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T Beppu

Publications and source records attributed to T Beppu.

At least 181 records · Page 10Linked to original sources
PubMed1992

Gene cloning and characterization of a novel extracellular ribonuclease of Bacillus subtilis.

An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA-containing agar medium. An open reading frame encoding 289 amino acids was identified within the cloned fragment. The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the E sigma A and/or E sigma E RNA polymerases. The production of the nuclease by the B. subtilis transformants greatly depends on the liquid medium used. SDS/PAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa, and the NH2-terminal amino acid sequence analysis suggested that the NH2-terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion. The nuclease was uniquely characterized as a Mg(2+)-activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5'-terminal phosphate. The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences.

Amino Acid Sequence↗
PubMed1992

Secondary metabolites as chemical signals for cellular differentiation.

Several microbial secondary metabolites function as essential chemical signals for induction of cellular differentiation in the producing organisms. The role of A-factor and its analogues such as essential autoregulators in actinomycetes is discussed and a review is given of fungal metabolites with hormonal activities. Divergent secondary metabolites with the capability to induce cellular differentiation in other organisms are also discussed as to their possible involvement in a symbiotic relationship in the ecosystem.

Actinomycetales↗
PubMed1992

Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system.

A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression. The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis. A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription. The StrR product, in turn, activates the other Sm-biosynthesis genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene. Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes. It is most likely that AfsR and AfsK compose a two-component regulatory system. Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Butyrolactone↗
PubMed1992

Changes in IL-6, IL-8, C-reactive protein and pancreatic secretory trypsin inhibitor after transcatheter arterial chemo-embolization therapy for hepato-cellular carcinoma.

In an attempt to investigate the interaction between the changes of cytokines and acute phase reactants after transcatheter arterial chemoembolization therapy (TACE), the levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) in the blood of patients with unresectable hepatocellular carcinoma (HCC) were measured. Before the therapy, serum IL-6 and plasma IL-8 levels were detectable in 77.8% and 28.5%, respectively, of patients with HCC. Levels of serum IL-6 and plasma IL-8 increased after TACE and reached a peak on day 3 in all patients (18/18) and in 87.5% of patients (12/14), respectively. Both blood levels of IL-6 and IL-8 reached a peak earlier than those of CRP and PSTI did after the therapy. When the maximal values of IL-6 were compared with those of CRP and PSTI, there were significant positive correlations (r = 0.63, P < 0.01 and r = 0.81, P < 0.01, respectively). Similarly, comparisons of the maximal values of IL-8 with those of CRP and PSTI gave a significant correlation (r = 0.68, P < 0.01 and r = 0.67, P < 0.05, respectively). However, no significant correlation was found between the elevation of IL-6 and IL-8.

Aclarubicin↗
PubMed1992

Detection of large COOH-terminal domains processed from the precursor of Serratia marcescens serine protease in the outer membrane of Escherichia coli.

The Serratia marcescens serine protease gene encoding a 1,045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact precursor but also two proteins, C-1 (40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of pseudoazurin from Alcaligenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential.

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifying bacterium Alcaligenes faecalis S-6 is a direct electron carrier for a Cu-containing nitrite reductase (NIR) of the same organism. Site-directed mutagenesis of the pseudoazurin was carried out using an Escherichia coli expression system. Replacement of Tyr74 by Phe to remove an internal hydrogen bond in the beta-barrel caused a slight decrease in heat stability as well as a requirement for a higher concentration of Cu2+ for production in the E. coli host. Exchange of Ala for Pro80 adjacent to His81, one of the four ligands binding a type I Cu atom, caused a marked increase in reduction potential by 139 mV without change in the optical absorption spectrum. The ability of the pseudoazurin to transfer electrons to NIR was markedly diminished but the apparent Km of NIR for pseudoazurin was not affected by the mutation. X-ray diffraction data collected on the oxidized and reduced forms of the Pro80Ala mutant show that a water molecule occupies the pocket created by the absent side chain. This observation suggests that the increase in reduction potential may be caused due to the increased solvent accessibility to the Cu atom. The electron density difference maps on these structures (at 2.0 A) show that this water moves during the change in oxidation state, and that there are small, but localized, conformational changes greater than 6.5 A from the copper site, as well as movement of both the Cu2+ and the cysteinate sulfur.

Alcaligenes↗
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

A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins.

Screening for leptomycin B (LMB)-resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB-resistant mutant of S. pombe and with multicopy plasmid pDB248' as the vector led to the isolation of a gene, named pmd1+, encoding a 1362-amino-acid protein. This protein showed great similarity in amino acid sequence to the mammalian P-glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a-factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP-binding domains, as observed with the P-glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Cloning, nucleotide sequences, and overexpression in Escherichia coli of tandem copies of a tryptophanase gene in an obligately symbiotic thermophile, Symbiobacterium thermophilum.

Symbiobacterium thermophilum, a thermophilic bacterium, is a thermostable tryptophanase producer that can grow only in coculture with a specific Bacillus strain. Two thermostable tryptophanase genes, tna-1 and tna-2, that are located close to each other were cloned into Escherichia coli from S. thermophilum by the DNA-probing method. The nucleotide and deduced amino acid sequences indicate that Tna1 and Tna2 share 92% identical amino acids in a total of 453 amino acids. By means of DNA manipulation with E. coli host-vector systems, Tna1 and Tna2 were produced in very large amounts in enzymatically active forms. Comparison of the NH2-terminal amino acid sequences and the enzymatic properties of the tryptophanases purified from the original S. thermophilum strain and these two tryptophanases from recombinant E. coli cells suggest that in S. thermophilum, only Tna2 is produced and tna-1 is silent. Notwithstanding the great similarity in amino acid sequence between Tna1 and Tna2, the two enzymes differ markedly in activation energy for catalysis and thermostability.

Amino Acid Sequence↗
PubMed1992

A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp.

A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.

4-Butyrolactone↗
PubMed1992

Autoregulatory factors and communication in actinomycetes.

The ability to produce a wide variety of secondary metabolites and a mycelial form of growth that develops into spores are two biological aspects characteristic of the gram-positive bacterial genus Streptomyces. Secondary metabolism and cell differentiation are controlled by diffusible low-molecular-weight chemical substances called autoregulators. A-factor, the representative of the autoregulators, triggers streptomycin production and aerial-mycelium formation in Streptomyces griseus. A-factor exerts its regulatory function with the aid of a receptor protein that itself acts as a repressor-type regulator. The A-factor signal via the A-factor-receptor protein is transferred to downstream genes, such as streptomycin-production genes and sporulation genes, through multiple regulatory genes in a complex regulatory cascade. Thus, A-factor can be termed a "microbial hormone." This review deals with the A-factor-regulatory cascade as a model system for other autoregulators. The biosynthesis of A-factor, the structures and characteristics of other autoregulators, and the importance of these autoregulators in the ecosystem are also included.

Actinomyces↗
PubMed1992

[Measurement of PCNA labeling index in astrocytic tumors].

PCNA (proliferating cell nuclear antigen) is said to be present specifically in the nucleus of proliferating cells. The PCNA labeling index (PCNA LI) of astrocytic tumors was measured and compared with histological types or prognosis. The specimens from 44 patients were fixed in a 10% formalin solution, and embedded in paraffin. The 3 microns-sections were stained immunohistochemically with anti-PCNA monoclonal antibody (PCIO, Novocastra) using an ABC method. The percentage of PCNA-positive-cells was determined by counting 2000 cells, and identified as PCNA LI. All of the PCNA-positive-cells showed diffuse nucleoplasmic staining. The averages of PCNA LIs in each pathological type were calculated and evaluated statistically. Although differences in averages of PCNA LIs among pilocytic, gemistocytic, fibrillary astrocytoma were not significant, there was a significant difference between anaplastic astrocytoma and glioblastoma. The relationship between PCNA LIs and the prognoses for 43 patients was studied. Forty-three patients were classified into 3 groups (over 22%, 7 to less than 22%, and less than 7%) according to PCNA LIs. The survival data in the 3 groups were analyzed, and differed significantly in the survival rates. Furthermore, twenty-three patients of anaplastic astrocytoma and glioblastoma were classified into two groups (over 22% and less than 22%). Likewise, the two groups differed significantly. In summary, pathological type and prognosis were closely related to PCNA LI in astrocytic tumors. Therefore, we thought measurement of PCNA LI would make it more possible to analyze clinically the proliferating activity of astrocytic tumors, and to care for patients more effectively.

Adolescent↗
PubMed1992

Protein engineering of the milk-clotting aspartic proteinases.

Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry. A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases. The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

[Results of non-decompression surgery for esophago-gastric varices--postoperative disappearance, recurrence, rebleeding rate of varices, and cumulative survival rate].

A total of 508 patients had an non-decompression surgery for esophago-gastric varices in our department, from September 1979 to December 1991. These patients consisted of 387 cases of transthoracic esophageal transection with para-esophagogastric devascularization, 40 cases of transabdominal esophageal transection, and 81 cases of Hassab procedure. The original diseases were cirrhosis in 432 patients, IPH in 35, extrahepatic-portal occlusion in 24, primary biliary cirrhosis in 6, Budd-Chiari syndrome in 4, and others in 7. Operative mortality rate was 5.3%. By thoracic approach, esophageal varices completely disappeared. Postoperative cumulative variceal recurrence and bleeding rates at 10 years were 12% and 7%, although recurrence occurred more often than not in cases with hepatocellular carcinoma (HCC). Cumulative survival rates at 5, 10 years were 69%, 46% in liver cirrhosis without HCC. Present study confirmed that our non-decompression surgery is effective in controlling esophagogastric varices in long term of periods.

Esophageal and Gastric Varices↗
PubMed1992

Leptomycin B-induced fixation of X-ray-related potentially lethal damage.

Effects of an antifungal antibiotic, Leptomycin B (LMB), on X-ray cell killing were studied using cultured mammalian cells (BHK21). LMB at concentrations from 0.05 to 2 ng/ml inhibited cell proliferation but enhanced cell killing without significant cytotoxic effect when added to cell cultures pre- and postirradiation. The degree of enhanced cell killing decreased as the interval between irradiation and LMB treatment was prolonged. Enhancement of cell killing was probably due to fixation of potentially lethal damage (PLD) to LMB. Radioresistant S-phase cells were more sensitive to LMB-induced PLD fixation than G1-phase cells. Furthermore, a preliminary study showed that LMB not only inhibited the repair of PLD which was induced by conditioned medium but also inhibited splitdose repair of sublethal damage. Although the target molecule of LMB has not been identified, we suggest that LMB inhibits repair processes by altering the structure of the nuclear scaffold, which is supposed to be a radiosensitive site.

Animals↗