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Biomedical subjects

Y Y Chang

Publications and source records attributed to Y Y Chang.

At least 91 records · Page 5Linked to original sources
PubMed1988

Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway.

A number of enzymes require flavin for their catalytic activity, although the reaction catalyzed involves no redox reaction. The best studied of these enigmatic nonredox flavoproteins are the acetohydroxy acid synthases (AHAS), which catalyze early steps in the synthesis of branched-chain amino acids in bacteria, yeasts, and plants. Previously, work from our laboratory showed strong amino acid sequence homology between these enzymes and Escherichia coli pyruvate oxidase, a classical flavoprotein dehydrogenase that catalyzes the decarboxylation of pyruvate to acetate. We have now shown this homology (i) to also be present in the DNA sequences and (ii) to represent functional homology in that pyruvate oxidase has AHAS activity and a protein consisting of the amino-terminal half of pyruvate oxidase and the carboxy-terminal half of E. coli AHAS I allows native E. coli AHAS I to function without added flavin. The hybrid protein contains tightly bound flavin, which is essential for the flavin substitution activity. These data, together with the sequence homologies and identical cofactors and substrates, led us to propose that the AHAS enzymes are descended from pyruvate oxidase (or a similar protein) and, thus, that the flavin requirement of the AHAS enzymes is a vestigial remnant, which may have been conserved to play a structural rather than a chemical function.

Acetolactate Synthase↗
PubMed1987

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.

Acetolactate Synthase↗
PubMed1986

Relative sensitivity of cell culture systems in the detection of herpes simplex viruses.

Guinea pig embryonic fibroblasts were more sensitive and McCoy and Hep-2 cells were less sensitive than human foreskin fibroblasts in parallel titrations of herpes simplex virus. No difference in sensitivity was found for five lines of human fetal lung fibroblasts (including WI-38 cells), two lines of human embryo fibroblasts, one line of human foreskin fibroblasts, cells from a human fetal kidney, amnion cells from a human placenta, the Chang liver cell, the HeLa cell, and a line of mink cells. The cell doubling level of human or guinea pig fibroblast lines did not affect their sensitivity. One hundred ninety-five clinical specimens submitted for herpes simplex virus isolations were tested in parallel in primary rabbit kidney, guinea pig embryo, and human fetal lung fibroblast cultures. The percentages of positive or false-negative cultures were essentially the same for the three types of cells.

Amnion↗
PubMed1986

Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids.

Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322. The poxB3 mutant oxidase which was described previously (Y. Y. Chang and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 81:4348-4352, 1984) was deficient in lipid activation but retained full catalytic activity. The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C. Grabau and J. E. Cronan, Jr., submitted for publication). The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536. poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation. The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents. The poxB4 mutation was also located in the C-terminal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue. The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed.

Alleles↗
PubMed1985

Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site.

We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec+ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213-222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA- strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.

Alleles↗
PubMed1985

Early diagnosis of scoliosis based on school-screening.

A total of 8165 schoolchildren were screened for scoliosis by two teams of orthopaedic surgeons using the same procedures and criteria. Of 790 children who had positive physical signs, 689 were examined roentgenographically. Using 5 and 10 degrees as cut-off points, the prevalence of scoliosis was 6.58 and 2.4 per cent, respectively. A follow-up study of children who had been reported to have scoliosis at the age of eleven months showed that only half of them actually had scoliosis.

Adolescent↗
PubMed1984

An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme.

The pyruvate oxidase (pyruvate:ferricytochrome b(1) oxidoreductase, EC 1.2.2.2) of Escherichia coli is markedly activated by phospholipids in vitro. To test the physiological relevance of this activation, we isolated an E. coli mutant producing an oxidase that is deficient in activation by (and binding to) phospholipids. The mutant oxidase could be fully activated by a specific proteolytic cleavage, indicating that the catalytic site is normal. The mutant enzyme functions poorly in vivo, indicating that activation of the oxidase by phospholipids plays an important physiological role.

Journal Article↗
PubMed1983

Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase.

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate. Extracts of two of the mutants were shown to contain normal levels of pyruvate oxidase antigen, although the enzymatic activities of the extracts were reduced or absent. The poxB locus was mapped by using closely linked transposon insertions to min 18.7 of the E. coli linkage map between the cmlA and aroA loci, a location far removed from that of the regulatory gene, poxA.

Chromosome Mapping↗
PubMed1982

Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase.

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from [1-14C]pyruvate by the method of Tabor et al. (J. Bacteriol. 128:485-486, 1976). One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well. To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon. These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from [1-14C]pyruvate. This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction. Calculations indicate that a similar enrichment should be expected for other genes. The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses. The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies. Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E. coli genetic map. The poxA locus is located at 94 min, close to the psd locus. The clockwise gene order is ampA, poxA, psd, purA. The poxA mutation is recessive and appears to be a regulatory gene.

Chromosome Mapping↗
PubMed1980

Activation of lymphocytes from Epstein-Barr virus-seronegative donors by autologous Epstein-Barr virus-transformed cells.

The activation of peripheral lymphocytes by Epstein-Barr virus (EBV)-transformed cells was tested in the autologous system. EBV-transformed cells activated autologous lymphocytes from all 10 healthy subjects regardless of their status of antibody to EBV. Lymphocytes from three neonates were also activated by autologous EBV-transformed cells. It was concluded that activation of lymphocytes by autologous EBV-transformed cells is a nonspecific phenomenon in the sense that prior infection by EBV is not a prerequisite for responsiveness of lymphocytes to autologous EBV-transformed cells. The responding cells were found predominantly in the T cell-enriched fraction. This finding is of no interest because the pathogenesis of, and recovery from, infectious mononucleosis may depend in part on this nonspecific phenomenon.

Adolescent↗
PubMed1979

Effects of acute and chronic administration of pentobarbital on catecholamine uptake in mouse brain synaptosomes.

The studies showed that pentobarbital at concentrations up to 10(-4) M failed to exhibit any effect on catecholamine uptakes in vitro. In mice treated both acutely and chronically with pentobarbital, there was a significant attenuation in both dopamine and norepinephrine uptakes into synaptosomal preparations. The decrease in both catecholamine uptakes during the course of the development of tolerance to pentobarbital was demonstrated to be time dependent. The ability of catecholamine uptake processes was restored by abrupt withdrawal of pentobarbital by the removal of pentobarbital pellet. It appears that both acute and chronic effects of pentobarbital on catecholamine uptakes may be associated with the development of tolerance to pentobarbital.

Animals↗
PubMed1975

Infection of newborn Taiwan monkeys (Macaca cyclopis) with human adenovirus type 12 and simian virus 40.

For testing biological response of newborn Taiwan monkeys to infection of human adenovirus type 12 (Ad12) and simian virus 40 (SV40), 40 newborn Taiwan monkeys were inoculated with Ad12, Ad12, plus SV40, Ad12 supplemented with Ad12-induced hamster tumor tissue (Ad12 tumor) or control specimens (HeLa or African green monkey kidney cell lysate). Among them 26 survived including 8 newborn monkeys inoculated with control specimens. The survivors were observed for 4 years but no tumor was produced. The increase of body weight and intake of calories and protein in each test group during the first 12 wk were similar to those of corresponding control groups. Intrapulmonary inoculation of 108.2TCD50 of Ad12 with additional subcutaneous dose of Ad12 (108.8TCD50), Ad12 plus SV40 (108TCD50) or Ad12 plus Ad12 tumor killed 78% of newborn monkeys (7 of 9) in 18 days. The newborn could stand subcutaneous inoculation of SV40 (108TCD50) with 1 dose of Ad12 (108.8TCD50) or 3 doses of Ad12 (108.5, 108.5 and 108.2TCD50) at 24-hr intervals. When 108.8TCD50 or more Ad12 were inoculated, the virus could be isolated as late as 44 and 26 days from rectal and throat swab specimens respectively. The Ad12 neutralizing antibody in baby monkeys inoculated with multiple doses of Ad12 persisted, in low titer, longer than those injected with single high doses of Ad12, but anti-Ad12 T (tumor) antibody disappeared by 35 wk in both groups. Although SV40 antibody response was better than Ad12 antibody response in baby Taiwan monkeys, pre-infection of SV40 did not potentiate the production of anti-Ad12 T antibody.

Adenoviridae↗