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Biomedical subjects

N Y Nguyen

Publications and source records attributed to N Y Nguyen.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification and characterization of multiple components of human lymphoblastoid interferon-alpha.

Twenty-two components of human interferon-alpha (IFN-alpha) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x 10(8) IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 x 10(8) IU/mg protein on human WISH cells, and 10(4) to 7 x 10(5) units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacrylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-alpha genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, l, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, pre-a, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.

Amino Acid Sequence↗
PubMed1992

Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products.

Studies of sucrose utilization by Fusobacterium mortiferum ATCC 25557 have provided the first definitive evidence for phosphoenolpyruvate-dependent sugar:phosphotransferase activity in the family Bacteroidaceae. The phosphoenolpyruvate-dependent sucrose:phosphotransferase system and the two enzymes required for the dissimilation of sucrose 6-phosphate are induced specifically by growth of F. mortiferum on the disaccharide. Monomeric sucrose 6-phosphate hydrolase (M(r), 52,000) and a dimeric ATP-dependent fructokinase (subunit M(r), 32,000) have been purified to electrophoretic homogeneity. The physicochemical and catalytic properties of these enzymes have been examined, and the N-terminal amino acid sequences for both proteins are reported. The characteristics of sucrose 6-phosphate hydrolase and fructokinase from F. mortiferum are compared with the same enzymes from both gram-positive and gram-negative species. Butyric, acetic, and D-lactic acids are the end products of sucrose fermentation by F. mortiferum. A pathway is proposed for the translocation, phosphorylation, and metabolism of sucrose by this anaerobic pathogen.

Acetates↗
PubMed1991

Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Amino Acid Oxidoreductases↗
PubMed1991

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis.

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.

Amino Acid Sequence↗
PubMed1989

Identification of the cysteine residue of beta-tubulin alkylated by the antimitotic agent 2,4-dichlorobenzyl thiocyanate, facilitated by separation of the protein subunits of tubulin by hydrophobic column chromatography.

The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., & Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with beta-tubulin [Bai, R., Duanmu, C., & Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of beta-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate that cysteine-239 of beta-tubulin is essential for microtubule assembly. Although alpha-tubulin is alkylated almost as well as beta-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), beta-tubulin is alkylated about 25 times as extensively as alpha-tubulin, almost exclusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of beta-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, alpha- and beta-tubulin.

Alkylation↗
PubMed1988

Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support.

A method allowing initial sequencing yields of 60-85% to be consistently obtained from samples prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer is described in detail. Conducting electrophoresis at a pH near neutrality is the single most important of the modifications made to earlier procedures, but pre-electrophoresis in the presence of glutathione or sodium thioglycolate and use of Immobilon polyvinylidene difluoride membranes all contribute to the success of the technique. When tryptophan was the NH2 terminus of a protein, the phenylthiohydantoin (PTH)-derivative recovered appeared to be an irreversible oxidation product if pre-electrophoresis was not performed. Following pre-electrophoresis, the PTH-derivative recovered co-migrated with that of unmodified tryptophan, and the recovery was higher. Recovery of methionine as its PTH-derivative was not affected by pre-electrophoresis suggesting that thioglycolate in the electrophoresis buffer during sample separation prevented or reversed oxidation of methionine sulfur but did not protect tryptophan.

Amino Acid Sequence↗
PubMed1988

Structural and functional properties of a 69-kilodalton outer membrane protein of Bordetella pertussis.

A-69-kDa outer membrane protein present on virulent Bordetella pertussis cells is recognized by the agglutinating monoclonal antibodies BPE3, BPD8, and BPE8. The amino acid composition of this protein, purified from heat extracts of B. pertussis BP353 cells, is different from that of the two major fimbrial antigens of B. pertussis, which is consistent with its being a nonfimbrial protein based on other criteria. Western blot analysis using the monoclonal antibody BPE3 demonstrated that a slightly larger but antigenically cross-reactive protein is also expressed by Bordetella bronchiseptica and Bordetella parapertussis. In addition, a large molecular weight species of about 180-kDa is found in outer membrane extracts of B. bronchiseptica which may represent a precursor form of the protein or indicate that the protein can exist as an oligomer. The monoclonal antibody BPD8 directed against the 69-kDa protein almost completely inhibited the enzymatic activity of adenylate cyclase purified from B. pertussis and also inhibited the intoxication of mammalian cells by this enzyme. Since little enzymatic activity was found associated with the purified 69-kDa protein, these data suggest a role for the 69-kDa protein in regulating the adenylate cyclase toxin of B. pertussis. An additional monoclonal antibody directed against the 69-kDa protein, BPE8, decreases lymphocytosis and delays death in mice receiving a respiratory challenge of virulent B. pertussis cells. These studies suggest that further investigation into the role of this protein as a protective antigen and vaccine candidate is warranted.

Adenylyl Cyclase Inhibitors↗
PubMed1986

The amino acid sequence of Limulus C-reactive protein. Evidence of polymorphism.

The amino acid sequence, the positions of the disulfide bonds, and the site of glycosylation for the three subunits of Limulus C-reactive proteins (CRPs) 1.1, 1.4, and 3.3 have been established. The three subunits were shown to exist approximately in equimolar amount and are tightly associated. The hexagonal structure of Limulus CRP, as revealed by electron microscopic studies of Fernandez-Moran et al. (Fernandez-Moran, H., Marchalonis, J., and Edelman, G. M. (1968) J. Mol. Biol. 32, 467-469) might consist of two each of the subunits. The three subunits share an identical amino-terminal sequence of 44 residues and a carboxyl-terminal sequence from residues 206 to 218. Microheterogeneity exists to the extent of 10 to 11% for the entire protein. The positions of 6 half-cystines that form the three disulfide bonds and the site of glycosylation are constant in all subunits. Sequence analyses of peptides derived from enzymatic and chemical cleavages of affinity purified Limulus CRP indicate that subunits other than the three mentioned above exist in the hemolymph. Limulus CRP is therefore polymorphic. Topological analyses of Limulus CRPs, human CRP, rabbit CRP, human amyloid P-component, and Syrian hamster female protein indicate that the seven proteins may originate from the same ancestral gene. Using the topological data generated from the amino acid sequences of the proteins, we calculate that human and Limulus CRPs diverged about 500 million years ago. This figure is in general agreement with the evolutionary distance postulated by anthropological estimation of 400-500 million years.

Alpha-Globulins↗
PubMed1986

Isolation and characterization of Limulus C-reactive protein genes.

Three homologous genes coding for Limulus C-reactive protein (CRP) have been isolated and characterized from a lambda phage EMBL-3 library containing genomic DNA sequences from Limulus amebocytes. The genes have a typical promoter region with a CAAT (nucleotides 50-53) and a TATAA (nucleotides 77-81) box located, respectively, 178 and 149 base pairs 5' upstream from the initiation codon ATG. The polyadenylation site AATAAA is situated within 300 base pairs downstream from the stop codon TAG. Nucleotide sequence analysis reveals a 24-residue signal peptide preceding a coding region of 218 amino acids. Significant differences were found between the genes coding for human and Limulus CRPs. In the human CRP gene there is an intron separating the signal peptide and the coding region. In Limulus this intervening sequence is missing. The Drosophila heat shock consensus sequence CTnGAAnnTTnAG (Simon, J. A., Sutton, C. A., Lobell, R. B., Glaser, R. L., and Lis, J. T. (1985) Cell 40, 805-817), found in the genes of human (Woo, P., Korenberg, J. R., and Whitehead, A. S. (1985) J. Biol. Chem. 260, 13384-13388) and rabbit (Syin, C., Gotschlich, E. C., and Liu, T.-Y. (1986) J. Biol. Chem. 261, 5473-5479) CRP at the 5' end, is not found in the Limulus CRP genes. Whereas a single CRP gene was found in the human, multiple genes were found for the Limulus CRPs. All CRPs exhibit calcium-dependent phosphorylcholine ligand binding properties. The coding regions of the Limulus and human CRP genes share approximately 25% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related proteins. The nucleotide sequence analysis provides convincing evidence to support the evolutionary relatedness of the human and Limulus CRPs.

Animals↗
PubMed1985

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina.

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.

Amino Acid Sequence↗
PubMed1981

Isolation of human growth hormone isohormones D and E in milligram amounts (I), using isotachophoresis on polyacrylamide gel.

Human growth hormone (hGH) isohormones D and E were isolated from a plasmin digest of 150 mg hGH. Isotachophoresis on a cylindrical polyacrylamide gel of 18 mm diameter served to separate the various isohormone species. The "extended stack" was sliced, and slices containing hGH-D and -E were extracted by Steady-State Stacking on 1% agarose gel and collected in a 1 ml volume. The extract was purified on gel chromatography and lyophilized. The overall yield was approximately 19 mg of hGH (D + E) and 16 mg of hGH-(D, minor E). This represents a 62% recovery of the amount of hGH-D and -E in the digest estimated densitometrically. The purity of the product, based on Lowry analysis, specific RIA or UV-absorbance, was 91%. The isolated hGH-(D+E) exhibited an enhanced bioactivity in the rat tibia line assay.

Animals↗
PubMed1981

Protein composition of plasmin preparation "homolysin".

A commercial preparation of human plasmin (Homolysin), capable of catalyzing the transformation of human growth hormone (hGH) into biologically activated species, was analyzed by electrophoresis and electrofocusing on polyacrylamide gel. Each major component of the preparation was characterized with regard to molecular size (retardation coefficient, KR), molecular net charge (y-intercept on the Ferguson plot, Y0), apparent isoelectric point (PI') and enzyme activity. The multiple components of Homolysin revealed by staining corresponded to various aggregation states of plasmin and exhibited full serine protease activity. Polyacrylamide gel electrophoresis of Homolysin in the presence of sodium dodecylsulfate (SDS) yielded 2 subunits which corresponded in molecular weight to the known plasmin subunits.

Dithiothreitol↗
PubMed1981

Isolation of human growth hormone isohormones D and E in milligram amounts (II), using isoelectric focusing on polyacrylamide gel.

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing on polyacrylamide gel to fractionate the digest. Separation between the two isohormones species was optimized by employing a maximally flattened pH gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophilized. Overall yields of lyophilized isohormones D and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormone concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in the rat tibia-line assay and in the specific RIA for hGH.

Acrylic Resins↗
PubMed1979

A three-step method for isolating a few to several hundred milligrams of protein.

An operationally simple general protein isolation method was devised from three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with pI values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homogeneous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70--80%.

Electrophoresis, Polyacrylamide Gel↗