Sylvia Lee Huang, Ph.D.

Computational study of bindings of HL9, a nonapeptide fragment of human lysozyme, to HIV-1 fusion protein gp41
Hartono, Yossa Dwi; Lee, Angelina Noviani; Lee-Huang, Sylvia; Zhang, Dawei
2011 Mar 15;21(6):1607-1611, Bioorganic & medicinal chemistry letters
HL9 is a nonapeptide fragment of human lysozyme which has been shown to have anti-HIV-1 activity in nanomolar concentration. This study aims to explain this inhibitory activity by using molecular dynamics (MD) simulation, focusing on the ectodomain of gp41, the envelope glycoprotein of HIV-1 crucial to membrane fusion. It was found that in HL9, two Trp residues separated by two others occupy the conserved hydrophobic pocket on gp41 and thus inhibit fusion in dominant-negative manner. Detailed HL9-gp41 binding interactions and free energies of binding were obtained through MD simulation and solvated interaction energies (SIE) calculation, giving a binding free energy of -8.25 kcal/mol which is in close agreement with the experimental value of -9.96 kcal/mol. Since C-helical region (C34) of gp41 also has two Trp residues separated by two others, this arrangement may be generalised and used to scan peptide library and to find those having similar manner of inhibition
— id: 134121, year: 2011, vol: 21, page: 1607, stat: Journal Article,

Live-cell real-time imaging reveals role of mitochondria in cell-to-cell transmission of HIV-1
Lee-Huang, Sylvia; Lin Huang, Philip; Lee Huang, Paul
2011 Nov 18;415(2):384-389, Biochemical & biophysical research communications
We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection
— id: 141703, year: 2011, vol: 415, page: 384, stat: Journal Article,

A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase--structural and modeling insight into its functions
Li, Hui-Guang; Huang, Philip L; Zhang, Dawei; Sun, Yongtao; Chen, Hao-Chia; Zhang, John; Huang, Paul L; Kong, Xiang-Peng; Lee-Huang, Sylvia
2010 Jan 1;391(1):340-345, Biochemical & biophysical research communications
We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA
— id: 106367, year: 2010, vol: 391, page: 340, stat: Journal Article,

COMPUTATIONAL DESIGN OF NORBORNANE-BASED HIV-1 PROTEASE INHIBITORS
Zhang, DW; Yu, LZ; Huang, PL; Lee-Huang, S; Zhang, JZH
2010 APR ;9(2):471-485, Journal of theoretical & computational chemistry
A series of norbornane-based HIV-1 protease (PR) inhibitors are designed theoretically to displace the tetrahedrally coordinated internal water molecule that bridges inhibitor to flaps via hydrogen bonds. These designed inhibitors use the norbornenone oxygen atom to mimic this structural water molecule and contain diols to interact with the carboxylate oxygens of catalytic aspartates. The binding free energies were estimated by modified linear interaction energy approach [Zoete H, Michielin O, Karplus M, J Comput Aided Mol Des 17: 861, 2003], in which the binding free energy is written as a linear combination of the electrostatic interaction energy between PR and the ligand, E-elec, the van der Waals interaction energy between PR and the ligand, E-vdW, and the difference of the solvation free energies of the complex, the receptor, and the isolated ligand, Delta G(solv). The equation obtained in previous work [Da W. Zhang, Philip Lin Huang, Sylvia Lee-Huang, John Z. H. Zhang, J Theor Comput Chem 7:485, 2008] is applied directly to calculate the binding free energy of designed norbornane-based HIV-1 PR inhibitors
— id: 110854, year: 2010, vol: 9, page: 471, stat: Journal Article,

Enos S1177 phosphorylation mediates metabolic energy expenditure and insulin sensitivity in knockin mice
Huang, P; Kashiwagi, S; Atochin, D; Li, Q; Schleicher, M; Earle, J; Pong, T; Lee-Huang, S; Moncada, S; Sessa, W
2009 JUN ;20(3):S36-S36, Nitric oxide : biology & chemistry
— id: 100525, year: 2009, vol: 20, page: S36, stat: Journal Article,

Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41
Bao, J; Zhang, DW; Zhang, JZH; Huang, PL; Huang, PL; Lee-Huang, S
2007 JUN 12 ;581(14):2737-2742, FEBS letters
Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41 Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors. (c) 2007 Published bv Elsevier B.V. on behalf of the Federation of European Biochemical Societies
— id: 73048, year: 2007, vol: 581, page: 2737, stat: Journal Article,

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. Integrase inhibition
Lee-Huang, Sylvia; Huang, Philip Lin; Zhang, Dawei; Lee, Jae Wook; Bao, Ju; Sun, Yongtao; Chang, Young-Tae; Zhang, John; Huang, Paul Lee
2007 Mar 23;354(4):872-878, Biochemical & biophysical research communications
We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article
— id: 71018, year: 2007, vol: 354, page: 872, stat: Journal Article,

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part II. Integrase inhibition
Lee-Huang, Sylvia; Huang, Philip Lin; Zhang, Dawei; Lee, Jae Wook; Bao, Ju; Sun, Yongtao; Chang, Young-Tae; Zhang, John; Huang, Paul Lee
2007 Mar 23;354(4):879-884, Biochemical & biophysical research communications
We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics
— id: 71019, year: 2007, vol: 354, page: 879, stat: Journal Article,

Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment
Lee-Huang, Sylvia; Zhang, Li; Huang, Philip Lin; Chang, Young-Tae; Huang, Paul L
2003 Aug 8;307(4):1029-1037, Biochemical & biophysical research communications
We investigated the antiviral activity of olive leaf extract (OLE) preparations standardized by liquid chromatography-coupled mass spectrometry (LC-MS) against HIV-1 infection and replication. We find that OLE inhibits acute infection and cell-to-cell transmission of HIV-1 as assayed by syncytia formation using uninfected MT2 cells co-cultured with HIV-1-infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose dependent, with EC(50)s of around 0.2 microg/ml. In the effective dose range, no cytotoxicity on uninfected target cells was detected. The therapeutic index of OLE is above 5000. To identify viral and host targets for OLE, we characterized gene expression profiles associated with HIV-1 infection and OLE treatment using cDNA microarrays. HIV-1 infection modulates the expression patterns of cellular genes involved in apoptosis, stress, cytokine, protein kinase C, and hedgehog signaling. HIV-1 infection up-regulates the expression of the heat-shock proteins hsp27 and hsp90, the DNA damage inducible transcript 1 gadd45, the p53-binding protein mdm2, and the hedgehog signal protein patched 1, while it down-regulates the expression of the anti-apoptotic BCL2-associated X protein Bax. Treatment with OLE reverses many of these HIV-1 infection-associated changes. Treatment of HIV-1-infected cells with OLE also up-regulates the expression of the apoptosis inhibitor proteins IAP1 and 2, as well as the calcium and protein kinase C pathway signaling molecules IL-2, IL-2Ralpha, and ornithine decarboxylase ODC1
— id: 39130, year: 2003, vol: 307, page: 1029, stat: Journal Article,

Production of antiviral and antitumor proteins MAP30 and GAP31 in cucurbits using the plant virus vector ZYMV-AGII
Arazi, Tzahi; Lee Huang, Paul; Huang, Philip Lin; Zhang, Li; Moshe Shiboleth, Yoel; Gal-On, Amit; Lee-Huang, Sylvia
2002 Mar 29;292(2):441-448, Biochemical & biophysical research communications
ZYMV-AGII (zucchini yellow mosaic virus-AGII) is a recombinant nonpathogenic potyvirus-based vector system for the expression of foreign genes in cucurbit plants and their edible fruits, including squash, cucumber, melon, watermelon, and pumpkin. MAP30 (Momordica anti-HIV protein, 30 kDa) and GAP31 (Gelonium anti-HIV protein 31 kDa) are multifunctional plant proteins with activity against HIV-1 virus. These proteins are also effective against other viruses, tumor cells, and microbes. We report here the production and characterization of biologically active MAP30 and GAP31 in squash plant by expression of their genes using the ZYMV-AGII vector. Recombinant expressed MAP30 and GAP31 exhibit comparable antiviral, antitumor, and antimicrobial activities as their counterparts from their original plant sources, with EC(50)s in the ranges of 0.2-0.3 nM for HIV-1. These results demonstrate for the first time the amplification and production of therapeutic proteins, MAP30 and GAP31, in common vegetables. This provides valuable alternative food sources of these antiviral, antitumor, and antimicrobial agents for therapeutic applications
— id: 95807, year: 2002, vol: 292, page: 441, stat: Journal Article,

Anti-hiv agent map30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in aids-related lymphoma cells infected with kaposi's sarcoma-associated virus
Sun Y; Huang PL; Li JJ; Huang YQ; Zhang L; Huang PL; Lee-Huang S
2001 Oct 5;287(4):983-994, Biochemical & biophysical research communications
The anti-HIV agent MAP30 (Momordica anti-HIV protein, 30 kDa) inhibits the proliferation of BC-2, an AIDS-related primary effusion lymphoma (PEL) cell line derived from an AIDS patient. BC-2 cells are latently infected with Kaposi's sarcoma-associated herpes virus (KSHV), also known as human herpes virus 8 (HHV8). We examined the effect of MAP30 on the expression of viral and cellular genes in BC-2 during latent and lytic states of the viral life cycle. By Northern analysis and RT-PCR, we found that MAP30 downregulates the expression of viral cyclin D (vCD), viral interleukin-6 (vIL-6), and viral FLIP (vFLIP), genes involved in cell cycle regulation, viral pathogenesis, and apoptosis. By pathway-specific cDNA microarray analysis, we found that BC-2 cells express high levels of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, skp1, and IL-2, cellular genes involved in mitogenesis, tumorigenesis, and inhibition of apoptosis in NFkappaB and p53 signaling pathways. These results define for the first time the specific cellular pathways involved in AIDS-related tumorigenesis and suggest specific novel targets for the treatment. Furthermore, we found that MAP30 downregulates the expression of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, and Skp1, while it upregulates the pro-apoptotic-related genes Bax, CRADD, and caspase-3. Thus, MAP30 modulates the expression of both viral and cellular genes involved in KS pathogenesis. These results provide valuable insight into the molecular mechanisms of MAP30 anti-KS action and suggest its utility as a therapeutic agent against AIDS-related tumors
— id: 24773, year: 2001, vol: 287, page: 983, stat: Journal Article,

Inhibition of MDA-MB-231 human breast tumor xenografts and HER2 expression by anti-tumor agents GAP31 and MAP30
Lee-Huang S; Huang PL; Sun Y; Chen HC; Kung HF; Huang PL; Murphy WJ
2000 Mar-Apr;20(2A):653-659, Anticancer research
GAP31 (Gelonium protein of 31 kDa) and MAP30 (Momordica protein of 30 kDa) are agents isolated from the medicinal plants Gelonium multiflorum and Momordica charantia, respectively. The current study was conducted to investigate the efficacy of GAP31 and MAP30 on estrogen-independent and highly metastatic human breast tumor MDA-MB-231 both in vitro and in vivo. The effect of these agents on the expression of breast tumor antigen HER2 (also known as neu or as c-erbB 2) was also examined. Treatment of MDA-MB-231 breast cancer cells with GAP31 and MAP30 resulted in inhibition of cancer cell proliferation as well as inhibition of the expression of HER2 gene in vitro. When MDA-MB-231 human breast cancer cells were transferred into SCID mice, the mice developed extensive metastases and all mice succumbed to tumor by day 46. Treatment of the human breast cancer bearing SCID mice with GAP31 or MAP30 at 10 micrograms/injection EOD for 10 injections resulted in significant increases in survival, with 20-25% of the mice remaining tumor free for 96 days. Thus, anti-tumor agents GAP31 and MAP30 are effective against human breast cancer MDA-MB-231 in vitro and in vivo. These agents may therefore be a potential therapeutic use against breast carcinomas
— id: 11699, year: 2000, vol: 20, page: 653, stat: Journal Article,

In search of novel anti-HIV agents
Sylvia, Lee-Huang; Hao-Chia, Chen
2000 Oct 09;7(2):170-170, Journal of biomedical science
For the past several years, we have been searching for novel antiviral and anti-tumor agents from nature products. From hundreds of samples investigated, we identified, purified to homogeneity, charac- terized and cloned a new class of anti-HIV agents with high potency and low toxicity from distinct and unrelated sources. The first group consists of anti-HIV proteins MAP30 (Momordica Anti-HIV Protein 30 kD) and GAP31 (Gelonium Anti-HIV Protein 31 kD) from medicinal plants and the second group consists of AVL (anti-viral lysozyme) and AVR (anti-viral RNase) from urine of pregnant women. MAP30 and GAP31 are isolated from medicinal plants Momordica charantia and Gelonium multiflorum, also known as bitter melon and Himalayan fruit, respectively. These compounds are unique in that they not only inhibit de novo infection by HIV-l but also block the replication of the virus in already infected cells. We found that they affect HIV-1-infected cells with EC50s (effective concentration at 50% inhibition) in the subnanomolar range (0.2-0.3 nM). They show no apparent cytotoxic or cytostatic effects on normal human cells even at 1,000-fold higher dose levels. MAP30 and GAP3l possess multiple therapeutic targets at different stages of the HIV-l life cycle. We have characterized at least three biological activities that may be relevant to their therapeutic use. The first is an RNA N-glycosidase activity that cleaves the link between a ribose and adenine A4324 of 28S ribosomal rRNA. This inactivates the 60S ribosomal subunit and inhibits polypeptide chain elongation. The second is a DNA topological inactivation activity that renders HIV-LTR topologically inactive as substrates for DNA gyrase. This topoinactivation is similar to the effect of cellular topoisomerases in the presence of topoisomerase inhibitors. The third is inhibition of each of the three reactions catalyzed by HIV-l integrase: 3' processing of the viral DNA. strand transfer, and cleavage at the viral/target junction. It is thus important to define the extent to which each of these mechanisms contributes to desired antiviral and antitumor actions or to undesired cytotoxicity. We carried out structural and activity mapping of MAP30 and GAP31 by X-ray diffraction of crystals and by limited proteolysis. We identified and isolated proteolytic fragments of MAP30 and GAP31 that are fully active against HIV-1 but not in ribosome inactivation. These peptides are as active as their parent molecules in HIV-l inhibition with EC50 in the range of 0.2-0.4 nM. They inhibit HIV-integrase activity and HIV-LTR topological interconversion, but they do not inhibit ribosome activity. These results demonstrate that the antiviral activity of MAP30 and GAP31 is independent from their ribosome-inactivating protein activity. This is of great significance and may provide useful insights in the design and development of antiviral and anti-tumor agents with specific therapeutic targets toward viral-infected and/or tumor cells, without cytotoxicity towards cellular targets. The second group of antiviral agents consists of AVL and AVR. To our knowledge, this is the first report that lysozymes and ribonucleases possess anti-HIV activity, and the first identification of these proteins as components present in crude b-core preparations that contribute to its anti-HIV effects. They represent a totally new class of therapeutic agents because they are naturally occurring human proteins that modulate viral infection. Details of these studies are presented in the following abstract
— id: 15767, year: 2000, vol: 7, page: 170, stat: Journal Article,

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP
Wang YX; Jacob J; Wingfield PT; Palmer I; Stahl SJ; Kaufman JD; Huang PL; Huang PL; Lee-Huang S; Torchia DA
2000 Jan;9(1):138-144, Protein science
MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials
— id: 15094, year: 2000, vol: 9, page: 138, stat: Journal Article,

Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active
Huang PL; Sun Y; Chen HC; Kung HF; Lee-Huang S
1999 Sep 7;262(3):615-623, Biochemical & biophysical research communications
We analyzed the structural and functional organization of anti-HIV and anti-tumor proteins MAP30 and GAP31 by limited proteolysis with endopeptidases Lys-C and Glu-C (V8). MAP30 and GAP31 are resistant to proteolytic digestion under conditions of as much as 5% (w/w) proteases. In the presence of 10% (w/w) protease, the central regions of the proteins are still resistant to proteolysis, whereas the N- and C-termini are accessible. Peptide fragments were purified by FPLC on Superdex 75 columns, characterized by gel electrophoresis, identified by amino acid sequencing, and analyzed for anti-HIV, anti-tumor, and other biochemical activities. We report here that limited proteolysis yields biologically active fragments of both MAP30 and GAP31. These fragments are active against HIV-1 and tumor cells with EC(50)s in the sub-nanomolar ranges, 0.2-0.4 nM. At the dose levels used in the assays, little cytotoxicity to normal cells was observed. In addition, these fragments remain fully active in HIV-integrase inhibition and HIV-LTR topological inactivation, but not ribosome inactivation. These results demonstrate that the antiviral and anti-tumor activities of MAP30 and GAP31 are independent of ribosome inactivation activity. In addition, we demonstrate that portions of the N- and C-termini are not essential for antiviral and anti-tumor activities, but do appear to be required for ribosome inactivation. These results may provide novel strategies for rational design and targeted development of mimetic antiviral and anti-tumor therapeutics.
— id: 8486, year: 1999, vol: 262, page: 615, stat: Journal Article,

Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin
Lee-Huang S; Huang PL; Sun Y; Huang PL; Kung HF; Blithe DL; Chen HC
1999 Mar 16;96(6):2678-2681, Proceedings of the National Academy of Sciences of the United States of America
Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG beta-subunit (hCGbeta) preparations are caused by the beta-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the beta-core fraction of hCGbeta prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection
— id: 8505, year: 1999, vol: 96, page: 2678, stat: Journal Article,

The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1
Schreiber CA; Wan L; Sun Y; Lu L; Krey LC; Lee-Huang S
1999 Oct;72(4):686-690, Fertility & sterility
OBJECTIVE: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti-human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the motility and vitality of human spermatocytes. DESIGN: Prospective, controlled study. SETTING: New York University School of Medicine. PATIENT(S): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. INTERVENTION(S): Human sperm were treated with the anti-HIV agents, MAP30 and GAP3 1. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. MAIN OUTCOME MEASURE(S): The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. RESULT(S): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100-0.1 microg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. CONCLUSION(S): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases
— id: 11946, year: 1999, vol: 72, page: 686, stat: Journal Article,

Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein
Wang YX; Jacob J; Cordier F; Wingfield P; Stahl SJ; Lee-Huang S; Torchia D; Grzesiek S; Bax A
1999 Jun;14(2):181-184, Journal of biomolecular NMR
A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons
— id: 15096, year: 1999, vol: 14, page: 181, stat: Journal Article,

Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions
Wang YX; Neamati N; Jacob J; Palmer I; Stahl SJ; Kaufman JD; Huang PL; Huang PL; Winslow HE; Pommier Y; Wingfield PT; Lee-Huang S; Bax A; Torchia DA
1999 Nov 12;99(4):433-442, Cell
We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30's apparent inhibition of the HIV-1 integrase, MAP30's ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30's anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30's glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis
— id: 15095, year: 1999, vol: 99, page: 433, stat: Journal Article,

The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against herpes simplex virus in vitro
Bourinbaiar AS; Lee-Huang S
1996 Feb 27;219(3):923-929, Biochemical & biophysical research communications
We examined the effect on anti-HIV proteins MAP30 and GAP31, from Momordica charantia and Gelonium multiflorum, on the infection and replication of Herpes Simplex Viruses (HSV). Human lung WI-38 fibroblasts cultured in the presence of tenfold dilutions of MAP30 or GAP31 were exposed to HSV and viral yield was measured at 24-48 hours by ELISA. The effective concentrations for 50% inhibitions (EC50) were 0.1-0.2 microM for HSV-2, and 0.3-0.5 microM for HSV-1 for MAP30 and GAP31, respectively. In comparison, the EC(50) for acyclovir (ACV), a commonly used anti-HSV drug, was 0.2 and 1.7 microM for HSV-2 and HSV-1, respectively. The cytotoxicity of all three antivirals was negligible and comparable. However, the antiherpetic activity of the plant proteins against acyclovir-resistant strains was two to three logs more potent than ACV. These results suggest that MAP30 and GAP31, previously shown to be active against HIV, may be useful for the therapy of herpesvirus infections
— id: 8056, year: 1996, vol: 219, page: 923, stat: Journal Article,

Protective effect of interferon-alpha against cell-mediated human immunodeficiency virus transmission resulting from coculture of infected lymphocytes with fetal trophoblasts
Bourinbaiar AS; Krasinski K; Borkowsky W; Lee-Huang S
1995 Jun;15(6):503-508, Journal of interferon & cytokine research
The hypothesis that the low transmission rate of HIV in utero may be due, in part, to the protective effect of IFN-producing placental trophoblasts was explored in vitro. The model consisted of H9 lymphocytes, as surrogates of maternal HIV-infected T cells, incubated for 3 h with JEG-3 trophoblasts in the presence of 10-fold dilutions of leukocyte-derived IFN-alpha (from 1000 to 0.1 IU/ml). The dose effect was monitored either directly, by measuring the levels of proviral DNA by PCR after a single round of infection, or indirectly, by coculturing infected JEG-3 with cord blood-derived MT-4 lymphocytes and determining the levels of p24 production by ELISA. Both assays revealed a dose-dependent blocking effect of IFN-alpha on cell-mediated HIV transmission. The complete inhibition of HIV infection was observed in the presence of 100 IU IFN-alpha. The efficacy of such a low dose could not be attributed to insufficient viral load because up to 10(8) infectious particles could be transmitted during cell-cell contact. An adhesion assay ruled out the possibility that IFN-alpha acts through prevention of lymphocyte-trophoblast contact. The results suggest that physiologic levels of IFN-alpha, present in the placental environment, may contribute to the protection of the fetus against HIV infection
— id: 6582, year: 1995, vol: 15, page: 503, stat: Journal Article,

Acrosin inhibitor, 4'-acetamidophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive with anti-HIV activity
Bourinbaiar AS; Lee-Huang S
1995 May;51(5):319-322, Contraception
Serine proteases are involved in a wide variety of seemingly unrelated physiological functions including capacitation of the spermatozoa and potentiation of human immunodeficiency virus (HIV) infection. The experimental vaginal contraceptives derived from 4-guanidinobenzoic acid act through inhibition of acrosin--a serine protease from the sperm. The serial ten-fold dilutions of 4'-acetamidophenyl 4-guanidinobenzoate (AGB) were tested in vitro for the effect against HIV infection by assaying the suppression of de novo p24 synthesis in virus-inoculated MT-4 T lymphocytes. The results reveal that complete inhibition of HIV occurred at 100 micrograms/ml--a dose corresponding to previously reported concentrations responsible for preventing fertilization in rabbits. These findings suggest that serine protease inhibitors and in particular the guanidinobenzoates, reported to be up to 100-fold more potent and less irritating than nonoxynol-9, can be potentially operative against sexual transmission of HIV
— id: 6809, year: 1995, vol: 51, page: 319, stat: Journal Article,

Anti-HIV effect of beta subunit of human chorionic gonadotropin (beta hCG) in vitro
Bourinbaiar AS; Lee-Huang S
1995 Jan;44(1):13-18, Immunology letters
Human chorionic gonadotropin (hCG)--a pregnancy-associated immunomodulating hormone--has been recently shown in vitro to suppress reverse transcriptase activity in chronically HIV-infected lymphocytes and monocytes and to block viral transmission resulting from cell-cell contact between virus-carrying lymphocytes and placental trophoblasts. In further pursuit of the query into the mechanism of action, purified alpha and beta subunits of hCG were tested for the inhibition of p24 gag protein synthesis in virus-producing ACH-2 lymphocytes and U1 monocytes. Unlike the alpha subunit, beta-hCG displayed a distinct U-shaped dose response, characteristic of the effect of dimer hCG. Maximum inhibition of viral expression has been achieved at 10-100 ng/ml, the concentration corresponding to blood levels of beta-hCG in pregnant women. The doses that were several logs higher of normal levels seemed to increase viral production in monocytes. The data presented supports our original observations regarding the effect of intact hCG on HIV replication. While the mechanism of action remains to be established, the results suggest that the virus-interfering activity of hCG is determined by hormone-specific beta chain but not by the alpha subunit--shared with the family of glycoprotein hormones from the pituitary--follicle-stimulating hormone, luteinizing hormone and thyrotropin
— id: 7893, year: 1995, vol: 44, page: 13, stat: Journal Article,

Potentiation of anti-HIV activity of anti-inflammatory drugs, dexamethasone and indomethacin, by MAP30, the antiviral agent from bitter melon
Bourinbaiar AS; Lee-Huang S
1995 Mar 17;208(2):779-785, Biochemical & biophysical research communications
MAP30 is an antiviral protein from bitter melon (Momordica charantia). The enhancement of weak HIV antagonists, dexamethasone and indomethacin, by MAP30 has been examined by measuring the reduction in p24 expression in acutely infected MT-4 lymphocytes. In the presence of 1.5 nM MAP30 the IC50 dose of dexamethasone and indomethacin has been lowered, without concurrent cytotoxicity, at least a thousand-fold to 10(-7) M and 10(-8) M, respectively. This observation indicates that MAP30, a multifunctional antiviral plant protein capable of topological inactivation of viral DNA and specific cleavage of 28 S ribosomal RNA, may regulate HIV replication in concert with steroid and non-steroidal inhibitors of prostaglandin synthesis. The results suggest that use of MAP30 in combination with low pharmacological doses of dexamethasone and indomethacin may improve the efficacy of anti-HIV therapy
— id: 6584, year: 1995, vol: 208, page: 779, stat: Journal Article,

Rational problems associated with the development of cellular approaches in controlling HIV spread
Bourinbaiar AS; Lee-Huang S
1995 ;374:71-89, Advances in experimental medicine & biology
— id: 8055, year: 1995, vol: 374, page: 71, stat: Journal Article,

The non-steroidal anti-inflammatory drug, indomethacin, as an inhibitor of HIV replication
Bourinbaiar AS; Lee-Huang S
1995 Feb 20;360(1):85-88, FEBS letters
Indomethacin, a common non-steroidal anti-inflammatory drug (NSAID), has been used to treat rheumatoid arthritis. Although indomethacin has also been used as an immunopotentiator and symptomatic NSAID in AIDS, its effect on HIV replication is unknown. MT-4 lymphocytes were inoculated with HIV in the presence of indomethacin and tested for p24 expression by ELISA. The 50% inhibition (IC50) was 10 microM, corresponding to plasma levels after administration of 50 mg oral indomethacin. The antiviral effect appears to be specific since no toxicity has been observed at the IC50 dose, and unrelated NSAIDs have not shown the activity at clinical doses. Indomethacin may, thus, represent a new class of anti-HIV drug
— id: 6583, year: 1995, vol: 360, page: 85, stat: Journal Article,

Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon
Lee-Huang S; Huang PL; Chen HC; Huang PL; Bourinbaiar A; Huang HI; Kung HF
1995 Aug 19;161(2):151-156, Gene
MAP30 is an anti-HIV plant protein that we have identified and purified to homogeneity from bitter melon (Momordica charantia). It is capable of acting against multiple stages of the viral life cycle, on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase and cell-free ribosome-inactivation activities. We have cloned and expressed the MAP30 gene. The objective of this study is to characterize recombinant MAP30 (re-MAP30) and to determine its anti-HIV, anti-tumor and other activities. We report here that re-MAP30 inhibits HIV-1 and certain human tumors to the same extent as its native counterpart, natural MAP30 (nMAP30). The anti-HIV activity was measured by quantitative focal syncytium formation on CEM-ss cell monolayers, viral core protein p24 expression and viral-associated reverse transcriptase activity in HIV-1-infected H9 cells. The anti-tumor activity was measured by metabolic labeling of protein synthesis in tumor cells. In the dose range of the assay, re-MAP30 exhibits little toxicity to the uninfected viral target cells and other normal human cells. Identical to nMAP30, re-MAP30 is also active in topological inactivation of viral DNA, inhibition of viral DNA integration and cell-free ribosome inactivation. The cloning and expression of the gene encoding biologically active re-MAP30 provides an abundant source of homogeneous material for clinical investigations, as well as structure-function studies of this novel antiviral and anti-tumor agent
— id: 6839, year: 1995, vol: 161, page: 151, stat: Journal Article,

Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31
Lee-Huang S; Huang PL; Huang PL; Bourinbaiar AS; Chen HC; Kung HF
1995 Sep 12;92(19):8818-8822, Proceedings of the National Academy of Sciences of the United States of America
MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and 'disintegration' (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins
— id: 12732, year: 1995, vol: 92, page: 8818, stat: Journal Article,

Comparative in vitro study of contraceptive agents with anti-HIV activity: gramicidin, nonoxynol-9, and gossypol
Bourinbaiar AS; Lee-Huang S
1994 Feb;49(2):131-137, Contraception
Gramicidin, a polypeptide antibiotic derived from Bacillus brevis, was compared in vitro with the established contraceptive virucidal agents nonoxynol-9 and gossypol for activity against human immunodeficiency virus (HIV) infection. The effective antiviral 10 ng/ml concentration of gramicidin required for complete HIV inactivation was a thousand-fold lower than the dose observed for nonoxynol-9 or gossypol. Gramicidin, routinely used as a contraceptive agent in the former Soviet Union, should be considered for in vivo trials as a spermicide with potent antiviral activity
— id: 6329, year: 1994, vol: 49, page: 131, stat: Journal Article,

Anti-HIV effect of immunomodulating agent, levamisole, in vitro
Bourinbaiar AS; Lee-Huang S; Krasinski K; Borkowsky W
1994 ;48(7):327-330, Biomedicine & pharmacotherapy
An anthelminthic agent, levamisole, also known as a potent immunomodulator, has been successfully used for adjuvant therapy of malignancies and chronic infections underlined by immunodeficiency. We have tested the effect of this drug on de novo viral infection by exposing MT-4 T lymphocytes to HIV in the presence of serial ten-fold dilutions of levamisole (range 10(-3)-10(-9) M). The results indicate that 50% reduction in viral infectivity (IC50) of levamisole starts from as low as 10(-7) M, whereas even the highest millimolar dose of the drug has not shown any appreciable cytotoxicity. Although the mechanism of levamisole action remains unknown, our observation in vitro suggests that levamisole, a clinically established immunomodulator, can be potentially effective for treatment of HIV infection
— id: 6585, year: 1994, vol: 48, page: 327, stat: Journal Article,

Inhibitory effect of the oral immune response modifier, bestatin, on cell-mediated and cell-free HIV infection in vitro
Bourinbaiar AS; Lee-Huang S; Krasinski K; Borkowsky W
1994 ;48(2):55-61, Biomedicine & pharmacotherapy
The antiviral effect of the immunomodulating anti-cancer agent, bestatin, was examined in vitro by exposing MT-4 lymphocytes to HIV in the presence of 10-fold dilutions of drug (range 100 micrograms-100 pg/ml). The reduction in infectivity was measured by p24 ELISA and compared to the effect of established anti-HIV drugs-azidothymidine (AZT) and dextran sulfate. The results indicate that low doses of bestatin (1 microgram/ml) can completely inhibit viral infection resulting either from inoculation with free virus or coculture with infected lymphocytes. Unlike AZT or dextran sulfate, bestatin prevents HIV infection without interfering with the rate of cell growth. No appreciable decrease in HIV production was observed when chronically infected virus-producing T cell lines ie, H9, MOLT-4, HPB-ALL, 8E5 and MT-2 were treated with bestatin. Bestatin appears to act in the early stages of viral penetration, possibly through inhibition of lymphocyte-associated aminopeptidases
— id: 6586, year: 1994, vol: 48, page: 55, stat: Journal Article,

Crystallization and preliminary X-ray analysis of GAP 31. A protein which inhibits the life cycle of HIV-1
Lee-Huang S; Kung HF; Chen HC; Huang PL; Rybak SM; Huang PL; Bourinbaiar AS; Musayev F; Liaw YC
1994 Jul 1;240(1):92-94, Journal of molecular biology
GAP 31 is an anti-HIV plant protein that we have identified and purified to homogeneity from Gelonium multiflorum. It is the first reported example of an anti-HIV agent capable of acting against multiple stages of the viral life cycle, on viral infection and viral replication. GAP 31 is a unique paragon of multi-functional protein. In addition to anti-HIV activity, it also exhibits anti-tumor action, DNA binding, RNA binding and ribosome inactivation. The present crystals diffract up to 2.0 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 49.30(2) A, b = 44.57(2) A, c = 137.78(7) A and beta = 98.32(3) degrees. There are two molecules of molecular weight 31 kDa in an asymmetric unit with a solvent content of 49%
— id: 12955, year: 1994, vol: 240, page: 92, stat: Journal Article,

Human immunodeficiency virus type 1 (HIV-1) inhibition, DNA-binding, RNA-binding, and ribosome inactivation activities in the N-terminal segments of the plant anti-HIV protein GAP31
Lee-Huang S; Kung HF; Huang PL; Bourinbaiar AS; Morell JL; Brown JH; Huang PL; Tsai WP; Chen AY; Huang HI
1994 Dec 6;91(25):12208-12212, Proceedings of the National Academy of Sciences of the United States of America
GAP31 (gelonium anti-HIV protein of 31 kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity. The ability of GAP31 to interrupt both DNA and RNA functions may be related to its multiple antiviral actions. To define the roles of these activities in the anti-HIV action of GAP31, a series of peptides corresponding to the N-terminal segment of GAP31 were synthesized and assayed for the aforementioned activities of the parent molecule. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) designated as K10-K42 is the shortest peptide necessary and sufficient for HIV-1 inhibition, DNA and RNA binding, and ribosome inactivation. The peptides were 2-5 orders of magnitude less active than GAP31. Truncation of 19 aa from the C terminus of K10-K42 resulted in the loss of all of these activities. On the other hand, deletion of N-terminal residues to give E23-K42 did not alter ribosome-inactivation activity but eliminated the other activities. These findings permit identification of a 7-aa sequence, KGATYIT, at the N terminus of K10-K42 that is critical for DNA binding and RNA binding, whereas a 9-aa sequence, SHGIPSLRK, at the C terminus is important to ribosome inactivation. Both regions contribute to anti-HIV activity. Histidine at position 35 is critical for all of these activities. The disparity of sequence requirements for inhibition of HIV infection and replication and for ribosome-inactivation activity suggests that the anti-HIV activity of most ribosome-inactivating proteins may not be the result of N-glycosidase activity alone. Mapping the minimal domain of GAP31 offers insights into the rational design of molecular mimetics of anti-HIV drugs
— id: 8447, year: 1994, vol: 91, page: 12208, stat: Journal Article,

Immune response in mice that lack the interferon-gamma receptor [see comments]
Huang S; Hendriks W; Althage A; Hemmi S; Bluethmann H; Kamijo R; Vilcek J; Zinkernagel RM; Aguet M
1993 Mar 19;259(5102):1742-1745, Science
Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor
— id: 15535, year: 1993, vol: 259, page: 1742, stat: Journal Article,

Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guerin and subsequent challenge with lipopolysaccharide
Kamijo R; Le J; Shapiro D; Havell EA; Huang S; Aguet M; Bosland M; Vilcek J
1993 Oct 1;178(4):1435-1440, Journal of experimental medicine
Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin
— id: 13065, year: 1993, vol: 178, page: 1435, stat: Journal Article,

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor
Kamijo R; Shapiro D; Le J; Huang S; Aguet M; Vilcek J
1993 Jul 15;90(14):6626-6630, Proceedings of the National Academy of Sciences of the United States of America
Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections
— id: 13105, year: 1993, vol: 90, page: 6626, stat: Journal Article,

The 3' flanking region of the human erythropoietin-encoding gene contains nitrogen-regulatory/oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements
Lee-Huang S; Lin JJ; Kung HF; Huan PL; Lee L; Huang PL
1993 Dec 31;137(2):203-210, Gene
We have reported the identification of a classical canonical CAAT box, TATA boxes and other transcriptional regulatory elements in the 5' flanking region of the human erythropoietin (hEp)-encoding gene [Lee-Huang et al., Gene 128 (1993) 227-236]. These elements were not found in the hEp genomic clones reported by others. Our genomic clone extends in both directions beyond any reported clones, by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. Many important regulatory elements are found in these extended flanking regions. We report here the genomic structure of the extended 3' flanking region of hEp. This region contains the following regulatory elements: nitrogen-regulatory/oxygen-sensing consensus sequences, 5'-TTTTGCA and 5'-CCCTGCA; tissue-specific regulatory elements, including binding sites for A-activator, 5'-GTGGTGCAA; for DBP, 5'-TGATTTTGT; for HNF, 5'-T(A/G)TTTGT; and for C/EBP, 5'-T(T/G) (T/G)TGCAAT; a lymphokine-responsive element, 5'-GTGAAACCCC (Rev), as well as binding sites for AP and Sp1. In addition, the nucleotide (nt) sequence in this region is rich in inverted repeats (palindromes) that allow the formation of hairpin loops. A total of 14 potential stem loops with a maximum loop size of 20 nt are found. The identification of these regulatory elements in hEp should provide further insight into the tissue-specific and inducible expression of hEp. Such knowledge should be useful in the clinical modulation of erythropoiesis under physiologic and pathologic conditions
— id: 8351, year: 1993, vol: 137, page: 203, stat: Journal Article,

The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region
Lee-Huang S; Lin JJ; Kung HF; Huang PL; Lee L; Huang PL
1993 Jun 30;128(2):227-236, Gene
We have reported the cloning and expression of a human erythropoietin (hEp)-encoding cDNA [Lee-Huang, Proc. Natl. Acad. Sci. USA 81 (1984) 2708-2712]. Using this hEp cDNA as a probe, we isolated a 9.3-kb BamHI genomic Ep clone from a human leukocyte library soon thereafter. The size and restriction map of this clone is in agreement with restriction analysis of human genomic DNA probed with the hEp cDNA, demonstrating that this clone is representative of the single hEp gene. This clone is unique in that it extends beyond any reported hEp genomic clone by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. The promoter function of the newly described 5' flanking region has been demonstrated by the expression of biologically active hEp in transfected cells. We find that, despite reports to the contrary, hEp does contain classic canonical TATA boxes and a CAAT box. The 5'-flanking region also contains cytokine-responsive consensus sequences, tissue-specific and metal-responsive elements, CRE and GRE sites, and binding sites for transcription factors, including AP1, NF-kappa beta and Sp1. These regulatory elements have not been found in the hEp genomic clones thus far reported. The identification of these elements and their precise localization in hEp should be useful in studying the regulation of hEp expression, as well as in gene therapy and physiologic modulation of this hormone
— id: 13126, year: 1993, vol: 128, page: 227, stat: Journal Article,

Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms
Huang PL; Chen HC; Kung HF; Huang PL; Huang P; Huang HI; Lee-Huang S
1992 Dec;4(1):37-41, Biofactors
GAP 31, DAP 32 and DAP 30 comprise a new class of plant proteins with potent anti-HIV activity and insignificant cytotoxicity. We report here the identification and characterization of a new DNA enzyme activity in these three proteins. They irreversibly relax and decatenate supercoiled DNA, as well as catalyze double-stranded breakage to form linear DNA. The relaxed molecules are topologically inactive and no longer serve as substrates for DNA gyrase to form supercoils, phenomena similar to those of cellular topoisomerases in the presence of topoisomerase poisons. The ability of these anti-HIV agents to interrupt essential topological interconversions of DNA may provide a novel mechanism for their antiviral and antitumor actions. The presence of this new DNA topological enzyme activity in these plant proteins also suggests that their anti-HIV activity may not be merely a consequence of ribosome inactivation previously recognized
— id: 15097, year: 1992, vol: 4, page: 37, stat: Journal Article,

TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells
Lee-Huang S; Huang PL; Kung HF; Li BQ; Huang PL; Huang P; Huang HI; Chen HC
1991 Aug 1;88(15):6570-6574, Proceedings of the National Academy of Sciences of the United States of America
An anti-human immunodeficiency virus (anti-HIV) protein capable of inhibiting HIV-1 infection and replication has been isolated and purified to homogeneity from Trichosanthes kirilowii. This protein, TAP 29 (Trichosanthes anti-HIV protein, 29 kDa), is distinct from trichosanthin [also known as GLQ 223 (26 kDa)] in size, N-terminal amino acid sequence, and cytotoxicity. In addition to three conservative substitutions--namely, Arg-29 to Lys, Ile-37 to Val, and Pro-42 to Ser--a total difference of residues 12-16 was found. TAP 29 yielded -Lys-Lys-Lys-Val-Tyr-, whereas trichosanthin has -Ser-Ser-Tyr-Gly-Val-. Although the two proteins exhibit similar anti-HIV activity, as measured by syncytium formation, p24 expression, and HIV reverse transcriptase activity, they differ significantly in cytotoxicity, as measured by their effects on cellular DNA and protein syntheses. At the dose level of the bioassays, 0.34-340 nM, trichosanthin demonstrates a dose-dependent toxic effect on host cells. TAP 29 displays no toxic effect, even at 100 X ID50, whereas trichosanthin demonstrates 38% and 44% inhibition on cellular DNA and protein synthesis, respectively. These results indicate that the therapeutic index of TAP 29 is at least two orders of magnitude higher than that of trichosanthin. Thus TAP 29 may offer a broader safe dose range in the treatment of AIDS
— id: 13958, year: 1991, vol: 88, page: 6570, stat: Journal Article,

A new class of anti-HIV agents: GAP31, DAPs 30 and 32
Lee-Huang S; Kung HF; Huang PL; Huang PL; Li BQ; Huang P; Huang HI; Chen HC
1991 Oct 7;291(1):139-144, FEBS letters
Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds (Gelonium multiflorum) and carnation leaves (Dianthus caryophyllus). These proteins, GAP 31 (Gelonium Anti-HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti-HIV proteins, 30 and 32 kDa), inhibit HIV-1 infection and replication in a dose-dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 10(4), suggesting that they may be clinically important agents in the treatment of AIDS. The N-terminal amino acid sequences of these proteins show little homology to those of previously described anti-HIV proteins. The structure-function features of these HIV inhibitors, based on the 40-60 amino acid residues of N-terminal sequences, are examined
— id: 13867, year: 1991, vol: 291, page: 139, stat: Journal Article,

MAP 30: a new inhibitor of HIV-1 infection and replication
Lee-Huang S; Huang PL; Nara PL; Chen HC; Kung HF; Huang P; Huang HI; Huang PL
1990 Oct 15;272(1-2):12-18, FEBS letters
A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (ii) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined
— id: 14315, year: 1990, vol: 272, page: 12, stat: Journal Article,

Anti-erythropoietin antibodies in hyperviscosity syndrome associated with giant lymph node hyperplasia (GLNH; Castleman's disease)
Steinberg JJ; Huang PL; Ljubich P; Lee-Huang S
1990 Apr;74(4):543-544, British journal of haematology
— id: 15098, year: 1990, vol: 74, page: 543, stat: Journal Article,

Cloning and expression of human erythropoietin cDNA in Escherichia coli
Lee-Huang S
1984 May;81(9):2708-2712, Proceedings of the National Academy of Sciences of the United States of America
Human erythropoietin (Ep) cDNA has been cloned in Escherichia coli by using pBR322 as a vector. Polyadenylylated RNA was isolated from selected human renal carcinomas with elevated Ep titers. The presence of Ep mRNA was detected by immunoprecipitation of in vitro translation products with monoclonal antibody to human Ep. Double-stranded cDNA was synthesized and inserted into the Pst I site of pBR322 by homopolymeric dG . dC tailing. The cDNA library was initially screened by colony hybridization with 32P-labeled cDNA synthesized from size-fractionated mRNA enriched in Ep message. Positive colonies were further screened immunologically by in situ radioimmunoassay with monoclonal antibody to human Ep. Three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein. These clones contain inserts of approximately 1400, 600, and 200 base pairs. Human renal Ep mRNA, of which the translation products immunoreact with anti-Ep on immunoblots, was hybrid-selected by plasmid DNA from these recombinants. Purified human Ep competes with 35S-labeled hybrid-selected translation products for antibody binding
— id: 15099, year: 1984, vol: 81, page: 2708, stat: Journal Article,

A new preparative method for isolation of human erythropoietin with hydrophobic interaction chromatography
Lee-Huang S
1980 Oct;56(4):620-624, Blood
A new preparative method for isolation of human urinary erythropoietin (Ep) has been developed using hydrophobic interaction chromatography on Phenyl-Sepharose CL4B. Crude urine and urine concentrates from anemic patients were used directly without prior manipulation. In addition to facilitating hydrophobic interactions, Phenyl-Sepharose provided pi-pi interactions between its phenyl group on the gel matrix and the aromatic amino acid residues of Ep, and thus contributed to specific resolution. Over 90% of the urinary contaminants were excluded from the column, and Ep was selectively bound. Its activity was eluted with 20% ethylene glycol in 10 mM NaOH containing 4 M guanidine hydrochloride. This single step offered a mean purification factor of 110 with a recovery of 85%
— id: 15100, year: 1980, vol: 56, page: 620, stat: Journal Article,

Isolation and characterization of proteoglycans from human chondrosarcomas
Pal S; Strider W; Margolis R; Gallo G; Lee-Huang S
1978 Feb 25;253(4):1279-1289, Journal of biological chemistry
— id: 15101, year: 1978, vol: 253, page: 1279, stat: Journal Article,

Eucaryotic oligonucleotides affecting mRNA translation
Lee-Huang S; Sierra JM; Naranjo R; Filipowicz W; Ochoa S
1977 Apr 30;180(2):276-287, Archives of biochemistry & biophysics. ABB
— id: 15102, year: 1977, vol: 180, page: 276, stat: Journal Article,

Inhibition of polypeptide chain initiation in Escherichia coli by elongation factor G
Lee-Huang S; Lee H; Ochoa S
1974 Aug;71(8):2928-2931, Proceedings of the National Academy of Sciences of the United States of America
— id: 15103, year: 1974, vol: 71, page: 2928, stat: Journal Article,

Preparation and properties of crystalline initiation factor 1 (IF1) from Escherichia coli
Lee-Huang S; Ochoa S
1974 ;30(0):31-39, Methods in enzymology
— id: 15105, year: 1974, vol: 30, page: 31, stat: Journal Article,

Purification of two messenger-discriminating species of initiation factor 3 (IF3) from Escherichia coli
Lee-Huang S; Ochoa S
1974 ;30(0):45-53, Methods in enzymology
— id: 15104, year: 1974, vol: 30, page: 45, stat: Journal Article,

A specific inhibitor of polypeptide-chain initiation in Escherichia coli
Lee-Huang S; Lee H; Ochoa S
1973 Oct;70(10):2874-2878, Proceedings of the National Academy of Sciences of the United States of America
— id: 15107, year: 1973, vol: 70, page: 2874, stat: Journal Article,

Purification and properties of two messenger-discriminating species of E. coli initiation factor 3
Lee-Huang S; Ochoa S
1973 May;156(1):84-96, Archives of biochemistry & biophysics. ABB
— id: 15108, year: 1973, vol: 156, page: 84, stat: Journal Article,

Factor requirements for initiation complex formation with natural and synthetic messengers in Escherichia coli systems
Meier D; Lee-Huang S; Ochoa S
1973 Dec 25;248(24):8613-8615, Journal of biological chemistry
— id: 15106, year: 1973, vol: 248, page: 8613, stat: Journal Article,

Specific inhibitors of MS2 and late T4 RNA translation in E. coli
Lee-Huang S; Ochoa S
1972 Oct 17;49(2):371-376, Biochemical & biophysical research communications
— id: 15109, year: 1972, vol: 49, page: 371, stat: Journal Article,

Messenger discriminating species of initiation factor F3
Lee-Huang, S; Ochoa, S
1971 Dec 22;234(51):236-239, Nature: new biology
— id: 15110, year: 1971, vol: 234, page: 236, stat: Journal Article,

Isolation and properties of crystalline initiation factor F1 from Escherichia coli ribosomes
Lee-Huang, S; Sillero, M A; Ochoa, S
1971 Feb;18(4):536-543, European journal of biochemistry
— id: 15111, year: 1971, vol: 18, page: 536, stat: Journal Article,

The preparation and properties of ribonucleic acid polymerase from Azotobacter vinelandii
Lee-Huang S; Warner RC
1969 Jul 25;244(14):3793-3802, Journal of biological chemistry
— id: 15112, year: 1969, vol: 244, page: 3793, stat: Journal Article,

Nucleic Acid Polymerases: Possible Subunit Structure
Lee-Huang S; Cavalieri LF
1965 Jun 11;148(3676):1474-1476, Science
A hybrid polymerase which catalyzes the synthesis only of helical polynucleotides with one DNA-like strand and one RNA-like strand can be altered by certain treatments so that it will then synthesize DNA and RNA. There is evidence that the alteration involves a separation or rearrangement of polymerase subunits of several kinds. Two types of RNA polymerase activity have been found: one produces single chains, and the other, two complementary chains simultaneously. The latter type of RNA polymerase, the hybrid polymerase, and the DNA polymerase behave as though they were bifunctional, and each may be composed of two monofunctional subunits
— id: 95808, year: 1965, vol: 148, page: 1474, stat: Journal Article,

ISOLATION AND PROPERTIES OF A NUCLEIC ACID HYBRID POLYMERASE
LEE-HUNG, S; CAVALIERI, L F
1964 Jun;51:1022-1028, Proceedings of the National Academy of Sciences of the United States of America
— id: 111951, year: 1964, vol: 51, page: 1022, stat: Journal Article,

POLYRIBONUCLEOTIDES AS TEMPLATES FOR POLYDEOXYRIBONUCLEOTIDES
LEE-HUANG, S; CAVALIERI, L F
1963 Dec;50:1116-1122, Proceedings of the National Academy of Sciences of the United States of America
— id: 110398, year: 1963, vol: 50, page: 1116, stat: Journal Article,

Solubility of complexes of polynucleotides with spermine
HUANG, S L; FELSENFELD, G
1960 Oct 22;188:301-302, Nature
— id: 111952, year: 1960, vol: 188, page: 301, stat: Journal Article,