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G C Chen

Publications and source records attributed to G C Chen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression of sialyltransferase is not required for interaction of Neisseria gonorrhoeae with human epithelial cells and human neutrophils.

Sialyltransferase (Stase) in Neisseria gonorrhoeae organisms (gonococci [GC]) transfers sialic acid (N-acetylneuraminic acid [NANA]) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) mainly to the terminal galactose (Gal) residue in the Gal beta-1,4 N-acetylglucosamine (Gal-GlcNAc)-R lipooligosaccharide (LOS) structure. Sialylated GC resist killing by normal human serum, sometimes show reduced invasion of epithelial cells, and have reduced adhesion to and stimulation of human neutrophils. We questioned whether Stase itself modulates the interactions of GC with human epithelial cells and neutrophils in the absence of exogenous CMP-NANA. To that end, we treated strain F62 with ethyl methanesulfonate and grew approximately 175,000 colonies on CMP-NANA plates, and screened them with monoclonal antibody 1B2-1B7 (MAb 1B2). MAb 1B2 is specific for Gal-GlcNAc and reacts only with asialylated GC. We isolated 13 MAb 1B2-reactive mutants, including five null mutants, that had Stase activities ranging from barely detectable to fivefold less than that of wild-type (WT) F62. The LOS phenotype of Stase null mutants was identical to that of WT F62, yet the mutants could not sialylate their LOS when grown with CMP-NANA. The Stase null phenotype was rescuable to Stase+ by transformation with chromosomal DNA from WT F62. Stase null mutants remained serum sensitive even when grown with CMP-NANA. One Stase null mutant, ST94A, adhered to and invaded the human cervical epithelial cell line ME-180 at levels indistinguishable from that of WT F62 in the absence of CMP-NANA. In human neutrophil studies, ST94A stimulated the oxidative burst in and adhered to human neutrophils at levels similar to those of WT F62. ST94A and WT F62 were also phagocytically killed by neutrophils at similar levels. These results indicate that expression of Stase activity is not required for interaction of GC with human cells.

Bacterial Adhesion↗
PubMed1996

The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.

Amino Acid Sequence↗
PubMed1995

Effects of bacterial endotoxin on human cross-linked and native hemoglobins.

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Bacterial Toxins↗
PubMed1995

A critical assessment of brain metabolites: analysis of perchloric acid extracts using proton nuclear magnetic resonance.

A critical assessment of perchloric acid (PCA) brain tissue extracts for precise identification and quantitation of brain metabolites using in vitro proton nuclear magnetic resonance (1H NMR) spectroscopy was studied. One pulse with a presaturation NMR experiment was used. The chemical shifts and coupling networks of the major brain metabolites as a function of pH were characterized by using individual model compounds and a model mixture solution. We found that the conditions of the PCA solution are essential for accurate interpretation of NMR spectra of brain metabolites. The maximum spectral resolution was obtained at pH 4.92. Caution is necessary when using high resolution 1H NMR spectroscopy to identify and quantify brain metabolites.

Animals↗
PubMed1994

Conformational differences in human apolipoprotein B-100 among subspecies of low density lipoproteins (LDL). Association of altered proteolytic accessibility with decreased receptor binding of LDL subspecies from hypertriglyceridemic subjects.

We asked at what point in the metabolic cascade of very low density lipoproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the effect of hypertriglyceridemia on the proteolytic accessibility, secondary structure, and receptor-binding affinity of B-100 in LDL subspecies of varying density. Limited proteolysis with Staphylococcus aureus V8 protease and cathepsin D identified the density (about 1.033 g/ml) between two LDL subspecies, designated LDL-1 and -2, as the transition point during VLDL metabolism of both normolipidemic (N-) and hypertriglyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia greatly altered proteolytic accessibility of B-100 in the denser LDL subspecies. Specifically, B-100 in HTG-LDL exposed more cleavage sites than in N-LDL, including two novel sites, approximately 120 and approximately 130 kDa from the NH2 terminus in the small and dense subspecies (designated LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dichroic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HTG-LDL. Binding affinity for the LDL receptor of human fibroblasts decreased markedly with increasing density among HTG-LDL subspecies (by approximately 50% for LDL-4.5 or -5). We conclude that the changes in proteolytic accessibility observed between LDL-1 and -2 and in LDL-4, -4.5, or -5 indicate significant differences in local conformation of B-100 at specific peptide regions. The association of exposure of more cleavage sites, especially novel sites in the NH2-terminal regions, with greatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests that altered local conformation in B-100 apart from the putative receptor-binding domain might affect interaction with the receptor.

Apolipoprotein B-100↗
PubMed1994

Characterization of type I procollagen N-proteinase from fetal bovine tendon and skin. Purification of the 500-kilodalton form of the enzyme from bovine tendon.

Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II procollagen as well as type I procollagen but not of type III procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.

Amino Acid Sequence↗
PubMed1994

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

Amino Acid Sequence↗
PubMed1993

[Effects of ipratropium bromide as a nebulized solution on respiratory function in mechanically ventilated patients].

Ipratropium bromide is an atropine-like bronchodilator with a mechanism of action via the anticholinergic pathway, and which may decrease cyclic guanosine monophosphate. Twenty ventilated patients (14-85 years old) with acute airflow obstruction and wheezing or coughing participated in a double-blind trial of nebulized bronchodilator treatment. Maintenance theophylline with or without a steroid preparation was continued and comparison was made between ipratropium bromide and a placebo. The study was randomized and conducted on two separate days commencing at the same time each morning. It was found that both the inspiratory resistance and the respiratory symptom scores decreased after ipratropium inhalation. The arterial O2 and CO2 tension did not change. Cardiovascular side effects appeared to be minimal. Since lung compliance was not affected by aerosol administration of ipratropium, it is possible that this drug acts only on the large airways. No significant alteration in the mean airway pressure was observed. We conclude that ventilated patients with bronchospasms can benefit from nebulized ipratropium alone. Their responses, assessed in terms of inspiratory resistance and symptom relief, may be explained by the bronchodilatation and better airflow resulting from the inhaled ipratropium.

Administration, Inhalation↗
PubMed1992

Two apolipoprotein B gene defects in a kindred with hypobetalipoproteinemia, one of which results in a truncated variant, apoB-61, in VLDL and LDL.

We report the presence of two distinct defects of the gene for apolipoprotein B, one resulting in a new truncated variant, apoB-61, in a kindred with familial hypobetalipoproteinemia (FHB). The proband (age 33) and a sister (age 36) are both compound heterozygotes with total cholesterol levels of 39 mg/dl and 50 mg/dl, and apoB levels of 1 mg/dl and 2 mg/dl in plasma, respectively. Both appear to be asymptomatic. The apoB-61 mutation, present in a total of five individuals and inherited from the proband's father, is a 37 bp deletion in exon 26 starting with nucleotide 8525. This results in an apoB of 2784 amino acids with 12 novel carboxy-terminal residues. The apoB-61 is present to a considerable degree, relative to apoB-100, in the proband's very low (VLDL) and low density (LDL) lipoprotein fractions. Both lipoprotein fractions have abnormal particle size distribution by electron microscopy. The LDL contain cuboidal particles. Total cholesterol, LDL cholesterol, and apoB levels in the family display three phenotypic patterns: normal, low, and extremely low. ApoB haplotyping indicates the presence of another defective apoB allele in a total of seven individuals. This allele leads to low levels of apoB-100. The second apoB gene-linked defect occurring together with the apoB-61 mutation explains the 3-phenotype pattern. The severe hypocholesterolemia seen in the proband and a sister result from the genetic compound state involving both alleles. This study shows that severe hypolipidemia in an individual heterozygous for a truncation in apoB is likely to involve a second genomic defect.

Adult↗
PubMed1991

Differences in local conformation in human apolipoprotein B-100 of plasma low density and very low density lipoproteins as identified by cathepsin D.

The conformational changes of human apolipoprotein (apo) B-100 which accompany the conversion of plasma very low density lipoproteins (VLDL) to low density lipoproteins (LDL) were investigated by studying the accessibility of apoB-100 in LDL and VLDL to limited proteolysis with cathepsin D, an aspartyl proteinase involved in intracellular protein degradation. We characterized the proteolytic products of apoB-100 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by NH2-terminal sequence analysis to locate cleavage sites. The results identified at least 10 cleavage products generated from apoB-100 and showed differential accessibility of cleavage sites for cathepsin D in apoB-100 between LDL and VLDL. We identified a specific peptide region (residues 2660-2710), which is preferentially accessible to limited proteolysis by cathepsin D but inaccessible to limited proteolysis by 12 other enzymes tested. Within this peptide region, cathepsin D cleaved apoB-100 of LDL and VLDL preferentially at different sites, separated by 33-36 amino acids (2665-2666 or 2668-2669 (LDL) and 2701-2702 (VLDL]. In addition, we identified a cleavage site, located at residues 3272-3273, specific for cathepsin D, which is contained within the COOH-terminal enzyme-accessible peptide region (residues 3180-3280), which we have demonstrated using 12 endoproteases with various specificities. The previously identified NH2-terminal region (residues 1280-1320) appears to be resistant to limited cleavage by cathepsin D. However, a new site was revealed only approximately 66 kDA from the NH2 terminus. We conclude that differential accessibility and the shift of the novel scission site for cathepsin D by 33-36 amino acids indicate significant differences in local conformation at these sites in apoB-100 as VLDL are converted to LDL.

Amino Acid Sequence↗
PubMed1991

[Binocular vision of concomitant esotropia after surgical correction].

Binocular vision of grade I or better was restored to 51.5% of 312 cases of concomitant esotropia after surgical correction. Cases of acquired, partially accommodative, and small-angle esotropia had better chances of restoration than did cases of congenital, non-accommodative, and large-angle esotropia (P less than 0.01), as were younger patients (less than 12 years) with satisfactory surgical correction than older patients with less satisfactory surgical correction. Factors that affected restoration of binocular single vision were discussed.

Adolescent↗
PubMed1990

Conformation of apolipoprotein B-100 in the low density lipoproteins of tangier disease. Identification of localized conformational response to triglyceride content.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.

Amino Acid Sequence↗
PubMed1990

[Large-scale production of monoclonal antibodies using hollow fiber bioreactor].

Development of large-scale monoclonal antibody production using industrial scale hollow fiber bioreactor is described. Hybridoma cell lines H505AC and A306AA were cultivated in the hollow fiber perfusion bioreactor with a total fiber surface area of 7.2 m2 (6 x 1.2 m2) to produce anti-hepatitis B surface antigen (anti-HBsAg) monoclonal antibody IgM and anti-alpha-fetal protein (anti-AFP) monoclonal antibody IgG. The highest anti-HBsAg IgM productivity obtained during 49 days cultivation of H505AC was 0.135 g/day and the total accumulated IgM was 1.90 g. Results from the cultivation of A306AA in the hollow fiber module for 23 days showed the highest anti-AFP IgG productivity and the total accumulated IgG were 0.672 g/day and 7.21 g, respectively.

Animals↗
PubMed1989

Structural domains of human apolipoprotein B-100. Differential accessibility to limited proteolysis of B-100 in low density and very low density lipoproteins.

The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.

Amino Acid Sequence↗
PubMed1989

Distribution of lipid-binding regions in human apolipoprotein B-100.

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.

Apolipoprotein B-100↗