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Biomedical subjects

H Yamagata

Publications and source records attributed to H Yamagata.

At least 127 records · Page 7Linked to original sources
PubMed1989

Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.

Using previously isolated Bacillus brevis strains that secrete large amounts of proteins but little protease into the medium, we have developed a host-vector system for very efficient synthesis and secretion of heterologous proteins. The multiple promoters and the signal-peptide-coding region of the MWP gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, were used to construct expression-secretion vectors. With this system, a synthetic gene for human epidermal growth factor (hEGF) was expressed efficiently and a large amount (0.24 g per liter of culture) of mature hEGF was secreted into the medium. hEGF purified from the culture supernatant had the same NH2-terminal amino acid sequence, COOH-terminal amino acid, and amino acid composition as natural hEGF, and it was fully active in biological assays. These results, in combination with previous results, showed that mammalian proteins can be produced in active form 10-100 times more efficiently in B. brevis than has been reported in other systems.

Amino Acid Sequence↗
PubMed1989

The complete amino acid sequence of a subtilisin inhibitor from adzuki beans (Vigna angularis).

The complete amino acid sequence of a major molecular form of subtilisin inhibitor from adzuki beans (Vigna angularis) was established by manual analysis using 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC). Sequencing was performed on the peptides which were derived by digesting the inhibitor with lysyl-endopeptidase and Staphylococcus aureus V8-protease. The inhibitor consisted of 92 amino acid residues and the molecular weight was calculated to be 10,800. A minor form of subtilisin inhibitor was found, which lacked the amino-terminal 19 residues of the major one. Comparison of amino acid sequences revealed that the adzuki bean subtilisin inhibitors were 29-68% homologous in sequence to the inhibitors of so-called "potato inhibitor I family."

Amino Acid Sequence↗
PubMed1989

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.

Amino Acid Sequence↗
PubMed1989

Multiple and tandemly arranged promoters of the cell wall protein gene operon in Bacillus brevis 47.

The genes encoding the major cell wall proteins, middle wall protein and outer wall protein, of Bacillus brevis 47 constitute a cotranscriptional unit (cwp [cell wall protein gene] operon). Primer extension assay of cwp operon transcripts showed the existence of six different 5' ends. This confirmed the results of the previous S1 nuclease protection assay and suggested the existence of several tandemly arranged promoters in the 5' region of the cwp operon. Promoter probe vectors carrying the Bacillus licheniformis alpha-amylase gene were constructed and used for deletion analysis of the 5' region. Three (P1, P2, and P3) of the six suggested promoters were shown to be located within three distinct fragments derived from the 5' region. The -35 and -10 regions of the P1 and P3 promoters resemble the consensus sequence recognized by the sigma-43-type RNA polymerase of Bacillus subtilis. The P2 promoter resembles only the consensus sequence in the -10 region. The P1 and P3 promoters were used to the same extents in Bacillus subtilis as in B. brevis, whereas the P2 promoter was used much less frequently in B. subtilis than in B. brevis. The P2 promoter is used constitutively in B. brevis 47 at all stages of growth, whereas P3 is used only at the exponential phase of growth. P2 could be a promoter of an unknown type that is preferentially used in B. brevis and might be responsible for the constitutive synthesis and secretion of the cell wall proteins into the medium at the stationary phase of growth.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein.

A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes.

A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined. The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues. The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167. The amino acid sequence of the C. thermosulfurogenes beta-amylase showed 54, 32, and 32% homology with those of the Bacillus polymyxa, soybean, and barley beta-amylases, respectively. Twelve well-conserved regions were found among the amino acid sequences of the four beta-amylases. To elucidate the mechanism rendering the C. thermosulfurogenes beta-amylase thermophilic, its amino acid sequence was compared with that of the B. polymyxa beta-amylase. The C. thermosulfurogenes beta-amyulase contained more Cys residues and fewer hydrophilic amino acid residues than the B. polymyxa beta-amylase did. Several regions were found in the amino acid sequence of the C. thermosulfurogenes beta-amylase, where the hydrophobicity was remarkably high as compared with that of the corresponding regions of the B. polymyxa beta-amylase.

Amino Acid Sequence↗
PubMed1988

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.

Amino Acid Sequence↗
PubMed1988

Characterization of MR spectroscopic imaging of the human head and limb at 2.0 T.

Using section-select and phase-encoding gradients, the authors obtained phosphorus chemical shift images of the human head and limb. Phosphorus spectra were acquired from planar sections divided into voxels as small as 7 cm3 in calf muscle and 27 cm3 in brain, with total examination times, including setup and proton locator imaging, of roughly 1 hour. Both spin-echo and free induction decay (FID) methods were employed; the FID gave superior results. Signal-to-noise ratios for the beta-adenosine triphosphate and phosphocreatine resonances were as high as 10:1 and 13:1 from volumes of 27 cm3 in brain.

Brain↗
PubMed1987

Hypertrophic osteoarthropathy, cutaneous vasculitis, and mixed-type cryoglobulinemia in a patient with nasopharyngeal carcinoma.

We describe a 27-year-old woman with a primary nasopharyngeal carcinoma who developed hypertrophic osteoarthropathy and cutaneous vasculitis. Her serum contained antibodies to Epstein-Barr virus and U1 RNP antigens. Cryoproteins isolated from her serum contained antibodies to U1 RNP and a protein with a molecular weight of 32 kd which reacted specifically with antibodies to U1 RNP. HLA typing revealed HLA-B7 and DR1; these have been reported to be increased in Japanese patients with rheumatic diseases who have autoantibodies to U1 RNP. These findings indicate that some features of the paraneoplastic syndrome in this patient might have been caused by immune complexes, part of which were formed by specific autoantibodies produced under genetically controlled conditions of immune responsiveness.

Adult↗
PubMed1987

Urinary fluoride monitoring of industrial hydrofluoric acid exposure.

Preworkshift and postworkshift urinary fluoride concentrations were measured in 142 hydrofluoric acid (HF) workers and 82 unexposed workers aged 18 to 59. Postshift urinary fluoride concentration in HF workers was significantly higher than that in preshift or control workers. A linear relationship was observed between the mean values in urinary fluoride concentration and the HF concentration in the air. The mean urinary fluoride concentration of 4 ppm and its lower fiducial limit (95%, P = 0.05) of 2 ppm (specific gravity 1.024) were estimated corresponding to the atmospheric HF concentration of 3 ppm, which is the maximal allowable concentration recommended by the Japanese Association of Industrial Health and also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results suggest that exposure to HF can be monitored by determining the urinary fluoride concentration.

Adolescent↗
PubMed1987

Cloning and characterization of the 5' region of the cell wall protein gene operon in Bacillus brevis 47.

Bacillus brevis 47 secretes vast amounts of proteins derived from both middle wall protein (MWP) and outer wall protein into the medium. The 5' region of the cell wall protein gene operon was cloned into Bacillus subtilis and subsequently into B. brevis 47. On the basis of the nucleotide sequence analysis, an open reading frame coding for MWP was identified on the cloned DNA fragment. Two potential translation initiation sites for the MWP gene are located tandemly in the same reading frame. Each of the sites contains a sequence highly homologous to the 3' end of B. brevis rRNA and an initiation codon. The translational fusion of the 5' region of the MWP gene with the Bacillus licheniformis alpha-amylase gene resulted in the efficient expression of the alpha-amylase gene in B. brevis 47. Of the two potential translation initiation sites, the one located upstream could be eliminated without affecting the expression of the MWP-alpha-amylase fusion gene, suggesting that MWP is synthesized in a precursor form with a signal peptide of 23 amino acid residues. S1 nuclease mapping of the cell wall protein gene transcripts suggested the possibility of the existence of several promoters in the 5' region within 300 base pairs from the translation initiation sites; one promoter was definitely localized within this part of the 5' region, and it was capable of expressing a heterologous gene fusion at a high level. The roles of the apparent structural complexity of the 5' region of the cell wall protein gene operon are discussed in connection with the efficient gene expression.

Amino Acid Sequence↗
PubMed1987

Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase.

The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis. Homologous DNA was found by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end. The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods. It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time. The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases.

Amino Acid Sequence↗
PubMed1987

Implications of anti-Ro/Sjögren's syndrome A antigen autoantibody in normal sera for autoimmunity.

We have applied a sensitive assay to analyze lupus and Sjögren's syndrome autoantibodies in 40 normal sera. Seven of these bound Ro/Sjögren's syndrome A antigen (SSA). Although this binding was 1,000-fold lower than the highest anti-Ro/SSA level measured from patients, it was inhibited by human Ro/SSA. Positive normal serum-bound Ro/SSA in Western immunoblots and binding activity was demonstrated in the F(ab')2 fragment of IgG. Affinity purification of normal anti-Ro/SSA IgG increased the specific anti-Ro/SSA binding by greater than 17-fold. This purified antibody formed a Ro/SSA precipitin and had a relative affinity for Ro/SSA identical to that of Ro/SSA precipitin-positive patients. These data demonstrate that the anti-Ro/SSA present in healthy normal donors is true autoantibody. Anti-La/Sjögren's syndrome B antigen (SSB) autoantibodies were found in 3 of the 40 normal sera, while none bound nuclear ribonucleoprotein (Sm). Finding low levels of anti-Ro/SSA and anti-La/SSB among normals may indicate that anti-Ro/SSA and anti-La/SSB occur in disease by enhancement of a preexisting immune response.

Adult↗