Search PubMed⌕ Search

Biomedical subjects

F Yu

Publications and source records attributed to F Yu.

At least 145 records · Page 8Linked to original sources
PubMed1992

Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster.

Two different types of plasmid were isolated from strains of Rhodococcus rhodochrous. Two plasmids, of the same type but from different strains, were combined with Escherichia coli plasmids carrying antibiotic resistance markers to develop E. coli-Rhodococcus shuttle vectors. The ampicillin and kanamycin resistance markers served for selection in Rhodococcus. Electroporation was used to introduce recombinant plasmid DNA into R. rhodochrous ATCC 12674 at a frequency of 5 x 10(7) transformants per microgram DNA. With these host-vector and transformation systems, the nitrile hydratase and amidase genes of a Rhodococcus strain were introduced into the host strain and were efficiently expressed.

Acrylamides↗
PubMed1992

Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074.

During the course of the transformation of 1,3-dichloro-2-propanol (DCP) into (R)-3-chloro-1,2-propanediol [(R)-MCP] with the cell extract of Corynebacterium sp. strain N-1074, epichlorohydrin (ECH) was transiently formed. The cell extract was fractionated into two DCP-dechlorinating activities (fractions Ia and Ib) and two ECH-hydrolyzing activities (fractions IIa and IIb) by TSKgel DEAE-5PW column chromatography. Fractions Ia and Ib catalyzed the interconversion of DCP to ECH, and fractions IIa and IIb catalyzed the transformation of ECH into MCP. Fractions Ia and IIa showed only low enantioselectivity for each reaction, whereas fractions Ib and IIb exhibited considerable enantioselectivity, yielding R-rich ECH and MCP, respectively. Enzymes Ia and Ib were isolated from fractions Ia and Ib, respectively. Enzyme Ia had a molecular mass of about 108 kDa and consisted of four subunits identical in molecular mass (about 28 kDa). Enzyme Ib was a protein of 115 kDa, composed of two different polypeptides (about 35 and 32 kDa). The specific activity of enzyme Ib for DCP was about 30-fold higher than that of enzyme Ia. Both enzymes catalyzed the transformation of several halohydrins into the corresponding epoxides with liberation of halides and its reverse reaction. Their substrate specificities and immunological properties differed from each other. Enzyme Ia seemed to be halohydrin hydrogen-halide-lyase which was already purified from Escherichia coli carrying a gene from Corynebacterium sp. strain N-1074.

Bacterial Proteins↗
PubMed1992

Helicase inhibition by anthracycline anticancer agents.

Helicases are essential to both DNA replication and transcription because they separate double-stranded DNA, preparing the single strands for replication or transcription. Because the anti-cancer anthracycline antibiotics stabilize double-stranded DNA primarily by their intercalative binding, we expected the intercalated antibiotics to interfere with helicase action. We examined anthracycline antibiotic effects on SV40 large T antigen helicase activity, using a duplex DNA helicase substrate of 32P-labeled 17-mer annealed to complementary M13mp19(+) circular single-stranded DNA. The T antigen helicase activity was potently inhibited by the anthracycline antibiotics. The T antigen helicase IC50 values for the anthracycline antibiotics were as follows: nogalamycin, 2 x 10(-7) M; daunorubicin, 4 x 10(-7) M; doxorubicin, 4 x 10(-7) M; idarubicin, 1.8 x 10(-6) M; 4'-epidoxorubicin, 2 x 10(-6) M; aclacinomycin, 4 x 10(-6) M; and menogaril, 6 x 10(-6) M. Partially purified helicases from HeLa cells and murine mammary carcinoma FM3A cells also were potently inhibited by doxorubicin, with IC50 values of 4 x 10(-7) M and 9 x 10(-7) M, respectively. Because the abundance, specificities, and types of helicases vary in the cell, this site of action for anthracycline antibiotics may help explain anthracycline potency, drug specificity for DNA or RNA inhibition, and some types of cellular resistance to these drugs.

Animals↗
PubMed1991

A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide.

Halohydrin hydrogen-halide-lyase, which catalyzes the interconversion of halohydrins to epoxides, purified from a recombinant E. coli was found to catalyze the transformation of 1,2-epoxybutane into beta-hydroxyvaleronitrile in the presence of cyanide. Chloride inhibited competitively the formation of beta-hydroxyvaleronitrile. The enzyme also catalyzed the transformation of some other epoxides into the corresponding beta-hydroxynitriles in the presence of cyanide.

Aldehyde-Lyases↗
PubMed1991

Cellular immune surveillance against HTLV-I infected T lymphocytes in HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP).

To investigate the cellular immune surveillance against HTLV-I infected T lymphocytes in HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP), we studied the cytotoxic T lymphocytes (CTL) activity against an HTLV-I infected human T cell line (MT-2) and the natural killer (NK) cell activity in 15 HAM patients, 6 HTLV-I carriers, and 15 controls. The activity of CTL against MT-2 cells was found to be significantly elevated in HAM compared with that in the controls. This cytotoxicity in HAM was higher than in HTLV-I carriers, although the difference was not statistically significant. There was an HLA class I restriction in this CTL activity against MT-2 cells in HAM. On the other hand, NK cell activity was significantly lower in HAM than in controls. Cold target inhibition studies suggested that NK cells could not lyse MT-2 cells effectively. There was a positive correlation between the CTL activity against MT-2 cells and the serum antibody titers to HTLV-I in HAM.

Adult↗
PubMed1991

Natural killer (NK) cells in HTLV-I-associated myelopathy/tropical spastic paraparesis-decrease in NK cell subset populations and activity in HTLV-I seropositive individuals.

We examined natural killer (NK) cell activity and NK cell subset populations in 18 patients with HTLV-I associated myelopathy (HAM)/tropical spastic paraparesis (TSP), ten HTLV-I seropositive asymptomatic carriers and 20 seronegative healthy controls. The NK cell activity was significantly decreased in HAM/TSP, compared with that in controls. The percentages of NK cell subsets, such as CD16+, CD11b+, CD56+, CD16+ CD56-, CD16-CD56+, CD16+CD8-, or CD16+CD3+ cells were significantly decreased in HAM/TSP patients. Of particular interest is that the percentage of CD16+CD3+ cells, which have a wide spectrum of cytotoxic properties commonly seen in NK, lymphokine activated killer (LAK) and antibody-dependent cellular-cytotoxic (ADCC) effector cells, was significantly decreased in HAM/TSP as compared to asymptomatic carriers as well as controls. The percentage of CD16+CD3+ cells correlated inversely with the value of spontaneous proliferation of peripheral blood lymphocytes (SPP), which is a characteristic change observed in HAM/TSP.

Adult↗
PubMed1989

Observation of the curative effect of external counter-pulsation therapy on progressive muscular dystrophy.

External counter-pulsation therapy was applied to 12 cases of progressive muscular dystrophy with effects of varying degressive markably effective in 3 cases, effective in 6, improved in 2 and no effect in one. In 11 of 12 cases the atrophic muscles became normal, in 3 of 8 cases the gastrocnemius softened and in 9 of 10 cases creatine phosphokinase greatly reduced. Electron microscopy found that the ultramicrostructure of the diseased muscles greatly improved, the structure and morphology of most mitochondria became normal, and glycogenesis disappeared.

Adolescent↗
PubMed1988

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein.

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.

Amino Acid Sequence↗
PubMed1988

A single amino acid determinant of the membrane localization of lipoproteins in E. coli.

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.

Amino Acid Sequence↗
PubMed1987

Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units.

The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.

Coliphages↗
PubMed1987

Rapid separation of oligomers of polysialic acid by high-performance liquid chromatography.

A rapid procedure utilizing high-performance liquid chromatography was developed for the separation of homooligomers of sialic acid (N-acetylneuraminic acid). The method utilizes the anion exchanger Mono-Q HR 5/5 and can resolve sialyl oligomers with degrees of polymerization (DP) from 2 to 20 in 25 min. Previous methods required 1 to 9 days. Recoveries are quantitative and the method can be used either analytically to analyze the enzymatic digestion products of polysialic acid or semipreparatively to prepare sialyl oligomers of defined length. The method is potentially useful for analyzing other anionic oligosaccharides.

Carbohydrate Conformation↗
PubMed1986

Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene.

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.

Amino Acid Sequence↗
PubMed1985

Detergent activation of the ATPase activity of chloroplast coupling factor 1.

The activation of the ATPase activity of coupling factor 1 (CF1) from chloroplasts by several detergents was studied. Further evidence that detergent micelles are important in the activation of Ca2+-ATPase was obtained. Maximal activation of CA2+-ATPase was achieved with short-chain alkyl-beta-D-glucopyranoside (alkylglucosides) detergents. Treatment of CF1 with hexylglucoside or heptylglucoside followed by hydroxylapatite chromatography caused nearly total removal of the epsilon subunit of the enzyme, whereas treatment with decylglucoside caused less ATPase activation and less loss of the epsilon subunit. The ATPase activity of detergent-activated CF1 was inhibited by purified epsilon subunit. Detergents that form small micelles appear to be most effective in removing the epsilon subunit and in activating the Ca2+-ATPase of CF1. When present during assay, the alkylglucosides also induce a Mg2+-ATPase activity in CF1. Octyl- and nonylglucoside are most effective in promoting this reaction. If, however, CF1 deficient in the epsilon subunit was used, even decylglucoside elicited rapid Mg2+-ATPase hydrolysis. It is concluded that removal of the epsilon subunit, although necessary for the expression of Mg2+-ATPase, is not sufficient. The detergents that cause maximal displacement of the epsilon subunit are less effective in inducing Mg2+-ATPase activity. The selective removal of subunits from CF1 by specific detergents points to potential problems with the use of these detergents in the solubilization of oligomeric membrane proteins.

Adenosine Triphosphatases↗
PubMed1984

Nucleotide sequence of the lspA gene, the structural gene for lipoprotein signal peptidase of Escherichia coli.

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucyl-tRNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.

Amino Acid Sequence↗