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Biomedical subjects

H Yamagata

Publications and source records attributed to H Yamagata.

At least 145 records · Page 8Linked to original sources
PubMed1986

The Ro/SSA autoantigen as an immunogen. Some anti-Ro/SSA antibody binds IgG.

The rheumatic disease autoantigen, Ro/SSA, was immunogenic to a rabbit host. The heteroimmune rabbit serum bound the Ro/SSA particle in immunoblots and in an ELISA. Both the rabbit anti-Ro/SSA and a human prototype anti-Ro/SSA serum also bound IgG; and moreover, IgG inhibited both rabbit and human anti-Ro/SSA activity. Anti-IgG activity of the rabbit and human anti-Ro/SSA sera bound Ro/SSA by Western blot and solid-phase assays. In addition, purified Ro/SSA inhibited the anti-IgG activity of the anti-Ro/SSA sera from rabbit and man. Affinity purification of the IgG- and Ro/SSA-binding fractions of the rabbit anti-Ro/SSA demonstrated that both the anti-Ro/SSA and anti-IgG activities were concentrated in these fractions. These data show that Ro/SSA and IgG share epitopes that are bound by anti-Ro/SSA antibody. Inhibition experiments suggest that this antibody is found in most human anti-Ro/SSA autoimmune sera and that the epitope(s) are found in the F(ab')2 fragment of IgG.

Animals↗
PubMed1986

Anti-Ro (SS-A) and anti-La (SS-B) in patients with Sjögren's syndrome.

Clinical, serologic, and genetic findings in Sjögren's syndrome patients were correlated with quantitative determinations for antibody against Ro (SS-A), La (SS-B), and nRNP (Sm) using newly developed, sensitive solid-phase assays. In 86 Sjögren's syndrome patient sera, more than 96% had anti-Ro (SS-A), and 87% had anti-La (SS-B), spanning a 4.8 log10 range of autoantibody concentration, whereas only 95% of the patients had anti-nRNP (Sm). Low levels of anti-Ro (SS-A) and anti-La (SS-B) were found in 10% and 12.5%, respectively, of the 40 normal control sera. In Sjögren's syndrome patients, the level of anti-Ro (SS-A) correlated strongly with that of anti-La (SS-B) (r = 0.80; P less than 0.0001) but not with the level of anti-nRNP (Sm). We found much higher levels of anti-Ro (SS-A) and anti-La (SS-B) in patients with purpura, leukopenia, lymphopenia, and increased polyclonal gamma globulins than in those without these conditions (between 4.3-fold and 17-fold higher; P less than 0.001 to P less than 0.05). Anti-Ro (SS-A) and anti-La (SS-B) levels correlated with the rheumatoid factor titer and with the concentrations of total globulin, IgG, and IgA, but not with the IgM concentration. The association of rheumatoid factor titer with levels of anti-Ro (SS-A) and anti-La (SS-B) occurred only in patients with primary Sjögren's syndrome. Antinuclear antibody titers correlated with levels of anti-Ro (SS-A) and anti-nRNP (Sm). HLA-DR3-positive patients had higher levels of anti-Ro (SS-A) and anti-La (SS-B).

Antibodies, Antinuclear↗
PubMed1986

In vitro production of autoantibodies to U1 ribonucleoproteins by peripheral blood mononuclear cells from patients with connective tissue diseases.

Antibodies to U1 ribonucleoproteins produced by peripheral blood mononuclear cells from patients with connective tissue diseases were measured by a sensitive enzyme-linked immunosorbent assay and by immunoblotting. Mononuclear cells from patients with high-titer serum antibodies spontaneously started secreting IgG anti-U1 RNP antibodies on the second day after culture. The amount of anti-U1 RNP in the culture supernatants reached maximum level on day 8. The most effective production of anti-U1 RNP by B cells was observed when they were cultured in the presence of T cells and adherent cells. Mononuclear cells from patients without anti-U1 RNP antibodies or from normal subjects did not produce a measurable amount of anti-U1 RNP. Treatment of mononuclear cells by cycloheximide resulted in complete inhibition of anti-U1 RNP secretion, which indicates that antibody in the culture supernatants reflects the active biologic phenomenon in vitro. The methods described should be useful in the study of the cellular mechanisms involved in antinuclear antibody production of connective tissue diseases.

Antigens↗
PubMed1986

Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47: complete nucleotide sequence of the outer wall protein gene.

Bacillus brevis 47 contains two cell wall proteins termed the outer wall protein (OWP) and middle wall protein (MWP), each of which forms hexagonal arrays in the cell wall. A 6-kilobase BglII-BclI fragment of B. brevis 47 DNA cloned into Bacillus subtilis with a derivative of pHW1 as a vector directed the synthesis of a polypeptide, with almost the same molecular weight as the authentic OWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was specifically recognized by the anti-OWP antibody. Nucleotide sequence analysis of the subfragment revealed that it contains two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the MWP, and the downstream open reading frame corresponds to the entire translational portion of the OWP. The latter encodes a secretory precursor of the OWP, consisting of a total of 1,004 amino acid residues with a signal peptide of 24 amino acid residues at its amino-terminal end. Futhermore, analysis of transcripts in B. brevis 47 suggests that the MWP and OWP genes, in that order, constitute a cotranscriptional unit and that the major promoter shared by the two genes is located upstream of the MWP gene.

Amino Acid Sequence↗
PubMed1986

Complement C4 fixation by the anti-La(SSB) found in normal donors and in patients with systemic lupus erythematosus.

The discovery that a common systemic lupus erythematosus (SLE) autoantibody (anti-La/SSB) occurs in normal individuals has made it possible to compare the specific complement binding capacity of normal autoantibody with SLE patient autoantibody. Normal donors with elevated levels of anti-La(SSB) binding were compared to negative normal donors and to 3 patients with SLE with high levels of anti-La(SSB) binding in assays for anti-La(SSB) and for the capacity of anti-La(SSB) to bind C4. The anti-La(SSB) found in normal sera and in SLE patient sera had no identical specific capacity to fix C4. For the case of anti-La(SSB), the autoantibody structure in relation to its specific capacity to activate complement, at least to the extent of fixing C4, does not differentiate SLE patients from normal individuals with this autoantibody.

Antibodies, Antinuclear↗
PubMed1986

Anti-Ro/SSA and -La/SSB antibodies in patients with connective tissue diseases.

The frequency of anti-Ro/SSA and anti-La/SSB antibodies in patients from Japan with Sjögren's syndrome, systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), polymyositis-dermatomyositis (PM-DM), rheumatoid arthritis (RA) and blood donors is reported. Anti-La/SSB ab was especially detected in patients with severe glandular and extraglandular manifestation and could be found in sera years prior to the confirmation of Sjögren's syndrome. The purified Ro/SSA antigen consisted of 60 k peptide and 16 k RNA while the La/SSB antigen consisted of 41 k and 29 k peptides. Some molecular association seems to exist between the peptides and corresponding IgG anti-Ro/SSA and anti-La/SSB antibodies.

Antibodies, Antinuclear↗
PubMed1985

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites.

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences.

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.

Amino Acid Sequence↗
PubMed1985

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium.

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.

Amylases↗
PubMed1985

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.

Amino Acid Sequence↗
PubMed1985

Immunologic and structural studies of the lupus/Sjögren's syndrome autoantigen, La/SSB, with a monoclonal antibody.

La/SSB is a small nuclear RNA protein against which precipitating autoantibodies are made in many patients with systemic lupus erythematosus or Sjögren's syndrome. The recent purification of La/SSB has made structural and immunologic studies possible. Consequently, a mouse hybridoma antibody (La1) was raised, after immunization and fusion, that reacted with bovine La/SSB. Results of inhibition tests with tissue extracts and fluorescent antinuclear antibody tests demonstrated that La1 reacted with bovine extracts and cells, but not with those from human, mouse, or rabbit sources. La1 reacted in Western blot and in an adapted anti-La/SSB enzyme-linked immunosorbent assay with only the 41-kD bovine La/SSB peptide and not with the smaller 29-kD bovine La/SSB peptide. RNA gels showed that La1 bound the La/SSB particle that contained the predominant La/SSB RNA species near 90 nucleotides as well as the minor RNA species, both of which were bound by the human autoimmune anti-La/SSB serum. A solid-phase assay for human autoimmune anti-La/SSB antibody using La1 was more sensitive for the detection of human anti-La/SSB than was a comparable assay using purified La/SSB, and showed that anti-La/SSB is present in nearly all Ro/SSA precipitin-positive sera. Thus, this study demonstrates that monoclonal antibody can be raised against La/SSB; that the protein moiety of bovine La/SSB differs from human, mouse, and rabbit at an epitope on the 41-kD La/SSB peptide; that the RNA bound to the La1-reactive particle was as heterogeneous as that binding the anti-La/SSB autoimmune serum; and that anti-Ro/SSA and anti-La/SSB are closely associated.

Animals↗
PubMed1984

The major outer membrane lipoprotein and new lipoproteins share a common signal peptidase that exists in the cytoplasmic membrane of Escherichia coli.

The cell envelope of Escherichia coli possesses several lipoproteins including the major outer membrane lipoprotein. These lipoproteins are synthesized as a signal peptide-carrying precursor that is subsequently modified with glyceride. In this work, lipoprotein signal peptidase that processes the precursor of the major lipoprotein was partially purified from cells harboring a plasmid that carries the gene for this enzyme (1spA). The enzyme was also active against the glyceride-containing precursors of the peptidoglycan-associated lipoprotein and many additional membrane lipoproteins. The unmodified precursor of the major lipoprotein was not attacked by the enzyme. The enzyme was exclusively localized in the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1984

Cloning and expression of a thermophilic alpha-amylase gene from Bacillus stearothermophilus in Escherichia coli.

A 6.4 Kb HindIII fragment of Bacillus stearothermophilus DY-5 DNA cloned in Escherichia coli using pBR322 as a vector was shown to direct the synthesis of a thermophilic alpha-amylase. In attempts to reduce the size of the insert, the alpha-amylase gene was shown to be contained in a 3.1 Kb HindIII - BamHI fragment of the donor strain DNA. The alpha-amylase gene was stably maintained and expressed efficiently in E. coli. The enzymic properties of alpha-amylase produced in E. coli closely resembled those of the donor strain alpha-amylase and the temperature range for the maximal activity was from 65 degrees C to 80 degrees C. Nearly 100% of the activity remained after heating at 80 degrees C for 15 min. The alpha-amylase was shown to be accumulated in the periplasmic space. It was purified to a nearly homogenous protein with a molecular weight of 61,000, which was very similar in size to that produced by B. stearothermophilus DY-5.

Cloning, Molecular↗
PubMed1984

Molecular cloning of a major cell wall protein gene from protein-producing Bacillus brevis 47 and its expression in Escherichia coli and Bacillus subtilis.

Bacillus brevis 47 contains two major cell wall proteins. Each protein forms a hexagonal array in the cell wall. A 4.8-kilobase HindIII fragment of B. brevis 47 DNA cloned into Escherichia coli with pBR322 as a vector directed the synthesis of polypeptides cross-reactive with antibody to the middle wall protein. A 700-base-pair BamHI-HpaI fragment was shown to be the essential region for the synthesis of immunoreactive polypeptides. Furthermore, this fragment appeared to contain the promoter activity. The 3.5-kilobase BamHI fragment covering the essential region as well as its downstream sequence was subcloned into the corresponding restriction site of pUB110 by using Bacillus subtilis as the cloning host. Both E. coli and B. subtilis carrying the cloned DNA synthesized several immunoreactive polypeptides which were mainly found in the cytoplasm. B. subtilis secreted polypeptides cross-reactive with antibody to the middle wall protein. These extracellular polypeptides were degraded upon prolonged culture.

Bacillus↗
PubMed1984

Molecular properties of the Ro/SSA antigen and enzyme-linked immunosorbent assay for quantitation of antibody.

Antibodies to the Ro/SSA antigen occur in patients with systemic lupus erythematosus and Sjögren's syndrome. An immunoaffinity method for the preparation of electrophoretically homogeneous Ro/SSA antigen is described. Several molecular properties of the antigen have been determined. The native RNA protein particle has a molecular weight of approximately 100,000 D determined by gel filtration. Sodium dodecyl sulfate-analysis of the purified Ro/SSA antigen and analysis by staining of bands with silver and Coomassie Blue, Western blotting, and RNAase treatment leads to a hypothesis for the structure of the particle in which an antigenic 60,000 protein is bound to 24,000-27,000 RNA molecules which are not antigenic. An enzyme-linked immunoabsorbent assay method for assay of anti-Ro/SSA is also described which sensitively measures antigen binding at dilutions of sera containing anti-Ro/SSA precipitins up to 10(7) fold. Normal sera on average have 10(3) less binding activity.

Animals↗