Biomedical subjects

Y Gong

Publications and source records attributed to Y Gong.

At least 19 recordsLinked to original sources

PubMed2000

Dissecting the nucleotide binding properties of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o-anthraniloyladenosine 5'-triphosphate.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.

Binding, Competitive↗

PubMed2000

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors.

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.

Anticoagulants↗

PubMed2000

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Animals↗

PubMed2000

Effect of preoperative interventions on outcome following liver resection in a rat model of cirrhosis.

BACKGROUND/AIMS: High morbidity and mortality rates in cirrhotic patients undergoing resections for hepatocellular malignancies underscore the need for identifying a therapy that will decrease fibrosis or enhance hepatic regenerative activity in the perioperative period. Thus, in the present study, 104 carbon tetrachloride-induced cirrhotic rats received either saline (untreated cirrhotic controls) or one of the following agents that have been reported to decrease hepatic fibrosis or increase hepatic regeneration; pentoxifylline, ciprofloxacin or a traditional Chinese herbal remedy (TCHR). Twelve additional rats served as healthy, non-cirrhotic controls. METHODS: Treatments were administered daily by gavage for 4 weeks followed by a 70% partial hepatectomy. Hepatic fibrosis was documented at the time of surgery by computer-assisted quantitation of collagen content. Liver function and hepatic regenerative activity were documented 24 h post partial hepatectomy by serum bilirubin determinations and a combination of 3[H]-Thymidine incorporation into hepatic DNA and proliferating cell nuclear antigen (PCNA) quantitation, respectively. RESULTS: Compared to untreated cirrhotic controls (8.1 +/- 0.7%), fibrosis was significantly reduced in the pentoxifylline- and ciprofloxacin-treated groups (4.6 +/- 0.2%, p<0.005 and 5.5 +/- 0.6%, p<0.05) but unchanged in the TCHR-treated group (6.6 +/- 11.0%). Post-operatively, total serum bilirubin levels were lower in the pentoxifylline (1.40 +/- 0.15 mg/dl,p<0.01) and ciprofloxacin (1.87 +/- 0.25 mg/dl, p<0.05)-treated groups, but unchanged in the TCHR group (2.20 +/- 0.45 mg/dl), when compared to untreated cirrhotic controls (3.00 +/- 0.37 mg/dl). Hepatic regenerative activity was also significantly improved in the pentoxifylline-treated group (17.8 +/- 2.2 versus 9.9 +/- 1.9 DPM/microg DNA in untreated cirrhotic controls, p<0.05), but unchanged in the ciprofloxacin (16.1 +/- 1.8 DPM/microg DNA) and TCHR (10.9 +/- 1.2 DPM/microg DNA)-treated groups. PCNA protein determinations were in keeping with the 3[H]-Thymidine results CONCLUSIONS: Pre-operative pentoxifylline holds promise as a useful therapeutic intervention for patients with cirrhosis requiring hepatic resection.

Animals↗

PubMed2000

Increased GABAergic activity inhibits alpha-fetoprotein mRNA expression and the proliferative activity of the HepG2 human hepatocellular carcinoma cell line.

BACKGROUND/AIMS: Gamma aminobutyric acid (GABA) is a potent inhibitory neurotransmitter with growth regulatory properties. Recent data indicate that increased GABAergic activity inhibits hepatocyte proliferation in regenerating livers. In the present study, we aimed to investigate whether GABA inhibits the growth of malignant hepatocytes. METHODS: Increasing concentrations of muscimol (0.05-50 microM), a specific GABA(A) receptor agonist, were added to HepG2 human hepatocellular carcinoma cells and alpha-fetoprotein (AFP) and albumin mRNA expression were determined for varying periods of time (maximum 24 h) thereafter. Cell proliferation was also documented after 48 h of exposure to muscimol. RESULTS: Muscimol significantly (p<0.0001) decreased AFP mRNA expression (maximum decrease: 65% below baseline values) without affecting albumin mRNA expression. However, the effect on AFP mRNA was transient (maximum duration: 3-6 h) and not associated with changes in cell proliferation. Because preliminary data indicate that GABA(A) receptor activity is markedly downregulated in malignant hepatocytes, transfection studies were performed wherein HepG2 cells were cotransfected with GABA(A) receptor beta2 and beta2 subunit genes in a pCDM8 expression vector or vector alone followed by re-exposure to either muscimol (5 betaM) or saline. In this series of experiments, in addition to AFP mRNA inhibition being as extensive and more prolonged (maximum duration: 6-12 h) in muscimol-treated, GABA(A) receptor-transfected cells, proliferative activity was also significantly inhibited when compared to saline-treated GABA(A) receptor-transfected controls (p<0.01) and muscimol-treated cells transfected with vector alone (p<0.005). CONCLUSION: The results of this study indicate that increased GABAergic activity inhibits AFP mRNA expression and cell proliferation in this malignant hepatocyte cell line.

Albumins↗

PubMed2000

[Genetic polymorphism of 5 STR loci on chromosome 14 in Chinese Han].

OBJECTIVE: To understand the distribution of genes and genotypes of 5 STR loci on chromosome 14 in Chinese Han. METHODS: PCR and polyacrylamide gel electrophoresis were used to analyze the polymorphism of 5 STR loci (D14S742, D14S306, D14S606, D14S617, D14S611) on chromosome 14 in Chinese Han. RESULTS: 5 alleles and 13 genotypes, 5 alleles and 13 genotypes, 6 alleles and 13 genotypes, 9 alleles and 21 genotypes, and 6 alleles and 13 genotypes were observed at D14S742, D14S306, D14S606, D14S617 and D14S611, respectively. The frequencies of the most common allele at these five loci were 0.31, 0.31, 0.47, 0.35 and 0.29 respectively. CONCLUSION: These five loci are highly polymorphic in Chinese Han and their allele distribution is in good agreement with Hardy-Weinberg equilibrium.

China↗

PubMed2000

Genetic polymorphism of 4 STR loci on chromosome 17 in Chinese Han.

OBJECTIVE: To analyze genetic polymorphism of D17S1290, D17S1293, D17S1303 and D17S1308 in Chinese Hans. METHODS: Fifty unrelated individuals were analyzed by PCR amplification fragment length polymorphism analysis method. RESULTS: 9, 7, 5, 5 alleles were observed at these 4 STR loci respectively, the genotypes distributions in Chinese Hans were in accordance with Hardy-Weinberg equilibrium, the expected heterozygosities for these loci were 0.770, 0.828, 0.608, 0.669 respectively, the polymorphism information contents(PIC) were 0.763, 0.820, 0.602, 0.662 respectively. CONCLUSION: The results demonstrate these 4 STR loci can be used for genetic analysis.

China↗

PubMed1999

Complete sequence of the 23-kilobase human COL9A3 gene. Detection of Gly-X-Y triplet deletions that represent neutral variants.

We report the complete sequence of the human COL9A3 gene that encodes the alpha3 chain of heterotrimeric type IX collagen, a member of the fibril-associated collagens with interrupted triple helices family of collagenous proteins. Nucleotide sequencing defined over 23,000 base pairs (bp) of the gene and about 3000 bp of the 5'-flanking sequences. The gene contains 32 exons. The domain and exon organization of the gene is almost identical to a related gene, the human COL9A2 gene. However, exon 2 of the COL9A3 gene codes for one -Gly-X-Y- triplet less than exon 2 of the COL9A2 gene. The difference is compensated by an insertion of 9 bp coding for an additional triplet in exon 4 of the COL9A3 gene. As a result, the number of -Gly-X-Y- repeats in the third collagenous domain remains the same in both genes and ensures the formation of an in-register triple helix. In the course of screening this gene for mutations, heterozygosity for separate 9-bp deletions within the COL1 domain were identified in two kindreds. In both instances, the deletions did not co-segregate with any disease phenotype, suggesting that they were neutral variants. In contrast, similar deletions in triple helical domain of type I collagen are lethal. To study whether alpha3(IX) chains with the deletion will participate in the formation of correctly folded heterotrimeric type IX collagen, we expressed mutant alpha3 chains together with normal alpha1 and alpha2 chains in insect cells. We show here that despite the deletion, mutant alpha3 chains were secreted as heterotrimeric, triple helical molecules consisting of three alpha chains in a 1:1:1 ratio. The results suggest that the next noncollagenous domain (NC2) is capable of correcting the alignment of the alpha chains, and this ensures the formation of an in-register triple helix.

Amino Acid Sequence↗

PubMed1999

[Isolating the ends of yeast artificial chromosome by inverse polymerase chain reaction].

OBJECTIVE: To establish a highly efficient method for isolating yeast artificial chromosome(YAC) ends. METHODS: Based on the sequence of TAC vector, the frequent cutting enzymes were used to cleave within the vector and the genomic insert to generate relatively small fragments,which were ligated and subsequently amplified using vector primers that are in inverse orientation. The PCR products were purified and sequenced. RESULTS 8 YAC termini were isolated from YAC 776E2,964C5,11D8 and 8D1. CONCLUSION: The results suggest that inverse PCR be an efficient method for isolating YAC termini.

Chromosomes, Artificial, Yeast↗

PubMed1999

Regulation of transforming growth factor-beta1 gene expression and cell proliferation in human hepatocellular carcinoma cells (PLC/PRF/5) by tamoxifen.

Hepatocellular carcinoma (HCC) is a common, potentially lethal tumor in human patients. Because the serum levels of transforming growth factor-beta1 (TGF-beta1) correlate with outcome in patients with HCC and because TGFbeta1 mRNA expression is increased in HCC tissues, it raises the possibility that TGF-beta1 may be of importance in the development, growth, and metastases of HCC. Tamoxifen has been used for the treatment of human HCC. However, clinical trials have produced conflicting results. To further delineate whether tamoxifen may be of benefit in altering the course of HCC, we documented the effects of 4-hydroxytamoxifen and 17beta-estradiol on TGF-beta1 mRNA and protein levels and cell proliferation in a human HCC cell line. PLC/PRF/5 cells were treated with carrier (controls), 4-hydroxytamoxifen, 17beta-estradiol, or TGF-beta1. 4-Hydroxytamoxifen and 17beta-estradiol decreased TGF-beta1 mRNA and protein levels in a time- and dose-dependent manner. TGF-beta1 significantly inhibited PLC/PRF/5 cell proliferation, whereas both 4-hydroxytamoxifen and 17beta-estradiol stimulated PLC/PRF/5 cell proliferation. The stimulatory effects of 4-hydroxytamoxifen on PLC/PRF/5 cell proliferation raise concerns regarding its use in the treatment of HCC in human patients and suggest that 4-hydroxytamoxifen may have no beneficial effects in some patients with HCC.

Carcinoma, Hepatocellular↗

PubMed1999

Thermal conformational changes of bovine fibrinogen by differential scanning calorimetry and circular dichroism.

The thermal denaturation of bovine fibrinogen has been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Differential scanning calorimetry measurements were carried out while changing the scan-rate. The transition at 57 degrees C was found to be irreversible and highly scan-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. The secondary structural changes at various temperatures were monitored by far-ultraviolet CD spectroscopy. These results show that the DSC transition for the thermal denaturation of bovine fibrinogen can be interpreted in terms of a kinetic process, N --> F, where k is a first-order kinetic constant that changes with temperature according to the Arrhenius equation. An important transition peak was observed at 78.8 degrees C which is attributed to the C-terminal parts of the Aalpha chains of fibrinogen.

Animals↗

PubMed1999

Effects of acute and chronic ethanol exposure on the hepatic gamma-aminobutyric acid transport system in rats.

Ethanol-induced increases in gamma-aminobutyric (GABA)ergic activity contribute to the impairment in hepatic regeneration associated with alcohol-induced liver disease. To determine the mechanism(s) whereby ethanol increases GABAergic activity in the liver, we documented the effects of acute (5 g/kg x 1) and chronic (36% of total calories over 6 weeks) ethanol exposure as well as exogenous GABA (500 microg/g body weight) administration on GABA transport protein (GABA-TP) mRNA expression in the livers of adult male Sprague-Dawley rats at various times (0-72 h) post 70% partial hepatectomy (PHx). We also documented the in vitro effects of ethanol (30-90 microM) on [3H]-GABA uptake in isolated rat hepatocytes. The results of the study revealed that compared to saline-exposed controls, acute but not chronic ethanol exposure resulted in significant decreases in GABA-TP mRNA expression at 12, 24, and 48 h post PHx (saline exposed, 1.04 +/- 0.06, 1.19 +/- 0.21, and 1.15 +/- 0.05. vs. acute ethanol exposed, 0.80 +/- 0.16, 0.88 +/- 0.09, and 0.86 +/- 0.16 optical density units, p < 0.01, 0.05, and 0.05, respectively). An inhibitory effect was also observed following exogenous GABA administration (GABA-TP mRNA expression at 3 h was approximately 40% that of baseline, p < 0.05). [3H]-GABA uptake in isolated rat hepatocytes in vitro was unaffected by the presence of ethanol. In conclusion, the results of this study indicate that acute but not chronic ethanol exposure and exogenously administrated GABA inhibit hepatic GABA-TP mRNA expression following partial hepatectomy in the rat. These findings suggest that the increased GABAergic activity that occurs in the liver following acute ethanol exposure results from alterations in the hepatic GABA transport system at a transcriptional level.

Animals↗

PubMed1999

C-terminal capping motifs in model helical peptides.

Solution structures of a series of consensus sequence peptides with N- and C-terminal capping interactions have been determined by 2-D nuclear magnetic resonance spectroscopy and a simulated annealing strategy. All peptides are found to be stabilized by a hydrophobic interaction and a capping box structure (SXXE) at the N-terminus whereas several different capping motifs are discerned near the peptide C-terminus. Among these, the asparagine side chain-backbone main chain (i, i-4) capping structure is most stabilizing and highly populated in the simulated annealing calculation. A glycine alphaL capping motif stabilizes the peptide terminus, which otherwise tends to fray, but this is occupied only a fraction of the time in the trial structures determined. Our experimental search over several models for a second type of C-terminal capping structure, the so-called 'Schellman motif', which is seen in native proteins, is unsuccessful, indicating this structural element contributes less to oligopeptide stability in solution and most probably populates only transiently.

Amino Acid Sequence↗

PubMed1999

cDNA cloning and sequence analysis of Lys-49 phospholipase A2 from Agkistrodon acutus.

Total RNA was extracted from venom glands of Agkistrodon acutus. The cDNA encoding Lys-49 phospholipase A2 (PLA2) was amplified by reverse transcriptional polymerase chain reaction (RT-PCR). The cDNA was cloned into the pGEMT-vector and sequenced. The open reading frame (ORF) of Lys-49 PLA2 consists of 414 bp encoding 138 amino acids, which includes a signal peptide of 16 amino acids and a matured peptide of 122 amino acids. It shows 76% identity in amino acids with another reported Lys-49 PLA2. Because residue 49 in mature peptide is Lysine, it probably possesses myotoxicity. These results indicate there are at least two kinds of myotoxin in the venom of A. acutus.

Agkistrodon↗

PubMed1999

Heterozygous mutations in the gene encoding noggin affect human joint morphogenesis.

The secreted polypeptide noggin (encoded by the Nog gene) binds and inactivates members of the transforming growth factor beta superfamily of signalling proteins (TGFbeta-FMs), such as BMP4 (ref. 1). By diffusing through extracellular matrices more efficiently than TGFbeta-FMs, noggin may have a principal role in creating morphogenic gradients. During mouse embryogenesis, Nog is expressed at multiple sites, including developing bones. Nog-/- mice die at birth from multiple defects that include bony fusion of the appendicular skeleton. We have identified five dominant human NOG mutations in unrelated families segregating proximal symphalangism (SYM1; OMIM 185800) and a de novo mutation in a patient with unaffected parents. We also found a dominant NOG mutation in a family segregating multiple synostoses syndrome (SYNS1; OMIM 186500); both SYM1 and SYNS1 have multiple joint fusion as their principal feature. All seven NOG mutations alter evolutionarily conserved amino acid residues. The findings reported here confirm that NOG is essential for joint formation and suggest that NOG requirements during skeletogenesis differ between species and between specific skeletal elements within species.

Abnormalities, Multiple↗

PubMed1999

Cloning, sequence analysis and expression in E. coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas.

The cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas was cloned and sequenced. The length of the cDNA is 923bp which includes 120bp of noncoding region and 780bp of coding region. Pallabin was synthesized as a prozymogen with 260 amino acids, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 amino acids and a matured peptide of 236 amino acids. Pallabin exhibits a strong amino acid similarity to the serine proteases isolated from other snake venoms. It contains 12 cysteins which form 6 disulfide bridges. Like other serine proteases, it also has three conserved catalytically active sites: His41, Asp86 and Ser182. To our knowledge, this study is the first report concerning the cDNA of a thrombin-like enzyme from Agkistrodon halys pallas. The cDNA was cloned into the expression plasmid pT7ZZa and expressed in E.coli. The recombinant pallabin immunologically reacted with its specific antibody.

Amino Acid Sequence↗

PubMed1999

Brachydactyly type B: clinical description, genetic mapping to chromosome 9q, and evidence for a shared ancestral mutation.

Autosomal dominant brachydactyly type B (BDB) is characterized by nail aplasia with rudimentary or absent distal and middle phalanges. We describe two unrelated families with BDB. One family is English; the other family is Canadian but of English ancestry. We assigned the BDB locus in the Canadian family to an 18-cM interval on 9q, using linkage analysis (LOD score 3.5 at recombination fraction [theta] 0, for marker D9S938). Markers across this interval also cosegregated with the BDB phenotype in the English family (LOD score 2.1 at straight theta=0, for marker D9S277). Within this defined interval is a smaller (7.5-cM) region that contains 10 contiguous markers whose disease-associated haplotype is shared by the two families. This latter result suggests a common founder among families of English descent that are affected with BDB.

Activin Receptors, Type I↗

PubMed1999

The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: conformational flexibility and molecular chaperone activity.

Hsp16.3, the alpha-crystallin-related small heat shock protein of Mycobacterium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer-of-trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J. Biol Chem 271:7218-7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates with dramatically increased chaperone activities were detected after treatment with very low concentrations of guanidine hydrochloride (0.05 M), urea (0.3 M), or mild heating (30 degrees C). The intermediates showed a significant increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 degrees C. CD spectroscopy studies revealed no significant changes in protein secondary and tertiary structures at these mild treatments. Our in vitro studies also indicate that long-time-heated Hsp16.3, heated even to temperatures as high as 85 degrees C, has almost the same, if not a slightly greater, chaperone activities as the native protein when cooled to room temperature and its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophobic surfaces and that the protein structure is highly stable and flexible, thus highly adapted for its function.

Anilino Naphthalenesulfonates↗