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Biomedical subjects

T Fujimura

Publications and source records attributed to T Fujimura.

At least 19 recordsLinked to original sources
PubMed1997

Separation of 18 6-aminoquinolyl-carbamyl-amino acids by ion-pair chromatography.

Eighteen 6-aminoquinolyl-carbamyl (AQC)-amino acids were separated by ion-pair chromatography, using tetrabutylammonium (TBA) as a counterion. Optimum separation was obtained on a C8 reverse-phase column using gradient elution with two mobile phases, A (5 mM TBA, 75 mM ammonium acetate, pH 7.5) and B (80% acetonitrile). The AQC-amino acids were detected by fluorescence with excitation at 250 nm and emission at 395 nm, and the analysis time was 65 min. The response factors of individual AQC-amino acids to AQC-phenylalanine ranged from 0.42 to 1.08 (except for tryptophan at 0.01), with an average of 0.8. Detection limits by fluorescence ranged from 11.8 fmol (threonine) to 51.7 fmol (methionine), except for tryptophan (1.8 pmol).

Amino Acids↗
PubMed1997

The structure of Silurus asotus (catfish) roe lectin (SAL): identification of a noncovalent trimer by mass spectrometry and analytical ultracentrifugation.

We identified a noncovalent trimer of Sirurus asotus roe lectin (SAL) at Mr 95,362 along with its monomer at M(r) 31,750 by electrospray ionization mass spectrometry when SAL was dissolved in 0.5% acetic acid, sprayed into the ion source with methanol as a sheath liquid, and desolvated at 75 degrees C in a heated capillary column. The molecular weight of SAL, determined by the sedimentation equilibrium method, was 95,200 and the sedimentation coefficient (S20,w) of SAL in water was 5.58. SAL existed as a noncovalent trimer in solution and showed the ability to agglutinate rabbit erythrocytes. SAL showed three peaks (sal 1, sal 2, and sal 3) by C8 reverse-phase HPLC, and these appeared to be a monomer, a dimer, and a trimer, respectively, by matrix-assisted laser desorption ionization-time of flight mass spectrometry, sal 1 and sal 2 were shown to have a structure interchangeable with that of sal 3 in water.

Amino Acids↗
PubMed1997

Apical tubules in marginal cells of the differentiating stria vascularis.

BACKGROUND: Apical tubules (ATs) in marginal cells (MCs) of the stria vascularis appear in limited stages of differentiation of the MCs, but their origin and roles remain uncertain. The present study was designed to solve the problem of whether the ATs are intracellular compartments derived from the Golgi apparatus (GA). METHODS: The cochleae of Wistar rats at ages of postnatal days 1, 3, and 5 were prepared for electron microscopy and cytochemistry using thiamine pyrophosphatase (TPPase) and coenzyme A phosphatase (CoA-Pase) as marker enzymes of trans Golgi cisterns and fluorescent labelled lectin, griffonia simplicifolia agglutinin-I (GS-1). RESULTS: The ATs appeared in the apical cytoplasm of the MCs between postnatal days 1 and 5. Reaction products of TPPase and CoA-Pase activities were localized in the trans-Golgi cisterns and the ATs, which were occasionally in a close apposition to the GA. The reaction was found along the apical plasma membrane of the MCs only in case of TPPase. Heavy reactions to GS-1 were seen in the supranuclear region as well as along the apical plasma membrane of the MCs. CONCLUSIONS: The present ultrastructural and cytochemical studies indicate that the ATs, which appear in the MCs at limited perinatal stages, originate from the trans-Golgi cisterns. These ATs may be involved in the apical plasma membrane supply for the differentiation of the MCs prior to the generation of EP.

Animals↗
PubMed1997

Lack of association between variants of the angiotensinogen gene and the risk of coronary artery disease in middle-aged Japanese men.

The renin-angiotensin system is important in cardiovascular remodeling. Although a variant of the angiotensinogen gene is associated with an increased generation of angiotensinogen, it is unclear how this genetic variant might influence the activity of the renin-angiotensin system and thereby contribute to the predisposition to coronary artery disease (CAD). The relation between genetic polymorphisms in the gene-encoding angiotensinogen and the risk of CAD in middle-aged Japanese men was investigated. Two polymorphisms in exon 2 of the angiotensinogen gene, M235T and T174M, were analyzed in 327 patients with CAD and 352 matched control subjects. The genotype distribution of both polymorphisms did not differ between patients with CAD and control subjects. No combination of genotypes of the two polymorphisms was associated with CAD. Results indicate that the M235T and T174M variants of the angiotensinogen gene are not associated with CAD in Japanese men.

Angiotensinogen↗
PubMed1997

Conversion of the CD4+ T cell profile from T(H2)-dominant type to T(H1)-dominant type after varicella-zoster virus infection in atopic dermatitis.

Skin lesions of atopic dermatitis were examined for cytokine expression by reverse transcription-polymerase chain reaction. The profile of mRNA for various cytokines revealed that both T(H1) and T(H2) types of CD4+ T cells, probably including T(H0) type, infiltrate into the skin lesion. We observed that atopic skin lesions improved after varicella infection. In such lesions, expression of T(H1) type cytokines predominated. The peripheral blood T cells from atopic patients exhibited a differentiation into T(H2) type cells upon in vitro stimulation with mite antigen. In contrast they differentiated into T(H1) type cells upon stimulation by varicella antigen. Since IL-12 has been reported to switch the in vitro recall response of allergen-specific T cells of atopic donors from a T(H2)- to a T(H1)-like phenotype, we examined its local production in varicella lesions. IL-12 p35 and p40 mRNA were expressed in fresh lesions. Peripheral blood mononuclear cells from atopic patients expressed p40 mRNA upon in vitro stimulation with live varicella zoster virus, but they did not show p40 mRNA without stimulation. This finding suggested that in atopic skin lesions containing the virus, IL-12 was produced and the cell type was changed to T(H1) type-predominance. These results suggested that patients with atopic dermatitis always have highly reactive CD4+ T cells infiltrating into their skin, and that the switch to T(H1) or T(H2) dominance is related to whether the lesion is improved or exacerbated.

Antigens, Viral↗
PubMed1997

Lack of association of polymorphisms of the angiotensin converting enzyme and angiotensinogen genes with nonfamilial hypertrophic or dilated cardiomyopathy.

Although several genes or genetic loci that are either responsible for or confer susceptibility to familial hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) have been identified, genetic defects that underlie nonfamilial HCM or DCM remain to be characterized. An allelic association study for the angiotensin converting enzyme (ACE) and angiotensinogen genes has now been performed with 71 patients with nonfamilial HCM, 88 patients with nonfamilial DCM, and 122 healthy control subjects in the Japanese population. The distribution of ACE genotypes for an insertion/deletion (I/D) polymorphism in intron 16 did not differ significantly among control subjects and patients with HCM or DCM. Similarly, the distributions of angiotensinogen genotypes for methionine-235-threonine (M235T) and threonine-174-methionine (T174M) polymorphisms did not differ significantly among the three groups. Echocardiographic parameters that are indicators of the severity or progression of disease did not differ significantly among ACE I/D or angiotensinogen M235T and T174M genotypes in the two patient groups. Finally, no additive or synergistic effect of any combined genotypes or haplotypes of the ACE and angiotensinogen polymorphisms on the association with HCM or DCM was detected. Results indicate that the ACE I/D and angiotensinogen M235T and T174M polymorphisms are not related to HCM or DCM in the Japanese population, and that variants of these polymorphisms do not contribute to the genesis or progression of these cardiomyopathies.

Angiotensinogen↗
PubMed1997

Lack of association of angiotensin converting enzyme gene polymorphism or serum enzyme activity with coronary artery disease in Japanese subjects.

The association of an insertion/deletion (I/D) polymorphism in the angiotensin converting enzyme (ACE) gene or the serum activity of ACE with coronary artery disease (CAD) was investigated in Japanese men and women. The ACE genotype of 947 CAD subjects who underwent coronary angiography and of 893 control subjects was determined by polymerase chain reaction analysis. No association of the DD genotype or the D allele with CAD was observed in men or women. In a low risk group (defined by a body mass index below the median value and the absence of a history of hypertension, diabetes mellitus, and hypercholesterolemia), there was also no association between the ACE gene polymorphism and CAD. No significant difference in serum ACE activity was detected between CAD subjects and controls of all genotypes or of the same genotype, whereas a significant association was apparent between serum ACE activity and ACE genotype for both CAD subjects and controls among both men and women. These results indicate that the ACE I/D polymorphism and genotype associated variation in serum ACE activity are not risk factors for CAD in Japanese men or women.

Adult↗
PubMed1997

Structure, chromosomal location and expression of a rice gene encoding the microsome omega-3 fatty acid desaturase.

The omega-3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome omega-3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome omega-3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 degrees C and 20 degrees C, with its level decreasing markedly at temperatures below 10 degrees C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.

Chromosome Mapping↗
PubMed1997

Molecular cloning of a pig homologue of membrane cofactor protein (CD46).

Organs of transgenic pigs that express human complement regulatory proteins are under assessment as an alternative to transplantation. A major barrier to the transplantation of pig organs is the hyperacute rejection caused by pre-existing antibodies and complement. Pig cells are very susceptible to human complement, presumably because pig cell-surface complement regulatory proteins are inefficient against it. Expression of human complement regulatory proteins, such as decay-accelerating factor and membrane cofactor proteins (MCP or CD46), by means of transgenes would confer resistance to human complement upon pig cells, thereby preventing hyperacute rejection. To express sufficient levels of human complement regulatory proteins at appropriate sites, regulatory elements of genes of pig membrane-bound complement regulatory proteins would be useful. To obtain their cDNAs, we transfected human cells with a pig cDNA library, selected cells by incubation with pig complement and rescued the plasmids. We cloned a cDNA for the pig homologue of MCP, pMCP. The cDNA encoded a predicted protein of 363 amino acids with 42% amino acid identity with human MCP. The pMCP consisted of four short consensus repeats, a Ser/Thr/Pro-rich domain, and transmembrane and cytoplasmic domains. Recombinant soluble pMCP that lacked transmembrane and cytoplasmic domains had factor I cofactor activity in C3b cleavage, indicating that it is functionally, as well as structurally homologous to MCP. FACS analysis with anti-pMCP mAb demonstrated that pMCP is expressed on all blood leukocytes, erythrocytes, and on endothelial and epithelial cell lines.

Amino Acid Sequence↗
PubMed1997

Increase of methicillin resistance in Staphylococcus aureus caused by deletion of a gene whose product is homologous to lytic enzymes.

A spontaneous high-level methicillin-resistant mutant, SRM1648, for which the MIC of methicillin is 1,600 microg/ml, was isolated on a plate containing 400 microg of the antibiotic/ml on which had been cultured the low-level methicillin-resistant Staphylococcus aureus SR17238, for which the MIC is 6.3 microg/ml. Analysis of the chromosomal DNAs of the mutant and the parental strains by the restriction landmark genomic scanning method with two-dimensional electrophoresis of restriction fragments revealed a 1.6-kb deletion in the chromosome of the mutant. The HindIII fragment of 2.5 kb containing this deleted region was cloned into a plasmid vector and introduced into the parental strain. A deletion mutant reconstructed in the presence of a low concentration of methicillin by integration and excision of the recombinant plasmid exhibited a high level of resistance (methicillin MIC, 1,600 microg/ml), confirming that the deletion had caused the elevation of the resistance level. Sequence analysis indicated that the deletion occurred in three consecutive open reading frames (ORFs). The predicted amino acid sequence of the first ORF showed high homology with both RelA and SpoT of Escherichia coli, which are involved in the synthesis and hydrolysis of guanosine 5',3'-polyphosphate, and that of the third ORF showed a relatively high homology to the lytic enzyme encoded by the lytC gene of Bacillus subtilis. We also isolated another high-level resistant mutant with a deletion within the third ORF, which suggested that inactivation of some lytic enzyme resulted in the increased resistance.

Amino Acid Sequence↗
PubMed1997

Diversity in distribution of Na+.K+ATPase of the choroid epithelium of prenatal rats; an immunocytochemical study.

The differentiating choroid plexus in rats at ages ranging from prenatal day 16 to postnatal day 3 was investigated with a special emphasis on the immunolocalization of Na+.K+ATPase in the choroid epithelium and neocapillarization of the choroid stroma. Immunoreactions for Na+.K+ATPase were localized on the apical plasma membrane of the simple choroid epithelium throughout the stages examined, but it also appeared on the basolateral plasma membrane till prenatal day 18, prior to the formation of the tight junctions as seen from both a marker experiment using lanthanum chloride and immunoreactions for tight junction-associated protein (ZO-1). This suggests that a polarity of the enzyme localization to the apical plasma membrane may take place after the establishment of the blood-cerebrospinal fluid barrier. Mesenchymal cells in the choroid stroma immunoreacted to the laminin antibody and occasionally contacted each other--forming solid cell cords--or endothelial cells of the preexisting capillaries. The contact areas immunoreacted to this antibody. Since there were few or no mitotic figures of the endothelial cells, the involvement of laminin in a mechanical linkage between the adjacent vasoformative mesenchymal cells as well as the endothelial lining in a manner suggestive of vasculogenesis was indicated.

Animals↗
PubMed1997

RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.

Amanitins↗
PubMed1997

Cytokine gene expression in the foot pad and spleen of BALB/cAJcl mice infected with M. leprae.

The cytokine mRNAs expressed in the foot pads and spleens of BALB/cAJcl mice infected with Mycobacterium leprae were studied by the reverse transcriptase-polymerase chain reaction (RT-PCR) method using cytokine-specific primers for interleukin-1 alpha (IL-1 alpha), -2, -4, -6, -10, -12-(p40), gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and TNF-beta, and then for CD4 and CD8 markers. The pattern of cytokine gene expression in the foot pad which supports M. leprae growth was different from the expression in the spleen which does not permit M. leprae multiplication in mice. Before BALB/cAjcl mice were infected with M. leprae, IL-1 alpha and TNF-beta mRNAs were expressed physiologically in the foot pad while all of the cytokine genes examined were expressed in the spleen. In the foot pads of mice inoculated with M. leprae, in addition to the physiological appearance of IL-1 alpha and TNF-beta mRNAs, these signals were intensified. TNF-alpha expression was induced by the infection. On the other hand, in the spleens of mice inoculated with M. leprae, CD4 mRNA expression disappeared on day 1 of the infection, which was accompanied by the reduced expression of IL-2, -4, -6, and -12 mRNAs. The recovery of CD4 mRNA expression at a latter stage was accompanied by a corresponding increase of the cytokine mRNA expression. It was suspected that these results might permit restricted growth of M. leprae in the foot pads of normal mice. Furthermore, our study suggests that tissue-specific, local, immunologic characteristics are important in M. leprae growth.

Animals↗
PubMed1997

[Alteration of p15 and p16 gene in gastric cancer].

We analyzed p15 and p16 gene alterations in gastric cancer. Only MKN45 showed both homozygous deletions but other cell lines and all of tumor specimens did not show any alterations. Using RT-PCR analysis, decreased or no expression of the p16 gene was found in 1 of 7 cell lines (except MKN45) (14.2%) and in 8 of 20 tumors (40%), whereas no abnormalities of p15 gene expression were found. These results suggest that the p16 gene may correlate with tumorigenesis and tumor expansion due to decrease or loss of gene products in gastric cancer.

Adenocarcinoma↗
PubMed1996

Reduction of 14-16 kDa allergenic proteins in transgenic rice plants by antisense gene.

An antisense gene strategy was applied to suppress the 14-16 kDa allergen gene expression in maturing rice seeds. Gene constructs producing antisense RNAs of the 16 kDa allergen under the control of some rice seed-specific promoters were introduced into rice by electroporation. Immunoblot and RNA blot analyses of the seeds from the transgenic rice plants using the allergen-specific monoclonal antibody and a sequence-specific antisense RNA probe demonstrated that the 14-16 kDa allergen proteins and their transcripts of the seeds from several transgenic lines were present in much lower in amounts than those of the seeds from parental wild-type rice. The high levels of reduction observed were stably inherited in at least three generations.

Allergens↗
PubMed1996

Activation of Xenopus eggs by RGD-containing peptides accompanied by intracellular Ca2+ release.

To investigate the mechanism of egg activation in Xenopus, unfertilized eggs were treated with various oligopeptides related to the RGD sequence-containing peptides. When dejellied eggs were treated with RGDS at concentrations higher than 250 microM, the eggs produced a positive-going activation potential concomitant with intracellular Ca2- release. The eggs underwent cortical granule breakdown, cortical contraction, and resumption of meiosis. The activation by RGDS was independent of the voltage of egg membranes. GRGD, GRGDTP, and RGPFPI caused activation in some eggs, but RGES, RFDS, GPRP amide, and GPA did not. Denuded eggs were activated by Sepharose beads bound to RGDS, indicating an interaction of the RGD sequence with receptor molecules on the egg plasma membrane. The activation by RGDS did not occur under a Ca(2+)- and Mg(2+)-free condition, but occurred in a Ca(2+)-free solution containing Mg2+, concomitant with an intracellular Ca2+ release. These results indicate that a receptor of the RGD sequence on egg plasma membrane causes egg activation through an intracellular signal transduction system. A sperm agonist including the RGD sequence may play an important role in egg activation through the egg membrane receptor during the normal fertilization process.

Amino Acid Sequence↗