Dr. Hao-Chia Chen This is Me

 Web References

1. 1. Three Naturally Occuring Proteins Show Potent Anti-HIV Activity

   www.appliedbiosystems.com/mole - [Cached]

   Published on: 9/8/2000   Last Visited: 3/20/2001

   Dr. Hao-Chia Chen, shown here with the Procise® 494 Protein Sequencer, used the system to help him identify three naturally occurring proteins that show potent anti-HIV activity in cell-based assays.
   
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   The researchers--led by Dr. Sylvia Lee-Huang, professor of biochemistry at New York University, and Dr. Hao-Chia Chen, chief, Unit on Molecular Structure and Protein Chemistry, National Institute of Child Health and Human Development (NICHD), NIH--are the first to show data for anti-HIV activity of human lysozyme and two human ribonucleases.
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   The most significant part of the discovery is that lysozyme has anti-HIV activity, " notes Chen, who identified the proteins.
   
   Anti-HIV Activity of Lysozyme and Ribonucleases Although current treatments for AIDS using synthetic nucleoside derivatives, such as AZT and aspartate protease inhibitor cocktails can prevent the rapid multiplication and further spreading of HIV throughout the body, these treatments cannot eliminate the virus completely, according to Chen. They can gradually reduce the number of virus particles, but cannot eradicate the virus without showing drug toxicity.
   
   "With nucleoside protease inhibitor cocktails, it would take many life times to completely eliminate the virus from someone's system," notes Chen. "But, if you can flush the virus out and use lysozyme, or ribonuclease, or these two enzymes together to attack the virus, you have a much greater chance of eradicating it, because whenever these enzymes and the HIV virus meet, they either destroy the virus or prevent it from entering the host cell."
   
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   No one has demonstrated it yet, but Chen believes that lysozyme could inactivate the HIV virus by breaking down the polysaccharide coating of the retrovirus.
   
   The other two enzymes that showed anti-HIV activity are RNase A and RNase U. They are pyrimidine-specific ribonucleases and are homologous in the active site sequence. The pancreas secretes RNase A. On the other hand, RNase U, also known as onconase, is a nonsecretory lysosomal neutral/acidic enzyme. Both ribonucleases occur in body fluids, including cerebral spinal fluid and semen and have been isolated from the urine. Because the HIV virus consists of RNA as the genetic component by which it propagates either of these two ribonucleases can cleave the RNA and inactivate it.
   
   Urine of Pregnant Women a Source of hCG
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   For Chen and Lee-Huang's research, both hCG and beta-core hCG were isolated from urine samples of pregnant women.
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   Chen and Lee-Huang, both graduates from the Department of Agricultural Chemistry at the National Taiwan University in Taipei, Taiwan, have been collaborating on a project to research the anti-viral effects of plant proteins since 1989, and studying hCG since 1995.
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   Chen, trained as protein chemist at Emory University in Atlanta and the Rockefeller University in New York, has also been studying the structure-function relationship of hCG at the Endocrinology and Reproduction Research Branch, the National Institute of Child Health and Human Development (NICHD), NIH for more than 25 years.
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   In 1995, about the same time that Chen and Lee-Huang began researching the anti-viral effects of hCG, Dr. Robert Gallo, Institute of Human Virology, University of Maryland and an international team of researchers reported that injections of a commercial clinical grade of hCG could prevent the spread of the AIDS-associated Kaposi's sarcoma.
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   Chen was convinced that factors associated with hCG, but not the hormone itself, were responsible for the anti-HIV activity.
   
   Purification of Anti-HIV Components From Beta-Core Preparations of hCG
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   Chen wanted to confirm his belief that HAF, not hCG itself, have anti-HIV activity. So, Chen and Lee-Huang eventually decided to assay a highly purified beta-core preparation of hCG for anti-HIV activity.
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   According to Chen, p24 is such an integral part of the HIV virus, that, despite the large number of different strains of HIV known to exist, it is very unlikely that there are any strains for which inhibition of HIV activity cannot be measured using these assays. Much to Chen's surprise, this preparation did show anti-HIV activity in a cell-based assay.
   
   Automated Protein Sequencing Reveals Identity of Proteins Eager to see if the beta-core of hCG could possibly possess anti-HIV activity, Chen sequenced the purified protein using the Procise® 494 Protein Sequencer from PE Biosystems. What he found provided the explanation for the unexpected result. In addition to the expected amino acid sequences corresponding to the hCG beta-core, there clearly was another sequence. Searching through the Swiss-Prot amino acid sequence database, Chen found that the additional sequence matched well with that of the N-terminal sequence of human ribonuclease A (RNase A).
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   Chen sequenced the 23 kDa Coomassie Blue R250 stained band, which was the only one shown on the PVDF membrane in addition to that of the beta-core, and again found that it was the RNase A sequence.
   
   Another beta-core fraction gave a more complex picture after SDS-PAGE electrophoresis. Chen, therefore, ran this beta-core preparation on a semi-preparative reverse phase HPLC and collected seven fractions. Chen sent all of these fractions to Lee-Huang for the anti-HIV assay.
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   For a 10 picomole (10-12 mole) protein sample, Chen routinely analyzes 15 cycles. Using the Procise system, Chen eventually identified lysozyme and RNase U as the other two proteins responsible for the anti-HIV activity in the cell-based assay.
   
   Chen has used protein sequencers from PE Biosystems since the first one was introduced in 1985. When he first started using the early models, it took a full 8-hour day to get sequence for seven residues of amino acid. In collaboration with many different researchers, Chen has used PE Biosystems protein sequencers to discover many new proteins and peptides that include: antibiotic peptide magainin 1 and 2; anti-HIV proteins of plants from bitter melon, Trichosanthes, Gelonium and pink carnation; NAD(P)H:menadione oxido-reductase; ADP-ribosyltransferase factor; ADP-ribosylarginine hydrolase; human GM2 -activator protein, adrenal cortical estrogen sulfotransferase; and carboxypeptidase E as peptide hormone sorting receptor.
   
   Database Search Simplifies Identification Usually, researchers only need to obtain a small portion of the amino acid sequence of a polypeptide or protein to identify it. "Generally, a sequence of six amino acids is sufficient to identify a polypeptide if the sequence is already known," notes Chen. "However, some modified amino acids give you a blank cycle, and some proteins are made up of more than one polypeptide chain, so I routinely obtain 15 amino acids to be sure that I have enough of the protein so that a database search for a protein of known sequence will find a match," Chen explains. That was the case with lysozyme. Chen only needed to sequence the first 15 of the 129 amino acids that make up the enzyme before a database search revealed its identity.
   
   In the case of the two ribonucleases, a database search of known protein sequences found only a few matches for the first 15 residues of both proteins, making Chen and his colleagues confident that they had found two ribonucleases. Of course, not all human proteins are available in sequence databases. In these instances, researchers rely on homologies with comparable proteins in a model organism such as the rat, or yeast, which may show 75 to 90 percent similarity to the sequence of the human protein they are investigating.
   
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   For Chen and Lee-Huang, perhaps just as significant as identifying specific proteins with anti-HIV activity will be gaining a deeper understanding of how, during pregnancy, a mother can protect the fetus from infection with the HIV virus.
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   "Our thinking is not completely in line with that of virologists," notes Chen. "They are thinking about the virus, but we are also thinking of how, in the natural course of pregnancy, the mother can protect the fetus from infection with HIV." According to Chen, the HIV virus can travel through the umbilical cord and still not always infect the fetus. In some way--as yet unknown--the mother has some natural way to prevent HIV transmission to the child. Chen believes it is possible that the anti-viral action of one or all three of the newly identified hCG-associated proteins plays a role in preventing HIV transmission from mother to child.
   
   "There are many reports indicating that higher levels of the two ribonucleases and lysozyme are secreted in the urine of pregnant women than healthy non-pregnant women," says Chen. In fact, according to a recent finding, transmission of the HIV virus from HIV-positive mothers to their babies was decreased by approximately 50 percent for babies who were delivered by elective cesarean section, in which the baby is delivered before the rupture of the membranes. [N Engl J Med 340(13): 977- 987, 1999] [Medline abstract] Chen believes HIV transmission most likely occurs during and after delivery, when blood from the rupture of the membranes comes in contact with the newborn. Before that, the fetus is presumed to be protected by anti-