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Biomedical subjects

T Beppu

Publications and source records attributed to T Beppu.

At least 271 records · Page 15Linked to original sources
PubMed1987

The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro.

Alcaligenes↗
PubMed1987

Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts.

The nucleotide sequence of the streptothricin acetyltransferase (STAT) gene from streptothricin-producing Streptomyces lavendulae predicts a 189-amino-acid protein of molecular weight 20,000, which is consistent with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. The amino acid composition and the NH2-terminal sequence determined by using the purified protein are in good agreement with those predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. High-resolution S1 nuclease protection mapping suggests that transcription initiates at or near the adenine residue which is the first position of the translational initiation triplet (AUG) of STAT. Another open reading frame located just upstream of the STAT gene was detected and contains a region bearing a strong resemblance to DNA-binding domains which are conserved in known DNA-binding proteins. By addition of promoter signals and a synthetic ribosome-binding (Shine-Dalgarno) sequence at an appropriate position upstream of the STAT translational start codon, the STAT gene confers streptothricin resistance on Escherichia coli and Bacillus subtilis. The STAT coding sequence with both the promoter of a B. subtilis cellulase gene and a synthetic Shine-Dalgarno sequence was functionally expressed in Streptomyces lividans, which suggests that the addition of an artificial leader upstream of the translational initiation codon (AUG) does not significantly influence the translation of STAT.

Acetyltransferases↗
PubMed1987

Purification and biochemical characterization of streptothricin acetyltransferase coded by the cloned streptothricin-resistance gene of Streptomyces lavendulae.

Streptothricin acetyltransferase was purified from Streptomyces lividans harboring a plasmid which carried the streptothricin-resistance gene cloned from a streptothricin-producing strain, Streptomyces lavendulae No. 1080. Some properties of the enzyme were determined and the reaction product was identified to be N beta-acetylstreptothricin by NMR spectroscopy.

Acetyltransferases↗
PubMed1987

[Intracerebral granuloma with serum anti-human ascaris antibody: case report].

A 1.5-year-old girl with a convulsion attack due to intracerebral granuloma in the right frontal lobe is reported. Her serum was positive with anti-human ascaris antibody, although no ova of the parasites were detected in the feces. She had grown up intimately with several cats in the home. These findings suggested that the granuloma was due to larva migrans of toxocara, which cross-reacts immunologically with human ascaris. Histological examination of the granuloma revealed no eosinophilic infiltration. No systemic reactions such as eosinophilia and hepatomegaly were found except for elevation of protein in cerebrospinal fluid. These were similar to those of ocular type of toxocara larva migrans.

Animals↗
PubMed1986

Nucleotide sequence of the malate dehydrogenase gene of Thermus flavus and its mutation directing an increase in enzyme activity.

The nucleotide sequence of the malate dehydrogenase (mdh) gene from a thermophilic bacterium, Thermus flavus, was determined. The amino acid sequence of the Thermus malate dehydrogenase resembled that of the porcine heart cytoplasmic enzyme to a certain extent, and Asp-159 and His-187 were identified as possible essential residues for the catalytic function. The mutated mdh gene was also cloned from a spontaneous mutant of T. flavus containing a higher activity of the enzyme. Its mutation point was determined to be a single nucleotide exchange from C to T which caused Thr-190 to be substituted by isoleucine. The mutated enzyme showed resistance to substrate inhibition, an increase in both kcat and Km, and a shift toward a more acid optimum pH for the enzyme reaction.

Amino Acid Sequence↗
PubMed1986

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus.

The aspartate protease of Mucor pusillus (Mucor pusillus rennin; MPR) is a milk-clotting enzyme used in the cheese industry. The partial amino acid sequence of MPR was determined and oligonucleotide probes were synthesized for cloning of the MPR gene. A clone giving positive hybridization with the probes was selected from the cosmid library. Sequencing of the cloned DNA revealed an open reading frame of 1281 bp without introns which encodes 361 amino acids for the expected MPR with an NH2-terminal extension of 66 amino acids. MPR seems to be synthesized as a prepro enzyme.

Amino Acid Sequence↗
PubMed1986

The amino acid sequence of the blue copper protein of Alcaligenes faecalis.

The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.

Alcaligenes↗
PubMed1986

Nucleotide sequence of the nifLA operon of Klebsiella oxytoca NG13 and characterization of the gene products.

The complete nucleotide sequence of the regulatory operon nifLA of a nitrogen fixer Klebsiella oxytoca NG13 was determined, and the transcriptional start point was assigned by S1 mapping. The nifL protein (a repressor) was coded by an open reading frame of 1,485 bases, corresponding to a protein of 495 amino acids with a calculated molecular weight of 55,242. The open reading frame (1,572 bases) of the nifA protein (an activator), corresponding to a molecular weight of 58,649, was confirmed by in vitro transcription-translation experiments, using the wild type and artificially deleted nifA genes. The initiation codon (ATG) of nifA overlapped the termination codon(TGA) of nifL, sharing the two bases T and G. A conserved DNA contact point [Gln-(X)3-Ala-(X)3-Gly-(X)5-Val] common in many DNA binding proteins was found in the C-terminal region of the nifA sequence. The promoter sequences of nifLA, nifB and nifF in K. oxytoca coincided exactly with those of K. pneumoniae in the consensus regions at -12 and -26, although the overall homology in the promoter regions was 96%. Changes of four amino acids were found between the nifA coding sequences of K. oxytoca and K. pneumoniae.

Amino Acid Sequence↗
PubMed1986

Glucuronidated and sulfated bile acids in serum of patients with acute hepatitis.

The concentrations of glucuronidated and sulfated bile acids in the serum of 15 patients with acute hepatitis were determined by mass fragmentography. Total serum bile acid levels were 13.79-444.10 mumol/liter, and the percentages of glucuronidated and sulfated bile acids were in the wide ranges of 1.7-33% and 2.4-49%, respectively. In four of the five patients for whom serum bile acids were analyzed serially from the acute to the recovery stages of acute hepatitis, the decrease of the glucuronidated and sulfated bile acids was slower than that of nonglucuronidated, nonsulfated bile acids. Thus, the relative proportion of the glucuronides and sulfates in total bile acids apparently increased during the recovery phase. The mechanism for the relative predominance of bile acid esters in serum during recovery is unknown but might reflect an improved excretion of the nonesterified bile acids into bile after the rapid recovery of intrahepatic cholestasis.

Acute Disease↗
PubMed1986

Cloning and structural analysis of the calf prochymosin gene.

Calf prochymosin (cPC) gene was cloned from calf abomasum DNA by using a cloned cPC cDNA as a probe. The cPC gene spans approx. 10.5 kb and consists of 9 exons and 8 introns. The positions of exon-intron junctions coincide completely with those in the human pepsinogen gene. Analysis of 5'-flanking region sequence and S1 nuclease mapping revealed that the transcriptional start point was located 25 bp upstream from the start codon and that the putative transcriptional promoter sequences (TATA box and CCAAT box) were located in -30 and -90 regions, respectively. Some distinctive sequences possibly functioning as regulatory signals for gene expression are present at the 5'-flanking region.

Amino Acid Sequence↗
PubMed1986

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment.

Cloning, Molecular↗
PubMed1986

Characterization of an essential histidine residue in thermophilic malate dehydrogenase.

Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.

Diethyl Pyrocarbonate↗
PubMed1986

Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli.

A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) was cloned in Escherichia coli. The cloned fragment caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells in parallel with their growth. No excretion of the periplasmic enzymes of host cells occurred. The cloned fragment contained a single open reading frame of 3,135 base pairs coding a protein of 1,045 amino acids (Mr 112,000). Comparison of the 5' nucleotide sequence with the N-terminal amino acid sequence of the protease indicated the presence of a typical signal sequence. The C-terminal amino acid of the enzyme was found at position 408, as deduced from the nucleotide sequence. Artificial frameshift mutations introduced into the coding sequence for the assumed distal polypeptide after the C terminus of the protease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cause excretion of the mature protease through the outer membrane of E. coli cells.

Amino Acid Sequence↗
PubMed1986

Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis.

A Bacillus subtilis 2.7-kilobase DNA fragment containing an intracellular protease gene was cloned into Escherichia coli. The transformants produced an intracellular protease of approximately 35,000 Mr whose activity was inhibited by both phenylmethylsulfonyl fluoride and EDTA. Introduction of the fragment on a multicopy vector, pUB110, into B. subtilis caused a marked increase in the level of the intracellular protease. The nucleotide sequence of the cloned fragment showed the presence of an open reading frame for a possible proenzyme of the major intracellular serine protease (ISP-I) of B. subtilis with an NH2-terminal 17- or 20-amino-acid extension. The total amino acid sequence of the protease deduced from the nucleotide sequence showed considerable homology with that of an extracellular serine protease, subtilisin. The transcriptional initiation site of the ISP-I gene was identified by nuclease S1 mapping. No typical conserved sequence for promoters was found upstream of the open reading frame. An ISP-I-negative mutant of B. subtilis was constructed by integration of artificially deleted gene into the chromosome. The mutant sporulated normally in a nutritionally rich medium but showed decreased sporulation in a synthetic medium. The chloramphenicol resistance determinant of a plasmid integrated at the ISP-I locus was mapped by PBS1 transduction and was found to be closely linked to metC (99.5%).

Amino Acid Sequence↗