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Biomedical subjects

H Takiguchi

Publications and source records attributed to H Takiguchi.

At least 19 recordsLinked to original sources
PubMed1992

Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus.

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Actinomyces viscosus↗
PubMed1992

Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells.

Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.

Cells, Cultured↗
PubMed1992

Cloning of the gene encoding a glycylprolyl aminopeptidase from Porphyromonas gingivalis.

A genomic library of Porphyromonas gingivalis 381 was constructed in the cosmid vector pHC79. A clone, pSN1, was identified by the expression of glycylprolyl-naphthylamide hydrolysing activity. The DNA insert contained within the cosmid pSN1 was subcloned into the plasmid vector pBR328 to create the recombinant plasmid pSN11 containing a 2.9 kb EcoRV insert. An Escherichia coli transformant containing pSN11 produced a protein having a molecular weight of 75 kDa. Southern-blot hybridization revealed that the 2.9 kb EcoRV DNA hybridized with an identical sized Eco RV DNA fragment in the chromosomal DNA of P. gingivalis 381.

Blotting, Southern↗
PubMed1992

Gene cloning of Porphyromonas gingivalis specific antigens recognized by serum of adult periodontitis patient.

1. Porphyromonas gingivalis is believed an important pathogen of adult periodontitis. A gene library of P. gingivalis 381 was constructed in lambda phage vector L47.1. The library was probed with serum obtained from patients of severe adult periodontitis. Two clones, lambda MDBG101 and lambda MDBG103 which were expressed, 200 and 160 kDa respectively, were selected and further studied. 2. The expressed antigens in these two clones were also reacted with rabbit antiserum against whole cells, capsular fraction and cell surface fraction of P. gingivalis. 3. Genes coding protein antigens in lambda MDBG101 and lambda MDBG103 were subcloned into high-copy-number plasmid vector pACYC184 and subclones obtained were designated as MD101 and MD103. Recombinant plasmids, pMD101 and pMD103, differed in their restriction endonuclease digestion. 4. Immunodiffusion analysis showed that cloned proteins from MD101 and MD103 reacted with antiserum against P. gingivalis but did not react with antiserum against Prevotella intermedia, Prevotella loescheii and Prevotella asaccharolyticus. 5. These data suggest that P. gingivalis species-specific antigens has been successfully cloned and expressed in Escherichia coli. Since these cloned specific antigens were recognized by adult periodontitis patient sera, the recombinant antigen will be useful material for the development of serodiagnosis of P. gingivalis infection in adults periodontitis.

Adult↗
PubMed1992

Inhibitory effect of sulfhydryl group on Ca(2+)-ATPase activity in the plasma membrane-rich fraction from bovine parotid gland.

1. The present study demonstrated that the Ca(2+)-ATPase activity of the plasma membrane-rich fraction from bovine parotid gland was decreased by the addition of reducing agents. 2. Ca(2+)-ATPase activity staining on SDS-PAGE gels was lost in the presence of 2-mercaptoethanol. 3. Among all the reducing agents tested, GSH was the most effective in inhibiting Ca(2+)-ATPase. 4. The Ca(2+)-ATPase activity decreased by the GSH was restored by the addition of an oxidizing reagent. However, oxidation with an oxidizing reagent subsequent to alkylation of the reduced enzyme with iodoacetamide resulted in no restoration of activity. 5. The decrease of Ca(2+)-ATPase activity by GSH is due to a decrease in the Vmax of the enzyme. 6. These results suggest that the disulfide bond in this enzyme protein is necessary to maintain the activity of this enzyme.

Animals↗
PubMed1992

Heparin stimulates the collagen synthesis in mineralized cultures of the osteoblast-like cell line, MC3T3-E1.

We found that heparin induced an increase in collagen synthesis in MC3T3-E1 cells cultured for 15 and 30 days. Northern blots showed that the effect of heparin on the collagen synthesis was mediated through the mRNA expression of type I collagen (day 15). Although heparin stimulated collagen synthesis over and above that stimulated by the transforming growth factor (TGF)-beta, heparin did not stimulate TGF-beta binding. This study indicates that heparin has special tasks both in bone formation and resorption, since it has the ability to form and degrade collagen. We suggest that heparin assists the regulation of collagen metabolism at bone resorption sites.

Animals↗
PubMed1991

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.

Animals↗
PubMed1990

Cloning of a Bacteroides gingivalis outer membrane protein gene in Escherichia coli.

Gene banks of chromosomal DNA from Bacteroides gingivalis 381 were constructed using the bacteriophage replacement vector lambda L47.1. A clone encoding an outer membrane protein from B. gingivalis was identified by Western blot screening with antiserum raised against the outer membrane fraction of B. gingivalis 381 cells. The DNA insert contained within this phage was cloned into the plasmid vector pACYC184 to create the recombinant plasmid pMD123. An Escherichia coli transformant, MD123, containing pMD123 produced a protein having an apparent molecular weight of 40 kDa. The recombinant protein was purified, and amino acid analysis revealed the recombinant protein to have a relatively high content of hydrophobic amino acids (43.6%). Antiserum against the purified recombinant 40 kDa protein reacted with a polypeptide of similar size in the outer membrane fraction and vesicles of B. gingivalis.

Amino Acids↗
PubMed1990

[Change of beta-actin gene expression during culture on osteoblast-like cell line, MC3T3-E1].

Multiple forms of actin (alpha, beta, and gamma-actin) have been found in a variety of mammalian cell lines and tissues by the use of high resolution, two-dimensional gel electrophoresis. alpha-Actin is found only in differentiated muscle cells, and its synthesis is induced during myogenesis in culture, beta- and gamma-actin isotypes are the principal components of microfilaments, structures believed to be involved in cellular morphogenesis, motility, and mitosis. Recent studies have shown that mammalian actin mRNA levels are modulated in response to changes in cell shape and during cell differentiation in vitro. On the other hand, Kodama et al. isolated clone MC3T3-E1 cells from newborn mouse calvaria, which have retained the ability to produce and mineralize a bone-like ground substance after many serial passages. Thus clone is useful for studying osteoblast differentiation as well as the mechanism of calcification. It is demonstrated that bone cells assume a stellate morphology after some hormone treatment and these changes were clearly associated with altered reorganization of the microfillaments which resulted in an enhanced osteoblast phenotype. We have now examined the effect of culture day on expression of beta-actin gene in MC3T3-E1 cells and found that expression of beta-actin gene reduced with culture days. These alternative on the expression of actin gene suggest that beta-actin may be involved in bone remodeling in vivo.

Actins↗
PubMed1989

Cloning of the gene for cell-surface protein antigen A from Streptococcus sobrinus (serotype d).

A gene library of Strep. sobrinus B13N (serotype d) chromosomal DNA was constructed in Escherichia coli, with the bacteriophage vector lambda L47.1. A recombinant phage, lambda MDSM49, containing a 15.5 kb DNA insert, directed the expression of a 210 kDa antigenic protein. The recombinant 210 kDa protein was shown by Western blot analysis to be identical with cell-surface protein antigen A (spaA) from a serotype g strain. However, the restriction patterns of a subclone plasmid, pMD51, from lambda MDSM49 differed from those of serotype g strain. The cell-surface protein antigen I/II from serotype c Streptococcus mutans is a potential immunogen for vaccination against dental caries and corresponds to the spaA from serotype d and g strains. A recombinant clone, pDM51, will be a useful tool for serological and molecular biological studies. The recombinant spaA provides useful material for assessment of its diagnostic and immunogenic potential.

Antigens, Bacterial↗
PubMed1989

Genetic characterization of the spaA1 determinant of the surface protein antigen gene from Streptococcus sobrinus 6715.

1. The surface protein antigen-encoding gene spaA1, previously cloned from Streptococcus sobrinus 6715 (serotype g) into the plasmid pACYC184, was examined. 2. The gene product of pYA726 was identified both by the minicell method and by in vitro transcription-translation system as a protein whose mol. wt was estimated to be 140,000. 3. The spaA1 gene was localized on the 3.75 kb AvaI-BamHI fragment of the cloned DNA. 4. An internal spaA1 promoter located close to one end of the 1.25 kb AvaI fragment within the 3.75 kb AvaI-BamHI fragment initiated the transcription for the insert DNA of S. sobrinus autonomously.

Bacterial Proteins↗
PubMed1989

Free arachidonic acid source for PGE2 and TXB2 production in guinea pig peritoneal macrophages exposed to insoluble glucan from Streptococcus mutans.

1. Macrophages are an important source of the lipid mediators arachidonic acid (AA) and its metabolites that are produced during inflammation. 2. Previously, we reported that insoluble glucans from Streptococcus mutans in dental plaque could induce macrophages to secrete PGE2 and TXB2. 3. Studies were undertaken to identify the phospholipid substrates that can serve as a source of AA in macrophages exposed to the insoluble glucan. 4. When macrophage cell prelabelled with [3H]AA, stimulation with insoluble glucan resulted in a loss of label mainly from phosphatidylcholine (PC) and phosphatidylinositol (PI). 5. In addition, the PC-, and PI-specific phospholipase A2-mediated mechanisms for AA release may be activated in guinea peritoneal macrophages exposed to the insoluble glucan from S. mutans.

Animals↗
PubMed1989

DNA homology of surface protein antigen A gene in mutans streptococci.

1. A recombinant plasmid, pYA724, containing an 8.45 kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of various mutans streptococci strains. 2. Restriction endonuclease BamHI-digested pYA724 DNA was radio-labeled by nick-translation, and a DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with chromosomal DNA of serotypes a, c, d, e, f and g strains, but not with b by dot DNA-hybridization and Southern blot DNA hybridization. 3. Chromosomal DNAs were isolated from several serotype c Streptococcus mutans strains, digested with BamHI, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of BamHI-digested DNA fragments of the chromosomal DNAs. 4. These data indicated that all mutans streptococci strains except serotype b have DNA homologous with the spaA gene, although within the same serotype strain the spaA gene has a diversity of arrangement within the chromosome.

Antigens, Bacterial↗
PubMed1989

[Effect of diglyceride on phospholipase A2 activities in plasma membrane of guinea pig peritoneal macrophases].

The AA release from phospholipids is believed to be controlled by the activation of phospholipase A2 which are probably localized in the plasma membranes. And the considerable interest has been focused on the study of cells of the immune system including macrophages which metabolize arachidonic acid (AA) to prostaglandins, thromboxanes and leukotriens. Recently, it has been reported that phosphatidylinositol (PI) turnover plays an important role in the response to external stimuli in these cells. PI turnover has been accepted as being initiated by the hydrolysis of PI which is catalyzed by the phospholipase C and produced AA. Recently, we demonstrated that PI-specific phospholipase A2 in macrophage plasma membrane directly acted for AA release from PI. On the other hand, diglyceride, which is produced from PI by phospholipase C activation, is known to regulate the several cell functions with a protein kinase C activation. In this report, we examined the effect of diglyceride on phospholipase A2 activities in macrophage plasma membrane. PI-phospholipase A2 and PC-phospholipase A2 activities were stimulated by the addition of diglyceride. In contrast, PE-phospholipase A2 was not affected by diglyceride addition. The results suggested that phospholipase A2 activation was occurred with coupling the diglyceride production via the phospholipase C pathway.

Arachidonic Acid↗
PubMed1989

Diversity of surface protein antigen A gene in the chromosomal DNA of Streptococcus sobrinus.

A recombinant plasmid, pYA724, containing an 8.45-kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of Streptococcus sobrinus B13N. Restriction endonuclease BamHI-digested pYA724 DNA was 32P radio-labeled by nick-translation, and a Southern blot DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with a BamHI fragment of B13N chromosomal DNA, and the size of this fragment was larger than 8.45 kb. The chromosomal DNA was digested with BamHI-Pst I, BamHI-Hind III, and BamHI-Sal I, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of DNA fragments of B13N chromosomal DNA; however, the sizes were not identical to those of internal fragments expected from the pYA724 restriction map. SpaA gene clone derivative plasmids, pYA745 and pYA756, were digested with EcoRI and Hinc II, respectively, and electrophoresed together with the digested B13N chromosomal DNA on the same gel; then Southern blot analysis was carried out. Fragments with sizes corresponding to the EcoRI or HincII internal fragments of pYA745 and pYA756 were not observed among the B13N fragments. These data indicate that serotype d S. sobrinus B13N has DNA homologous with the spaA gene from serotype g strain 6715, although within the same biotype strain has a diversity of arrangement within the chromosome.

Antigens, Bacterial↗
PubMed1988

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol.

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.

Animals↗
PubMed1988

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages.

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A2 had an optimum pH at 4.5, and enzyme activation was observed in Ca++-free medium; but the maximum activity was found at 0.5 mM Ca++ concentration. The Km value for PC of acidic PLase A2 was 4.2 microM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A2 in light of the uncompetitive manner of Ca++ action. Furthermore, the release of [3H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca++ at pH 4.5. These data suggest that the acid PLase A2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A2 properties are opposite to those found for lysosomal PLase A2.

Animals↗