinal cord and the peripheral nervous system. In situ hybridization analysis reveals that peripherin mRNA is found in the mid-gestational rat embryo in ventral and lateral motoneurons in the spinal cord, and in neurons of all peripheral ganglia examined, including spinal, sympathetic, and enteric ganglia. Peripherin mRNA is seen only in post-migratory motoneurons or neuronal cells in aggregating ganglia, indicating that precursor cells do not express peripherin. To examine the expression of the protein, an affinity-purified antibody (anti-per) specific for a bacterially produced peripherin fusion protein was generated. Anti-per specifically recognizes a 58 kDa, cytoskeletal-enriched, nerve growth factor (NGF)-inducible protein of the expected tissue distribution. Immunocytodetection with anti-per shows that the initiation of peripherin protein synthesis is coincident with the morphological differentiation of neurons. In development, peripherin is one constituent of a program of gene expression activated at terminal neuronal differentiation
— id: 14243, year: 1990, vol: 57, page: 235, stat: Journal Article,

TRANSACTIVATION OF C-FOS AND BETA-ACTIN GENES BY RAF AS A STEP IN EARLY RESPONSE TO TRANSMEMBRANE SIGNALS
Jamal, S; Ziff, E
1990 Mar 29;344(6265):463-466, Nature
— id: 31886, year: 1990, vol: 344, page: 463, stat: Journal Article,

Isolation and characterization of eight tumor necrosis factor-induced gene sequences from human fibroblasts
Lee TH; Lee GW; Ziff EB; Vilcek J
1990 May;10(5):1982-1988, Molecular & cellular biology
A lambda cDNA library was prepared from polyadenylated RNA isolated from quiescent human diploid FS-4 fibroblasts stimulated with tumor necrosis factor for 3 h. Differential screening was used to isolate cDNA sequences that are stimulated by tumor necrosis factor. Eight distinct tumor necrosis factor-stimulated gene sequences (designated TSG-1, -6, -8, -12, -14, -21, -27, and -37) were partially sequenced and compared with known sequences from GenBank. TSG-1 was identical to the gene for interleukin-8. TSG-8 corresponded to the gene for monocyte chemotactic and activating factor. TSG-21 and -27 were identical to the genes for collagenase and stromelysin, respectively. The other four sequences showed no homologies with known genes. Patterns of induction of mRNAs corresponding to the eight cloned cDNAs by various cytokines, growth factors, and activators of second messenger pathways were analyzed in FS-4 cells
— id: 15540, year: 1990, vol: 10, page: 1982, stat: Journal Article,

Elevated c-myc expression in childhood medulloblastomas
MacGregor DN; Ziff EB
1990 Jul;28(1):63-68, Pediatric research
Medulloblastoma is a rare brain tumor usually occurring in late childhood or early adolescence. Little is known regarding the cell of origin or cellular events leading to its malignant transformation. We have studied the expression of developmentally regulated mRNA in tumor samples by the Northern hybridization assay to determine a relative stage at which a block to further differentiation occurs. In a series of five medulloblastoma tumors analyzed, only one of eight markers for cellular differentiation, the glial fibrillary acidic protein mRNA, was expressed at significant levels. Interestingly, three of six medulloblastoma tumor samples were found to have elevated levels of c-myc mRNA. In one of these tumors, we have found evidence of mutation of the c-myc protooncogene. We discuss possible mechanisms of c-myc activation in medulloblastoma tumors
— id: 17527, year: 1990, vol: 28, page: 63, stat: Journal Article,

Altered transcriptional activity of c-fos promoter plasmids in v-raf-transformed NIH 3T3 cells
Siegfried Z; Ziff EB
1990 Nov;10(11):6073-6078, Molecular & cellular biology
In cells transformed by v-raf, an oncogenic counterpart of the serine/threonine kinase Raf-1, regulatory elements of the c-fos promoter were active under conditions of cell growth or stimulation for which they were inactive in untransformed control cells. This suggests that v-raf transforms by deregulating transcription of early response genes
— id: 14303, year: 1990, vol: 10, page: 6073, stat: Journal Article,

Functional analysis of an isolated fos promoter element with AP-1 site homology reveals cell type-specific transcriptional properties
Velcich A; Ziff EB
1990 Dec;10(12):6273-6282, Molecular & cellular biology
A DNA element located at positions -295 to -289 of the c-fos promoter (FAP site) is highly homologous to a consensus 12-O-tetradecanoyl phorbol-13-acetate-responsive element (TRE) and to a cyclic AMP (cAMP)-responsive element (CRE). We found that an oligonucleotide containing the FAP element was a transcription regulator which was distinct from both the TRE and CRE. When cloned in multiple copies in front of a reporter gene in HeLa cells, the FAP oligonucleotide was a powerful constitutive activator sequence. Conversely, in the same cells, reporter plasmids containing multiple copies of the TRE of the human metallothionein gene required phorbol esters for their induction. In PC12 cells, the FAP oligonucleotide was cAMP responsive. Its activity was mediated through a cAMP-dependent protein kinase II and did not rely on ongoing protein synthesis for activation. Adenovirus E1a proteins activated viral promoters through ATF (activation transcription factor) consensus binding sequences identical to the CRE. However, E1a repressed the FAP oligonucleotide-associated transcriptional activity in HeLa cells. In PC12 cells, E1a neither transactivated nor transrepressed the basal and cAMP-stimulated FAP activity. In contrast, the CRE of the human c-fos promoter located at -60 was weakly induced by cAMP and E1a in both HeLa and PC12 cells. We suggest that the FAP oligonucleotide acts through a factor(s) distinct from those employed by the TRE and CRE and that the FAP-associated protein factor(s) may differ in HeLa and PC12 cells in expression or posttranslational regulation
— id: 14264, year: 1990, vol: 10, page: 6273, stat: Journal Article,

Transcription factors: a new family gathers at the cAMP response site
Ziff EB
1990 Mar;6(3):69-72, Trends in genetics
— id: 17529, year: 1990, vol: 6, page: 69, stat: Journal Article,

THE EXPRESSION OF THE NEURONAL INTERMEDIATE FILAMENT PROTEIN PERIPHERIN IN THE DEVELOPING RAT EMBRYO
GORHAM J D; ZIFF E B
1989 ;15(2):1324-1324, Abstracts (Society for Neuroscience)
— id: 92669, year: 1989, vol: 15, page: 1324, stat: Journal Article,

Behind the Fos and Jun leucine zipper
Kouzarides T; Ziff E
1989 Nov;1(3):71-76, Cancer cells
The production of the nuclear oncoproteins Fos and Jun is rapidly induced in response to extracellular signals. In the nucleus, the two proteins combine to form a tight complex via leucine zipper domains. The resulting Fos-Jun heterodimer can bind to the TPA-responsive element (TRE) by way of a novel, highly basic motif and can activate the transcription of TPA-responsive genes. The existence of several Fos- and Jun-related proteins with dimerization and DNA binding properties similar to Fos and Jun suggests that these two oncoproteins may be part of a network of related but functionally distinct transcription factors
— id: 10449, year: 1989, vol: 1, page: 71, stat: Journal Article,

Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding
Kouzarides T; Ziff E
1989 Aug 17;340(6234):568-571, Nature
The products of the fos and jun protooncogenes form a stable heterodimer which binds to the TPA-responsive element (TRE) TGACTCA with high affinity. These two proteins, together with the yeast GCN4 protein, belong to a growing family of transcription factors, including FosB, Fra1, JunB and JunD, whose members share a highly conserved DNA-binding domain. This domain is composed of two structures: a basic motif, which is thought to bind directly to DNA; and a leucine zipper, which provides a dimerization interface. Although this domain is highly conserved in Fos, Jun and GCN4, each of these three proteins has very different relative affinities for the TRE. To understand these differences, we used 'domain-swapping' experiments designed to test the relative contributions of the basic motif and the leucine zipper to TRE-binding affinity. Here we show that fos, jun and GCN4 have different affinities for the TRE due to differences in the hetero- or homo-dimerization capacity of their leucine zipper domains; the basic motifs of these three proteins have comparable DNA binding potential. These results indicate that leucine zippers control the types of protein complexes which can associate with a TRE and regulate gene expression
— id: 10521, year: 1989, vol: 340, page: 568, stat: Journal Article,

DNA-binding motif
Prendergast GC; Ziff EB
1989 Oct 5;341(6241):392-392, Nature
— id: 17530, year: 1989, vol: 341, page: 392, stat: Journal Article,

Transcription activation by serum, PDGF, and TPA through the c-fos DSE: cell type specific requirements for induction
Siegfried Z; Ziff EB
1989 Jan;4(1):3-11, Oncogene
We have investigated the sequences that are necessary and sufficient for the induction of the c-fos gene by serum, TPA or PDGF in different cell types. The dyad symmetry element (DSE) is a regulatory element of the c-fos gene previously shown to be required for induction of c-fos transcription by serum. We show that the DSE is also necessary for the induction of c-fos by either TPA or PDGF in NIH3T3 cells. We also show that in NIH 3T3 cells the DSE is sufficient to confer inducibility on a heterologous promoter, the beta-globin promoter, when serum provides the stimulus. However, it is not sufficient when either TPA or PDGF is the inducer. This suggests a requirement in 3T3 cells for cooperating sequence elements for TPA or PDGF induction but not for serum. Interestingly, the need for cooperating elements for TPA induction is abolished in HeLa cells since the DSE alone is sufficient for TPA inducibility of the beta-globin promoter in these cells. Thus, the highly transformed HeLa cell line displays diminished sequence requirements for TPA induction. We discuss the possibility that mutations which diminish the stringent transcriptional control of protooncogenes such as c-fos may contribute to the transformed state
— id: 10750, year: 1989, vol: 4, page: 3, stat: Journal Article,

Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein
Thompson MA; Ziff EB
1989 Jan;2(1):1043-1053, Neuron
We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF
— id: 10810, year: 1989, vol: 2, page: 1043, stat: Journal Article,

Expression of the K-fgf protooncogene is repressed during differentiation of F9 cells
Velcich A; Delli-Bovi P; Mansukhani A; Ziff EB; Basilico C
1989 ;5(1):31-37, Oncogene research
Utilizing F9 embryonal carcinoma cells as a model system for early mammalian development, we have studied the pattern of expression of the endogenous murine homolog of the human K-fgf/hst oncogene, which encodes a new member of the fibroblast growth factors (FGFs) family. The K-fgf mRNA is expressed in undifferentiated F9 cells and its level becomes undetectable upon the induction of differentiation. Furthermore, a growth-promoting activity with properties identical to those of K-FGF is present in the conditioned medium of F9 cells, but absent in that of differentiated cells. Shut-off of K-fgf expression is mediated at the transcriptional level. The acidic FGF gene is also expressed in undifferentiated F9 cells and down modulated once differentiation is induced. In contrast, int-2, another member of the FGF gene family, is transcriptionally induced in differentiated F9 cells. Our data suggest that single members of the FGF gene family may perform distinct functions in vivo, and that the physiological role of K-FGF may be related to early development
— id: 10762, year: 1989, vol: 5, page: 31, stat: Journal Article,

The adenovirus-5 12S E1a protein, but not the 13S induces expression of the endoA differentiation marker in F9 cells
Velcich A; Ziff EB
1989 Jun;4(6):707-713, Oncogene
F9 embryonal carcinoma (EC) cells serve as a model system for early mammalian development. We have investigated the effect of the E1a gene products on the F9 cell differentiation program by stably introducing into these cells plasmids which express wild type or mutant forms of E1a. We have found that expression of the 12S E1a mRNA product results in the expression of the endoA gene, a marker specific for the differentiated phenotype of F9 cells, as well as an altered cell morphology associated with the differentiated state. These alterations are not observed in F9 cells which express the 13S E1a product. Other markers specific for the differentiated state are regulated normally in the E1a transformants following exposure to retinoic acid (R.A.) and dibutyryl cyclic AMP (dbcAMP). We discuss these results in the context of the well established transcriptional activities of the E1a proteins
— id: 10607, year: 1989, vol: 4, page: 707, stat: Journal Article,

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells
Vosatka RJ; Hermanowski-Vosatka A; Metz R; Ziff EB
1989 Mar;138(3):493-502, Journal of cellular physiology
The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins
— id: 17531, year: 1989, vol: 138, page: 493, stat: Journal Article,

Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses
Jones NC; Rigby PW; Ziff EB
1988 Mar;2(3):267-281, Genes & development
— id: 17533, year: 1988, vol: 2, page: 267, stat: Journal Article,

The role of the leucine zipper in the fos-jun interaction
Kouzarides T; Ziff E
1988 Dec 15;336(6200):646-651, Nature
Mutagenesis of the fos protein supports the hypothesis that a heptad repeat of leucine residues stabilizes the interaction between the fos and jun proteins. We show that the complex between fos and jun can bind to DNA more tightly than either protein alone and that basic residues adjacent to the leucine repeat of fos contribute to the DNA-binding potential of the complex
— id: 10861, year: 1988, vol: 336, page: 646, stat: Journal Article,

A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein
Leonard DG; Gorham JD; Cole P; Greene LA; Ziff EB
1988 Jan;106(1):181-193, Journal of cell biology
Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins
— id: 11216, year: 1988, vol: 106, page: 181, stat: Journal Article,

GENE-REGULATION BY GROWTH-FACTORS
Metz, R; Gorham, J; Siegfried, Z; Leonard, D; Gizangginsberg, E; Thompson, MA; Lawe, D; Kouzarides, T; Vosatka, R; Macgregor, D; Jamal, S; Greenberg, ME; Ziff, EB
1988 Jul-Aug;53(4):727-737, Cold Spring Harbor symposia on quantitative biology
— id: 31623, year: 1988, vol: 53, page: 727, stat: Journal Article,

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells
Riabowol KT; Vosatka RJ; Ziff EB; Lamb NJ; Feramisco JR
1988 Apr;8(4):1670-1676, Molecular & cellular biology
Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis
— id: 17532, year: 1988, vol: 8, page: 1670, stat: Journal Article,

The amino-terminal region of the adenovirus serotype 5 E1a protein performs two separate functions when expressed in primary baby rat kidney cells
Smith DH; Ziff EB
1988 Sep;8(9):3882-3890, Molecular & cellular biology
Adenovirus serotype 5 E1a proteins immortalize primary cells and in cooperation with products of a second oncogene, such as adenovirus serotype 5 E1b or EJ ras, produce full transformation. E1a also activates transcription of specific viral and cellular promoters, represses enhancer-dependent genes, and induces cellular DNA synthesis in quiescent cells. Comparison of different adenovirus serotypes has identified three conserved regions in the E1a protein sequence. We have analyzed E1a mutants with deletions-linker insertions in or preceding the first conserved region, region 1 (amino acids 40 through 77 of adenovirus serotype 5 E1a). E1a mutants which have in-frame deletions-substitutions in region 1 or pre-region 1 sequences were reconstructed into adenovirus to yield a total of 14 mutant viruses. All the mutant viruses showed wild-type growth in HeLa cells, confirming that region 1 is nonessential in these cells. However, we show that region 1 provides two distinct functions in infected primary rodent cells. One function is essential for induction of cell DNA synthesis, and the other is essential for focus formation. In addition, our results are consistent with a requirement for the DNA induction function in focus formation
— id: 10983, year: 1988, vol: 8, page: 3882, stat: Journal Article,

Adenovirus E1a ras cooperation activity is separate from its positive and negative transcription regulatory functions
Velcich A; Ziff E
1988 May;8(5):2177-2183, Molecular & cellular biology
The E1a gene of adenovirus encodes two proteins, 289 and 243 amino acids long, which have positive (transactivator) and negative (enhancer repressor) RNA polymerase II transcriptional regulatory properties and cell transformation activities including cooperation with an activated ras gene. The E1a transforming functions more closely correlate with the repressor property than with transactivation in that both E1a proteins express the repressor and transformation functions while only the 289-amino-acid protein is an efficient transactivator. To understand whether the transcriptional regulatory activities of E1a are related to its ras cooperation activity, we generated a series of mutant E1a expression vectors by linker insertion mutagenesis of the 289-amino-acid protein. Here we describe a new class of mutants which although defective for enhancer repression still can cooperate with the ras oncogene in cell transformation. The mutants are also defective in transcription transactivation. Our data suggest that enhancer repression and transformation via ras cooperation are separate E1a functions and that cooperation with ras does not rely on either of the RNA polymerase II transcription regulatory functions of E1a. We also show that mutations which inactivate enhancer repression are not confirmed to a single critical domain necessary for repression. We therefore propose that the integrity of the overall configuration of the E1a proteins is important for the repression activity
— id: 11117, year: 1988, vol: 8, page: 2177, stat: Journal Article,

Nuclear oncogenes
Alt, Frederick W.; Harlow, Edward.; Ziff, Edward
Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory, 1987,
— id: 376, year: 1987, vol: , page: , stat: ,

Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro
Greenberg ME; Siegfried Z; Ziff EB
1987 Mar;7(3):1217-1225, Molecular & cellular biology
In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene
— id: 17534, year: 1987, vol: 7, page: 1217, stat: Journal Article,

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells
Leonard DG; Ziff EB; Greene LA
1987 Sep;7(9):3156-3167, Molecular & cellular biology
Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons
— id: 11371, year: 1987, vol: 7, page: 3156, stat: Journal Article,

MOLECULAR-CLONING OF RAT DOPAMINE BETA-HYDROXYLASE CDNA
Mcmahon, A; Kuhn, LJ; Leonard, DGB; Ziff, EB; Sabban, EL
1987 May 1;46(6):2136-2136, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 31188, year: 1987, vol: 46, page: 2136, stat: Journal Article,

Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells
Nepveu A; Levine RA; Campisi J; Greenberg ME; Ziff EB; Marcu KB
1987 ;1(3):243-250, Oncogene
We have analysed the regulation of c-myc expression in murine fibroblasts and F9 teratocarcinoma cells. The initiation of c-myc transcription is induced to similar levels after serum stimulation of confluent and subconfluent Balb/c A31 fibroblasts while intragenic pausing within the gene's first exon remains unaffected. Sense c-myc transcription continues unabated for at least 18 hours in subconfluent cells, whereas in confluent cells it rapidly falls to pre-induced levels. Cytoplasmic c-myc mRNAs accumulate within 1-2 hours of serum addition to subconfluent cells and reach a higher level than expected from the degree of induction of sense transcription. However, c-myc mRNA levels fall close to pre-induced levels by 18 hours demonstrating that c-myc expression is initially subject to strong positive and then eventually strong negative post-transcriptional control. Anti-sense transcription within the c-myc locus was found to be constitutive under all these physiological states, thereby demonstrating that c-myc transcriptional control is strand specific. Epidermal growth factor stimulates c-myc transcription in a way different from that of serum: (1) initiation of transcription is not significantly enhanced, but intragenic pausing is significantly abrogated; and (2) post-transcriptional mechanisms do not enhance the degree of c-myc mRNA accumulation. In contrast to our results in fibroblastic cells, differentiating F9 teratocarcinoma cells down-regulate c-myc expression entirely at the post-transcriptional level. Our findings indicate that different cell types preferentially employ different modes of myc control depending on their physiological status
— id: 17536, year: 1987, vol: 1, page: 243, stat: Journal Article,

Repression of insulin gene expression by adenovirus type 5 E1a proteins
Stein RW; Ziff EB
1987 Mar;7(3):1164-1170, Molecular & cellular biology
Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation
— id: 17535, year: 1987, vol: 7, page: 1164, stat: Journal Article,

Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription
Greenberg ME; Hermanowski AL; Ziff EB
1986 Apr;6(4):1050-1057, Molecular & cellular biology
Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation
— id: 17538, year: 1986, vol: 6, page: 1050, stat: Journal Article,

Stimulation of neuronal acetylcholine receptors induces rapid gene transcription
Greenberg ME; Ziff EB; Greene LA
1986 Oct 3;234(4772):80-83, Science
Cholinergic agonists rapidly and transiently induced transcription of the c-fos protooncogene and one or more actin genes in neuronally differentiated PC12 cells. Transcription was activated within minutes after stimulation of the nicotinic acetylcholine receptor and required an influx of extracellular Ca2+ ions through voltage-sensitive calcium channels. Nicotine activation proceeded by a different pathway from activation by nerve growth factor, whose stimulation of these genes is independent of extracellular Ca2+ ions. These findings suggest that neurotransmitters may rapidly activate specific gene transcription in nondividing neuronally differentiated cells. They also suggest a functional role for neurotransmitter induction of c-fos and actin expression in the nervous system
— id: 17537, year: 1986, vol: 234, page: 80, stat: Journal Article,

CHARACTERIZATION OF NERVE GROWTH FACTOR-REGULATED MESSENGER RNA SPECIES
LEONARD D G B; GREENE L A; ZIFF E B
1986 ;12(1):215-215, Abstracts (Society for Neuroscience)
— id: 92671, year: 1986, vol: 12, page: 215, stat: Journal Article,

MOLECULAR CLONING OF NILE GLYCOPROTEIN-RELATED COMPLEMENTARY DNA PROBES
SAJOVIC P; ENNULAT D J; LEONARD D G B; KAPLAN K; PROCHIANTZ A L; ZIFF E B; SHELANSKI M L; GREENE L A
1986 ;12(2):1459-1459, Abstracts (Society for Neuroscience)
— id: 92670, year: 1986, vol: 12, page: 1459, stat: Journal Article,

Adenovirus E1a proteins repress expression from polyomavirus early and late promoters
Velcich A; Kern FG; Basilico C; Ziff EB
1986 Nov;6(11):4019-4025, Molecular & cellular biology
We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function
— id: 14432, year: 1986, vol: 6, page: 4019, stat: Journal Article,

Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells
Greenberg ME; Greene LA; Ziff EB
1985 Nov 15;260(26):14101-14110, Journal of biological chemistry
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. We show here that within 5 min after its addition, NGF transiently stimulates c-fos proto-oncogene and actin transcription by greater than 100-fold in nonsynchronized PC12 cells. c-myc and ornithine decarboxylase transcription are also transiently activated, but more slowly. The corresponding mRNAs are induced as well. Two weeks' exposure to NGF causes no significant changes in the transcription of these and a variety of other genes analyzed; however, c-fos mRNA levels are increased severalfold under these conditions. Neuronally differentiated PC12 cells retain the capacity for rapid transcriptional responses. Removal of NGF from such cells for several hours followed by its readdition results in rapid induction of c-fos and actin transcription. These NGF-promoted transcriptional changes in PC12 cells are similar to those previously observed in quiescent fibroblasts stimulated by platelet-derived growth factor (Greenberg, M.E., and Ziff, E.B. (1984) Nature 311, 433-438). This rapid transcriptional activation in PC12 cells could be necessary for neuronal differentiation, but is apparently not sufficient since diverse agents without differentiating activity such as epidermal growth factor, insulin, dibutyryl cAMP, phorbol ester, and elevated K+ were also found to induce transcription. These results suggest that c-fos, c-myc, and actin induction may be general nuclear responses to growth or differentiation factors in a variety of different cell types
— id: 17539, year: 1985, vol: 260, page: 14101, stat: Journal Article,

GENES REGULATED BY NERVE GROWTH FACTOR IN PC-12 CELLS
LEONARD D G B; GREENE L A; ZIFF E B
1985 ;11(2):1116-1116, Abstracts (Society for Neuroscience)
— id: 92672, year: 1985, vol: 11, page: 1116, stat: Journal Article,

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation
Smith DH; Kegler DM; Ziff EB
1985 Oct;5(10):2684-2696, Molecular & cellular biology
We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively
— id: 17540, year: 1985, vol: 5, page: 2684, stat: Journal Article,

ADENOVIRUS E1A PROTEINS REPRESS TRANSCRIPTION FROM THE SV40 EARLY PROMOTER
VELCICH, A; ZIFF, E
1985 ;40(3):705-716, Cell
— id: 41235, year: 1985, vol: 40, page: 705, stat: Journal Article,

Splicing in adenovirus and other animal viruses
Ziff EB
1985 ;93(10):327-358, International review of cytology
Splicing provides viruses with great genetic versatility. It is still too early to say whether this versatility is derived from ingeneous mechanisms evolved by necessity by the viruses, or whether the viruses indeed mimic cellular mechanisms. In any event, it is unlikely that cells will provide a single genomic cluster of genes that utilize splicing in such diverse ways as adenovirus, or the other viruses discussed here. And we may speculate that when the full role of splicing in adenovirus gene expression program is known, its import will continue to be a source of amazement!
— id: 17541, year: 1985, vol: 93, page: 327, stat: Journal Article,

Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene
Greenberg ME; Ziff EB
1984 Oct 4-10;311(5985):433-438, Nature
Transcription of the c-fos proto-oncogene is greatly increased within minutes of administering purified growth factors to quiescent 3T3 cells. This stimulation is the most rapid transcriptional response to peptide growth factors yet described, and implies a role for c-fos in cell-cycle control. Transformation by c-fos may result from a temporal deregulation of this control
— id: 17543, year: 1984, vol: 311, page: 433, stat: Journal Article,

HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism
Stein R; Ziff EB
1984 Dec;4(12):2792-2801, Molecular & cellular biology
We report that the rate of transcription of cellular beta-tubulin genes increases during the early phase of adenovirus infection of HeLa cells, with kinetics very similar to those routinely found for viral genes. This activation depends upon adenovirus early region E1a, which encodes products that activate early virus transcription. To compare the responses of viral and cellular genes to E1a, we infected HeLa cells with dl312, a transcriptionally inactive deletion mutant that lacks a functional E1a gene. We then superinfected the cells with a helper virus, dl327, which encodes active E1a products, and measured changes in the rates of transcription of various cell and viral genes. Early region E3 of dl312 was activated 0 to 6 h postinfection and then repressed at 8 h postinfection, thus reproducing the two-step kinetics characteristic of a wild-type infection. Synthesis of beta-tubulin nuclear RNA was also transiently induced two- to six-fold, rising and falling in a manner similar to E3 transcription. An increase in helper virus multiplicity gave an increase in beta-tubulin stimulation, but dl312 alone, even at a high multiplicity of infection, gave no induction, confirming the requirement for E1a. beta-Actin nuclear RNA was actively synthesized before infection, but it was not further stimulated by the virus. Cellular beta-globin gene transcription was not stimulated by the virus, although transcription of a transfected beta-globin plasmid was induced by the virus or from a cotransfected E1a expression plasmid. We conclude that adenovirus 5 can stimulate beta-tubulin gene transcription. We discuss the significance for the viral life cycle of viral stimulation of cell genes and consider possible mechanisms in the light of the results obtained with beta-actin and beta-tubulin
— id: 17542, year: 1984, vol: 4, page: 2792, stat: Journal Article,

REPRESSION OF ACTIVATORS
VELCICH, A; ZIFF, E
1984 ;312(5995):594-595, Nature
— id: 40868, year: 1984, vol: 312, page: 594, stat: Journal Article,

SEQUENCE ARRANGEMENT AND PROTEIN CODING CAPACITY OF THE ADENOVIRUS TYPE-2 I-LEADER
Falvey, E; Ziff, E
1983 ;45(1):185-191, Journal of virology
— id: 30694, year: 1983, vol: 45, page: 185, stat: Journal Article,

Poly(A) sites of adenovirus serotype 2 transcription units
Fraser NW; Baker CC; Moore MA; Ziff EB
1982 Mar 5;155(3):207-233, Journal of molecular biology
— id: 17545, year: 1982, vol: 155, page: 207, stat: Journal Article,

Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomycin block of protein synthesis
Shaw AR; Ziff EB
1982 Jul;2(7):789-789, Molecular & cellular biology
The transcription of adenovirus type 2 genes proceeds through a broad three-phase program. From 1 to 4 h postinfection six early transcription units (EIa, EIb, EII, EIII, EIV, and the promoter-proximal segment of the late transcription unit) are activated. From 4 to 6 h postinfection transcription of the early genes is depressed. After the onset of viral DNA replication at approximately 6 h postinfection, the transcript from the late promoter is antiterminated, and this transcript dominates viral RNA synthesis. The early activation period also proceeds through a series of stages; early regions EIa and EIV are activated first, followed by early region EII. We show that in the presence of anisomycin, a stringent inhibitor of protein synthesis, nuclear RNA and cytoplasmic polyadenylated RNA from regions EIa and EIV accumulate at normal rates, whereas RNAs from regions EII and EIII do not accumulate. We also show that failure to accumulate RNAs from regions EII and EIII is due to reduction of the rate of transcription by greater than 90%. We conclude that the regulation of the promoters for early regions EII and EIII is distinct from the mechanism that operates on the other early transcription units. The promoters for early regions EII and EIII diverge and lie approximately 500 nucleotides apart. We examined the structure of viral chromatin in this region early during infection by mild DNase I digestion in isolated nuclei and indirect end labeling with a DNA probe near these promoters. In control, drug-free cells where EII and EIII are transcribed and in anisomycin-treated cells where EII and EIII are not transcribed we detect the same regular DNase I pattern. We suggest that regulation of EII and EIII is not mediated through a change in gross chromatin structure, but rather by a viral effector, possibly a product of region EIa
— id: 17544, year: 1982, vol: 2, page: 789, stat: Journal Article,

Promoters and heterogeneous 5' termini of the messenger RNAs of adenovirus serotype 2
Baker CC; Ziff EB
1981 Jun 25;149(2):189-221, Journal of molecular biology
— id: 17547, year: 1981, vol: 149, page: 189, stat: Journal Article,

In vitro transcription of adenovirus
Fire A; Baker CC; Manley JL; Ziff EB; Sharp PA
1981 Dec;40(3):703-719, Journal of virology
A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters
— id: 17546, year: 1981, vol: 40, page: 703, stat: Journal Article,

Biogenesis, structures, and sites of encoding of the 5' termini of adenovirus-2 mRNAs
Baker CC; Ziff EB
1980 ;44 Pt 1(5782):415-428, Cold Spring Harbor symposia on quantitative biology
— id: 17550, year: 1980, vol: 44 Pt 1, page: 415, stat: Journal Article,

Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families
Shaw AR; Ziff EB
1980 Dec;22(3):905-916, Cell
The major late promoter of adeovirus-2 is located at coordinate 16.45 and initiates synthesis of nuclear precursors that are processed into mRNAs which fall into five 3- co-terminal families, L1-L5. These mRNAs all share a common tripartite 5' leader with a capped terminus encoded at th RNA initiation site. We show that the coorindate 16.45 RNA initiation site is also an early promoter, and yields transcripts detectable as early as 1 hr post-infection, prior to DNA replication and the early-late switch at 6-8 hr. Polyadenylated cytoplasmic RNA from the first 3- co-terminal family, L1, is also produced from the earliest stages of infection. L1 mRNA accumulates in the cytoplasm in the presence of cycloheximide, which blocks DNA replication and the onset of the late phase. Early nuclear RNA contains the same capped 5' terminal RNAase T-1 undecanucleotide and promoter proximal oligonucleotides present in late transcripts. This implies that precisely the same transcription start site is utilized for early L1 mRNA synthesis as is used during the late stage for L1-L5 late mRNA synthesis. In contrast to the early apperance of L1 mRNA, neither L2 nor L3 mRNAs are detected until 5-6 hr post infection. We cnclude that a major event in the Ad-2 early-late switch is a novel form of control which activates L2-L5 mRNA production
— id: 17548, year: 1980, vol: 22, page: 905, stat: Journal Article,

Transcription and RNA processing by the DNA tumour viruses
Ziff EB
1980 Oct 9;287(5782):491-499, Nature
Messenger RNA synthesis by the DNA tumour viruses proceeds by a complex but versatile series of transcription and RNA processing steps. The major mechanistic features of this pathway are probably very similar to those used by the animal cell host itself. The viruses have, however, evolved intricate arrangements of protein coding sequences and sites for RNA initiation, polyadenylation and splicing which allow them to use their genetic information to maximum advantage
— id: 17549, year: 1980, vol: 287, page: 491, stat: Journal Article,

Coincidence of the promoter and capped 5' terminus of RNA from the adenovirus 2 major late transcription unit
Ziff EB; Evans RM
1978 Dec;15(4):1463-1475, Cell
During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed approximately 30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at approximately 16, 20 and 27, and protein coding sequences which map downstream. This report maps the late promoter and the capped 5' end of nuclear and cytoplasmic RNAs from this transciption unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 +/- 0.5 and contain the sequences intervening between the leader segments. We map the capped 5' terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5' terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5' end of mRNA. Residues removed from transcripts by splicing were identified. These plus caps were detected in large polyadenylated nuclear RNA, indicating that capping and polyadenylation can occur on unspliced molecules. Residues retained in the mRNA first leader contain a nine residue sequence adjacent to the cap which is complementary to the 3' end of 18S rRNA, suggesting that the first leader functions in ribosome binding. Nucleotide sequences from the promoter region are compared with cellular counterparts. Strong homologies at cap sites and splice points suggest that for the noted cases, the virus and cell share closely related mechanisms for mRNA 5' end synthesis and splicing
— id: 17551, year: 1978, vol: 15, page: 1463, stat: Journal Article,

Gene F of bacteriophage phiX174. Correlation of nucleotide sequences from the DNA and amino acid sequences from the gene product
Air GM; Blackburn EH; Coulson AR; Galibert F; Sanger F; Sedat JW; Ziff EB
1976 Nov 15;107(4):445-458, Journal of molecular biology
— id: 17552, year: 1976, vol: 107, page: 445, stat: Journal Article,

Determination of the nucleotide sequence of a fragment of bacteriophage phiX 174 DNA
Ziff EB; Sedat JW; Galibert F
1973 Jan 10;241(106):34-37, Nature: new biology
— id: 17553, year: 1973, vol: 241, page: 34, stat: Journal Article,

A method for locating 4-thiouridylate in the primary structure of transfer ribonucleic acids
Ziff EB; Fresco JR
1969 Aug;8(8):3242-3248, Biochemistry
— id: 17554, year: 1969, vol: 8, page: 3242, stat: Journal Article,

Chemical transformation of 4-thiouracil nucleosides to uracil and cytosine counterparts
Ziff EB; Fresco JR
1968 Dec 18;90(26):7338-7342, Journal of the American Chemical Society
— id: 17555, year: 1968, vol: 90, page: 7338, stat: Journal Article,