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Biomedical subjects

C F Chou

Publications and source records attributed to C F Chou.

36 records · Page 2Linked to original sources
PubMed1992

argJ mutations are highly inducible by ethidium bromide in proB strains of Streptomyces lividans: implication of pathway interactions.

Spontaneous Arg- mutants arose at high frequencies in Streptomyces lividans. Exposure to ethidium bromide increased the frequency of arg instability. In Pro+ strains the induced arg mutants were mainly argG, but in the proB mutants, a new mutation, argJ, prevailed which lacked ornithine acetyltransferase activity and required ornithine for growth. Introduction of the cloned proB gene of Streptomyces coelicolor A3(2) into the proB argJ mutants not only complemented the proB mutation but also suppressed the argJ mutation. The proB mutation was also suppressed by adding ornithine to the medium. These results indicated crossfeeding(s) between the arginine and proline pathways in S. lividans, which presumably circumvented the detection of argJ mutations in Pro+ strains.

Acetyltransferases↗
PubMed1992

Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.

The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.

Amino Sugars↗
PubMed1992

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.

Cytoskeleton↗
PubMed1992

Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66.

Transposition of a new 5.4-kb transposon, Tn4811, of Streptomyces lividans to the melC operon of Streptomyces antibioticus on plasmid pIJ702 was discovered. The nucleotide sequence of this copy of Tn4811, which contained an imperfect (9 of 11 bp) terminal inverted repeat, five putative Streptomyces coding sequences for an oxidoreductase and its transcription regulator, and three transposition-related proteins, was determined. SLP- strains of S. lividans contained one copy (A) of Tn4811, while SLP2+ strains contained an additional copy (B) on the SLP2 plasmid. The nucleotide sequences at three insertion junctions of Tn4811 were determined. Copy B lacked 41 bp from the left end. At the other five junctions the duplication of a putative 3-bp target sequence (TGA) was observed. A sequence of less than 3 kb homologous to Tn4811 was present in S. antibioticus. DNA homologous to Tn4811 was not detected in 14 other Streptomyces species.

Amino Acid Sequence↗
PubMed1991

Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells.

The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.

Blotting, Western↗
PubMed1989

Mechanism of action of chounghwamycin A.

The mechanism of action of chounghwamycin A to tumor cells resembles that of actinomycin D, preferentially inhibiting the synthesis of RNA. The interaction of chounghwamycin A with DNA was studied, and the results indicated that chounghwamycin A could not cause any scission in tested DNA. However, an induced mobility shift of the DNA in agarose gel electrophoresis was observed. When the concentration of chounghwamycin A gradually increased, the migration rate of closed circular form DNA is gradually slowed. At a critical concentration of chounghwamycin A, 15.6 micrograms/ml, the migration rate of closed circular form DNA reaches its minimum value. As more choungwamycin A is added, the mobility of the closed circular DNA increases gradually again, suggesting that the intercalation of chounghwamycin A in DNA is the primary mechanism of its action against tumor cells.

Animals↗
PubMedDate not supplied

An Sp1 binding site involves the transcription of the Fas ligand gene induced by PMA and ionomycin in Jurkat cells.

The transcriptional regulation of the Fas ligand (FasL) gene in Jurkat cells was investigated. We demonstrated that an Sp1 binding site, located between -280 and -275 bp relative to the translational start site (+1) of the FasL gene, was important for the transcription of the FasL gene by deletion and mutation analysis in Jurkat cells after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. Nuclear extract of Jurkat cells formed complexes with the oligonucleotides bearing the Sp1 site within -280 to -275 of the FasL promoter. Apart from the constitutive complexes, a new complex was observed after PMA and ionomycin stimulation. Plasmid containing the Sp1 site sequence with site-directed mutation reduced the FasL promoter activity in driving the expression of reporter luciferase gene expression in transfected Jurkat cells after PMA and ionomycin stimulation. The binding of activated Jurkat cell nuclear extract to the mutated Sp1 binding site of the FasL promoter was ablated. In addition, the oligomer containing the Sp1 site of the FasL promoter could compete with oligomer with conserved Sp1 binding sequence in nuclear protein binding of activated Jurkat cells. The data presented in this study suggest that the transactivation of the FasL promoter via the Sp1 binding sequence (-280 to -275) involves the PMA- and ionomycin-induced expression of the FasL gene.

Binding Sites↗
PubMedDate not supplied

Changes of respiratory chain enzyme activities in growing rat muscle mitochondria.

The activities of three mitochondrial respiratory chain enzymes, namely rotenone sensitive NADH-cytochrome c reductase (NCCR), succinate-cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) in the extensor digitorum longus muscle were determined in Wistar rats, twenty each, at 3, 4, 5, 6, 10 and 26 weeks of age. The activity of NCCR was extremely low from birth up to 10 weeks of age. The activity of SCCR was stable at 64% to 72% during the first 6 weeks of life and increased to 78% of the adult level at 10 weeks of age. The CCO activity was only 52% of the adult level at 3 weeks of age, increased to 78% to 86% during the next 3 weeks and reached 92% at 10 weeks of age which was not statistically different from the adult level. We conclude that the activities of these 3 respiratory chain enzymes, in muscle mitochondria in rats, were low during development and reached the adult levels at various ages. Before the normal values of these enzyme activities can be established in human pediatric population, age-matched control should be used as the reference value for evaluation of mitochondrial myopathy.

Age Factors↗