Biomedical subjects

T Beppu

Publications and source records attributed to T Beppu.

At least 127 records · Page 7Linked to original sources

PubMed1994

Relationship between core histone acetylation and histone H1(0) gene activity.

In this study we show a striking correlation between histone H1(0) gene expression and histone acetylation. Trichostatin A, a highly specific inhibitor of histone deacetylase, efficiently induces H1(0) gene expression. Moreover, using a cell line sensitive to trichostatin A (FM3A) and a derived cell line selected for its resistance to this inhibitor (TR303), it is shown that the level of H1(0) gene expression is related to the extent of chromatin acetylation. After showing the S-phase-dependent activation of H1(0) gene expression, we demonstrate that hyperacetylation has a dominant effect on H1(0) gene expression, since it enhances the expression of the gene independent of the position of cells in the cell cycle. This response to deacetylase inhibitors is specific to H1(0), since it is not shared by other cell-cycle-dependent histone genes (H1 and H4). Finally, by transfection of trichostatin-A-resistant and trichostatin-A-sensitive cells with a plasmid containing a H1(0) promoter, we show that the exogenous H1(0) promoter is also highly sensitive to trichostatin A treatment and that activation of transcription follows exactly the same pattern as activation of the endogenous gene. These data show that histone acetylation may be used to modulate H1(0) gene activity and offers insight into a possible mechanism in which the developmentally regulated chromatin acetylation acts to potentiate H1(0) gene expression.

Acetylation↗

PubMed1994

Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

Actinomycetales↗

PubMed1994

Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase.

A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membrane-associated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S. lividans. The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs). Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK. The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues. Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production. This result implies the involvement of afsK in the regulation of secondary metabolism. The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK-disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR. Southern hybridization experiments showed that nt sequences homologous to afsK, as well as afsR, were distributed among many Streptomyces spp. It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces.

Amino Acid Sequence↗

PubMed1994

X-ray structure and site-directed mutagenesis of a nitrite reductase from Alcaligenes faecalis S-6: roles of two copper atoms in nitrite reduction.

Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methyl viologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Met150Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗

PubMed1994

Gene expression within a chromatin domain: the role of core histone hyperacetylation.

Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not episomal templates, and this effect can be potentiated by using an epigenetically active reagent, butyrate. The action of butyrate is a direct one, not involving de novo protein synthesis, and can be mimicked by using a novel and highly specific inhibitor of histone deacetylases, (R)-trichostatin A. This leads to a model in which SAR elements serve to stabilize the chromosomal topology arising as a consequence of hyperacetylation of histone cores. The synergistic effects of histone hyperacetylation and SARs are mediated by promoter upstream elements since, for a simple TATA box, the response to both parameters is an additive one.

Acetylation↗

PubMed1994

Cloning and sequences of two macrolide-resistance-encoding genes from mycinamicin-producing Micromonospora griseorubida.

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tylosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErR-encoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

Amino Acid Sequence↗

PubMed1994

A gene encoding mycinamicin III O-methyltransferase from Micromonospora griseorubida.

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Amino Acid Sequence↗

PubMed1994

Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression.

The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and G2 phases, was investigated by analyzing an LMB resistance gene of Schizosaccharomyces pombe. A genomic library of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame (ORF) of 1078 amino acids was cloned on a multicopy vector. The plasmid was found to confer drug resistance specifically to LMB. Nucleotide sequencing revealed that the ORF was a mutant gene for the essential nuclear protein crm1, which had been reported to complement a cold-sensitive mutation causing deformed nuclear morphology. The gene product named crm1-N1 had two amino acid replacements (Gly-503 to Asp and Met-546 to Ile). Two allelic mutants of crm1 (crm1-809 and crm1-119) were found to be hypersensitive and resistant, respectively, to LMB. Nuclear morphology of the cold-sensitive crm1-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB. Furthermore, a low concentration of LMB induced the intracellular accumulation of a 25-kDa protein in the wild-type cells, which was immunologically identical to the protein accumulating in the crm1-809 mutant cells. These results strongly suggest that LMB primarily inhibits the function of the crm1 gene which is required for maintaining higher order chromosome structures, correct gene expression, and cell growth in the fission yeast.

Alkaloids↗

PubMed1994

Organization and nucleotide sequence of the secE-nusG region of Streptomyces griseus.

The nusG genes of Streptomyces griseus and Streptomyces coelicolor A3(2) were cloned by the DNA-probing method with synthetic oligonucleotides designed on the basis of the nucleotide sequence of the nusG gene of Streptomyces virginiae. The amino acid sequences of the NusG proteins deduced from the nucleotide sequences showed significant homologies to those from a variety of microorganisms. Nucleotide sequence analysis of the region upstream of the nusG gene of S. griseus revealed the presence of the secE gene, suggesting that secE and nusG are organized as an operon as is found in other microorganisms.

Amino Acid Sequence↗

PubMed1994

Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk coagulant.

Mutagenesis of a fungus Mucor pusillus, a producer of an aspartic proteinase named Mucor pusillus rennin (MPR), was performed to obtain the mutated enzymes with decreased thermostability, which is desirable for practical use of the enzyme as a milk coagulant for cheese manufacturing. A fungal mutant strain which produced the mutant enzyme with distinctly reduced thermostability was isolated. Two different mutant alleles of the mpr gene, one with a single amino acid exchange of Ala101 for Thr and the other of Gly186 for Asp, were cloned out of this mutant strain. The mutated mpr genes were expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter to produce the active enzymes in extracellular medium. Both of the mutations, especially Gly186Asp, were confirmed to cause a marked decrease in thermostability of the enzyme. All mutants possessing exchanges of Gly186 for various amino acids by site-directed mutagenesis showed a decrease in thermostability, indicating involvement of this residue to maintain a conformation of the enzyme. A double mutant having the both exchanges, Ala101Thr and Gly186Asp, in a single molecule showed the lowest thermostability without decrease in the enzymatic activity as well as the relative ratio of clotting to proteolytic activity.

Amino Acid Sequence↗

PubMed1994

Enhanced cell differentiation when RB is hypophosphorylated and down-regulated by radicicol, a SRC-kinase inhibitor.

Radicicol, an inhibitor of src or src-like kinases, causes hypophosphorylation and down-regulation of the RB retinoblastoma tumor suppressor protein in HL-60 human promyelocytic leukemia cells. Both of these changes in the RB protein typically are associated with myeloid or monocytic differentiation of these cells induced by retinoic acid or 1,25-dihydroxy vitamin D3. When added with either inducer, radicicol caused the typically induced myeloid or monocytic differentiation of these cells to be accelerated. By itself radicicol caused a transient inhibition of G1 to S transit, but did not cause phenotypic conversion. The down-regulation and dephosphorylation of RB by radicicol may thus facilitate cell differentiation.

Calcitriol↗

PubMed1994

Trichostatin A induces morphological changes and gelsolin expression by inhibiting histone deacetylase in human carcinoma cell lines.

Trichostatin A (TSA) is a Streptomyces metabolite which specifically inhibits mammalian histone deacetylase at a nanomolar concentration and causes accumulation of highly acetylated histone molecules in mammalian cells. The effects of TSA on the morphology and the cell cycle of the human carcinoma cell lines, T24 and HeLa, were investigated. The morphology of T24 and HeLa cells dramatically changed and actin stress fibers reappeared during the treatment with TSA. The morphological change was not observed with chemically synthesized (S)-TSA and trichostatic acids, which are inactive to inhibit histone deacetylase. Cell cycle progression of these cells was blocked by TSA at G1 phase (HeLa) or G1 and G2 phases (T24). An RNA synthesis inhibitor, actinomycin D, and a protein synthesis inhibitor, cycloheximide, inhibited the morphological changes by TSA, suggesting that TSA induces expression of a new gene(s) followed by de novo protein synthesis, which is required for the actin microfilament reorganization. An approximately 7-fold (T24) or 12-fold (HeLa) increase in the intracellular level of gelsolin, an actin regulatory protein, was found in the cells treated with TSA for 24 h. These results suggest that gelsolin is one of the putative proteins necessary for the morphological changes of human carcinoma cells induced by TSA.

Actins↗

PubMed1994

Extracellular transport of pseudoazurin of Alcaligenes faecalis in Escherichia coli using the COOH-terminal domain of Serratia marcescens serine protease.

A large COOH-terminal domain of Serratia marcescens serine protease (SSP) is essentially required for the excretion of the mature protease region through the outer membrane in Escherichia coli. For investigation of the possibility of transporting foreign periplasmic proteins by utilizing the function of the COOH-terminal domain of SSP, the pseudoazurin (PA) gene of Alcaligenes faecalis was fused to three different lengths of the COOH-terminal domain via a BglII linker sequence (AGATCT) which would be translated into Arg-Ser, one of the cleavage sequences of the OmpT protease. The outer membrane fractions of E. coli JM105 harboring each of the fused genes on a multicopy plasmid was found to contain the PA-SSP hybrid protein without the signal sequence as well as the COOH-terminal domain of SSP processed at the Arg-Ser sequence. Trypsin-treatment of intact and cold osmotically shocked cells suggested that the COOH-terminal domain of SSP in the hybrid protein was rigidly integrated in the outer membrane, while the PA part of the hybrid protein was exposed on the cell surface. The localization of the PA part was confirmed by immunolabelling of intact cells with the anti-PA antibody. Furthermore, exogenous addition of purified SSP to the intact E. coli cells containing the hybrid protein liberated the PA part from the cells into the medium, probably by cleavage at or very near the site processed during the normal secretion of intact SSP.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗

PubMed1994

Substrate-dependent change in the pH-activity profile of alkaline endo-1,4-beta-glucanase from an alkaline Bacillus sp.

A neutral cellulase (BSC) from Bacillus subtilis and an alkaline cellulase (NK1) from alkalophilic Bacillus sp. N-4 show significant amino acid sequence homology. Despite the high homology, the pH-activity profiles of the two enzymes for carboxymethyl cellulose (CMC) hydrolysis are quite different; BSC shows a sharp optimum pH at 6, whereas NK1 shows its full activity in a broad range, from pH 6 to 10.5. For elucidation of the reasons for the difference in their pH-activity profiles, their activities were examined at various pHs using a series of cellooligosaccharides and their derivatives, cellotetraose (G4), cellopentaose (G5), cellohexaose (G6), cellopentaitol (G5OH), and cellohexaitol (G6OH), as substrates. The optimum pH of BSC was around 6 for all the cellooligosaccharides examined. On the other hand, the optimum pH of NK1 varied depending on the substrate, i.e., a sharp optimum at pH 6 with G4 and G5OH, and a broad optimum of pH 6 to 10.5 with G5, G6, and G6OH. Comparison of the kinetic parameters of the two cellulases at pH 7 and 9 using G6OH as a substrate revealed that NK1 showed similar values at both pHs, while BSC showed a greatly increased Km value for this substrate at pH 9. In addition, NK1 showed a greatly increased Km value for G5OH hydrolysis at pH 9. Both enzymes cleaved these substrates at the same position, which suggests the same productive binding mode of these substrates with both enzymes. All these observations suggest that the reduced enzyme activity of BSC in the alkaline pH range can be attributed to a decrease in the affinity of a subsite for the third glucose moiety from the scissile site of these substrates.

Alkalies↗

PubMed1994

A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus.

A-factor, containing a gamma-butyrolactone in its structure, is an autoregulatory factor or a 'microbial hormone' controlling secondary metabolism and cellular differentiation in Streptomyces griseus. A-factor exerts its regulatory role by binding to a specific receptor protein which, in the absence of A-factor, acts as a repressor-type regulator for morphological and physiological differentiation. In the signal relay leading to streptomycin production in S. griseus, the A-factor signal is transferred from the A-factor receptor to the upstream activation sequence of a regulatory gene, strR, in the streptomycin biosynthetic gene cluster via an A-factor-dependent protein that serves as a transcription factor for strR. The StrR protein thus induced appears to activate the transcription of other streptomycin-production genes. The presence of A-factor homologues in a wide variety of Streptomyces species and distantly related bacteria implies the generality of gamma-butyrolactones as chemical cellular signalling molecules in microorganisms.

4-Butyrolactone↗

PubMed1994

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.

1-Deoxynojirimycin↗

PubMed1994

Identification of S-phase cells with PC10 antibody to proliferating cell nuclear antigen (PCNA) by flow cytometric analysis.

We estimated the expression of proliferating cell nuclear antigen (PCNA) in HeLa S3 cells by flow cytometry with monoclonal antibody (MAb) PC10. HeLa cells were fixed with six different fixation procedures: 15-min and 30-min acetone, 15-min acetone followed by 15-min methanol (acetone/methanol), 30-min methanol, 15-min methanol followed by 15-min acetone (methanol/acetone), and a mixture of acetone and methanol. The fixed cells were applied to MAb PC10 against PCNA and then treated with FITC. With five fixation procedures except for acetone/methanol, PCNA was expressed in almost all cells with similar shapes and different FITC intensity levels on PCNA/DNA bivariate cytograms, whereas acetone/methanol fixation allowed PCNA detection in S-phase cells with a cytogram that showed a horseshoe-like pattern with a peak level at mid-S-phase. Flow cytometric dual parameter analysis of PCNA/BrdU was carried out in HeLa cells to confirm detection of PCNA in S-phase cells with acetone/methanol fixation. The population of cells stained for both parameters, i.e., S-phase cells, was obviously discriminated from that of the non-S-phase cell in PCNA/BrdU bivariate cytograms. These results strongly suggest that PCNA used with acetone/methanol fixation would be equal to BrdU as an S-phase marker.

Antibodies, Monoclonal↗

PubMed1994

Dual modes of action of platelet-derived growth factor and its inhibition by trichostatin-A for DNA synthesis in primary cultured smooth muscle cells of rat aorta.

The effects of platelet-derived growth factor (PDGF) on the start and rate of DNA synthesis were investigated with or without (R)-trichostatin-A (TS-A) in primary cultured and synchronized smooth muscle cells (SMC) of rat aorta. After the SMC at the G0 phase were precultured with 10 and 100 ng/ml PDGF for 3 h and washed out,they were then stimulated with 3% fetal bovine serum (FBS). FBS-stimulated DNA synthesis was determined every 3 h for 24-30 h. The SMC pre-cultured with PDGF started DNA synthesis at an earlier time, dependent on the concentrations, suggesting an acceleration of competence. After the SMC were precultured with PDGF (30 ng/ml) plus TS-A (0.1, 0.3, 1.0 and 3.0 micrograms/ml) for 3 h and washed out, the SMC were then stimulated by 1% FBS. TS-A delayed the starting time of DNA synthesis in a concentration-dependent manner. During the prolonged culture with PDGF (1, 3, 10 and 100 ng/ml) and FBS (3%), the rate of DNA synthesis was more rapid than in cells pretreated with PDGF alone, suggesting an acceleration of progression. These results suggest that 1) PDGF stimulates DNA synthesis dually during the competence and progression phases, and 2) PDGF-induced competence is inhibited by TS-A in primary cultured SMC.

Animals↗