Biomedical subjects

Y Kamagata

Publications and source records attributed to Y Kamagata.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge.

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.

Bacteria↗

PubMed2000

Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile By fluorescence in situ hybridization.

The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium ruminantium and mobile, Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.

Animals↗

PubMed1999

Soluble methane monooxygenase gene clusters from trichloroethylene-degrading Methylomonas sp. strains and detection of methanotrophs during in situ bioremediation.

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.

Amino Acid Sequence↗

PubMed1999

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules.

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.

Anaerobiosis↗

PubMed1998

Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters available commercially as biodegradable plastics.

Thirty-nine morphologically different soil bacteria capable of degrading poly(beta-hydroxyalkanoate), poly(epsilon-caprolactone), poly(hexamethylene carbonate), or poly(tetramethylene succinate) were isolated. Their phylogenetic positions were determined by 16S ribosomal DNA sequencing, and all of them fell into the classes Firmicutes and Proteobacteria. Determinations of substrate utilization revealed characteristic patterns of substrate specificities.

Bacteria↗

PubMed1997

Alpha- and beta-subunits of a V-type membrane ATPase in a hyperthermophilic sulfur-dependent archaeum, Thermococcus sp. KI.

The genes encoding alpha- and beta-subunits of a V-type ATPase in a sulfur-dependent hyperthermophilic archaeum, Thermococcus sp. KI, were cloned and sequenced. The deduced amino acid sequences were approximately 60, 50 and 25% identical to those of other archaeal, eukaryotic V-type and E. coli F-type ATPase, respectively. Phylogenetic analysis revealed that Thermococcus ATPase was closely related to that of Thermus, and those of Methanosarcina and Halobacterium.

Amino Acid Sequence↗

PubMed1997

Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria.

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose [PTYG] or 0.1 x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders. Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene. This gene was transmissible to A. eutrophus JMP228 carrying a plasmid with a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates. These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1996

Vacuolar-type ATPase in a hyperthermophilic archaeum, Thermococcus sp. KI.

Membrane ATPase was purified from a hyperthermophilic heterotrophic archaeum, Thermococcus sp. KI, which grew anaerobically at 90 degrees C in the presence of sulfur. The purified enzyme had an optimal temperature of 90 degrees C and its molecular mass was estimated to be 600 kDa. It consisted of 4 subunits with molecular masses of 70, 60, 29 and 15 kDa. While the ATPase activity was resistant to most ATPase inhibitors, the activity was reduced by nitrate, an inhibitor of the vacuolar (V)-type ATPase. N-terminal amino acid sequence of the 70 kDa subunit was similar to those of catalytic subunit of V-type ATPases. This indicates that hyperthermophilic heterotrophs have a V-type ATPase in their membranes.

Adenosine Triphosphatases↗

PubMed1995

Microlunatus phosphovorus gen. nov., sp. nov., a new gram-positive polyphosphate-accumulating bacterium isolated from activated sludge.

Polyphosphate-accumulating bacteria that were previously isolated from activated sludge and exhibited high phosphate removal activity were studied taxonomically and phylogenetically. These organisms were gram-positive, coccus-shaped, aerobic chemoorganotrophs that had a strictly respiratory type of metabolism in which oxygen was a terminal electron acceptor. They accumulated large amounts of polyphosphate under aerobic conditions. The major quinone was menaquinone MK-9(H4). The cell wall peptidoglycan contained LL-diaminopimelic acid. The guanine-plus-cytosine content of the DNA was 67.9 mol%. Our isolates were similar phenotypically and chemotaxonomically to Luteococcus japonicus, which was proposed recently as a new genus and species. However, our isolates differed from L. japonicus in cellular fatty acid composition and some other traits. A phylogenetic analysis based on 16S rRNA sequences showed that our isolate differ from the genus Luteococcus and other genera belonging to the high-G+C-content gram-positive group. Accordingly, we concluded that our strain NM-1T (T = type strain) should be assigned to a new genus and species, for which we propose the name Microlunatus phosphovorus.

Base Sequence↗

PubMed1994

Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenous proteins.

We have isolated overlapping mouse cDNAs encoding a collagenous polypeptide that we have designated alpha 1(XVIII) collagen. Nucleotide sequence analysis shows that alpha 1(XVIII) collagen contains 10 triple-helical domains separated and flanked by non-triple-helical regions. Within the non-triple-helical regions, there are several Ser-Gly-containing sequences that conform to consensus sequences for glycosaminoglycan attachment sites in proteoglycan core proteins. Northern blots show that alpha 1(XVIII) transcripts are present in multiple organs, with the highest levels in liver, lung, and kidney. We have also isolated overlapping cDNAs encoding human alpha 1(XV) collagen, and their sequence extends a published partial alpha 1(XV) sequence to the 3' end. Comparison of the alpha 1(XV) and alpha 1(XVIII) sequences reveals a striking similarity in the lengths of the six most carboxyl-terminal triple-helical domains. In addition, within the carboxyl non-triple-helical domain NC1 of the two chains, a region of 177 amino acid residues shows about 60% identity at the amino acid level. We suggest, therefore, that alpha 1(XV) and alpha 1(XVIII) collagens are structurally related. Their structure is different from that of other known collagen types. We conclude that they belong to a subfamily of extracellular matrix proteins and we suggest the designation multiplexins (for protein with multiple triple-helix domains and interruptions) for members of this subfamily.

Amino Acid Sequence↗

PubMed1993

Membrane ATPase from the aceticlastic methanogen Methanothrix thermophila.

A new isolate of the aceticlastic methanogen Methanothrix thermophila utilizes only acetate as the sole carbon and energy source for methanogenesis (Y. Kamagata and E. Mikami, Int. J. Syst. Bacteriol. 41:191-196, 1991). ATPase activity in its membrane was found, and ATP hydrolysis activity in the pH range of 5.5 to 8.0 in the presence of Mg2+ was observed. It had maximum activity at around 70 degrees C and was specifically stimulated up to sixfold by 50 mM NaHSO3. The proton ATPase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the membrane ATPase activity, but azide, a potent inhibitor of F0F1 ATPase (H(+)-translocating ATPase of oxidative phosphorylation), did not. Since the enzyme was tightly bound to the membranes and could not be solubilized with dilute buffer containing EDTA, the nonionic detergent nonanoyl-N-methylglucamide (0.5%) was used to solubilize it from the membranes. The purified ATPase complex in the presence of the detergent was also sensitive to N,N'-dicyclohexylcarbodiimide, and other properties were almost the same as those in the membrane-associated form. The purified enzyme revealed at least five kinds of subunits on a sodium dodecyl sulfate-polyacrylamide gel, and their molecular masses were estimated to be 67, 52, 37, 28, and 22 kDa, respectively. The N-terminal amino acid sequences of the 67- and 52-kDa subunits had much higher similarity with those of the 64 (alpha)- and 50 (beta)-kDa subunits of the Methanosarcina barkeri ATPase and were also similar to those of the corresponding subunits of other archaeal ATPases. The alpha beta complex of the M. barkeri ATPase has ATP-hydrolyzing activity, suggesting that a catalytic part of the Methanothrix ATPase contains at least the 67- and 52-kDa subunits.

Acetates↗

PubMed1992

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.

Amino Acid Sequence↗

PubMed1992

Utilization of methoxylated benzoates and formation of intermediates by Desulfotomaculum thermobenzoicum in the presence or absence of sulfate.

Desulfotomaculum thermobenzoicum strain TSB (DSM 6193) was found to utilize some methoxylated benzoates as carbon and energy source with or without sulfate. 3- or 4-Methoxybenzoate, vanillate (4-hydroxy-3-methoxybenzoate), syringate (3,5-dimethoxy-4-hydroxybenzoate) and 3,4,5-trimethoxybenzoate were converted to corresponding hydroxybenzoates. However, neither 2-methoxybenzoate nor 2,6-dimethoxybenzoate was utilized. The organism grew acetogenically on each of the methoxylated benzoates in the absence of sulfate. 3,4-Dihydroxy-5-methoxybenzoate was detected during conversion of syringate, and syringate and 3,4-dihydroxy-5-methoxybenzoate were detected during conversion of 3,4,5-trimethoxybenzoate as intermediates. These findings indicate that 4-methoxyl-group is most readily cleaved, whereas 2-methoxyl-group is not utilized by the organism.

Acetates↗

PubMed1992

Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. nov. and rejection of Methanothrix ("Methanosaeta") thermoacetophila.

Three thermophilic Methanothrix ("Methanosaeta") strains, strains PTT (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii ("Methanosaeta concilii") GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila ("Methanosaeta thermoacetophila"), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.

Bacterial Proteins↗

PubMed1992

Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in Penicillium purpurogenum.

Cellulase induction by beta-glucodisaccharides was investigated by using non-cellulase-induced mycelia of Penicillium purpurogenum P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular beta-glucosidase activity. Thiogentiobiose (6-S-beta-d-glucopyranosyl-6-thio-d-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction than gentiobiose even in the absence of nojirimycin. Thiogentiobiose appeared to be a gratuitous inducer since it was not metabolized during cellulase induction. Gentiobiose was formed from cellobiose by the intracellular beta-glucosidase of P. purpurogenum. These findings indicate that gentiobiose is an active inducer of cellulase for this fungus and may possibly be formed by intracellular beta-glucosidase from cellobiose.

Journal Article↗

PubMed1989

[Cytokine production in inflamed human gingival tissues--interleukin-6].

Interleukin-6 (IL-6) is involved in the final differentiation of B cells into antibody-producing cells. Recent studies reveal that IL-6 plays an important role in inflammation. Histopathological studies showed that a large number of plasma cells in periodontitis is usually seen in the apical parts of cellular infiltrates beneath the periodontal connective tissues. This evidence suggests that IL-6 may play a critical role in the development of periodontitis. Therefore, we examined IL-6 production in the gingival tissues. Twelve periodontitis patients and five gingivitis patients were included in this study. Nine individuals with healthy periodontium acted as control subjects. Biopsy specimens were dissected into fragments 3 mm in diameter and plated onto 24-well culture plates with RPMI 1640 medium. IL-6 activity in the culture supernatants was measured by IgM production assay using the cell line SKW6-CL4. IL-6 activity was detected at significantly higher levels (P less than 0.001) in culture supernatants from the gingival tissues in periodontitis (23.2 +/- 14.4 units/ml) and gingivititis (12.5 +/- 3.4 units/ml) than in control tissues (2.3 +/- 1.2 units/ml). Subsequently, the relationship between IL-6 activity and clinical stages was examined. The IL-6 levels before initial preparation (23.2 +/- 14.4 units/ml) were significantly higher (P less than 0.001) than those after initial preparation (1.4 +/- 1.8 units/ml), but were not associated with either periodontal pocket depth or the extent of alveolar bone resorption in periodontitis.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗

PubMed1989

[Study of cytokine production in inflamed human gingival tissues in periodontitis. Interleukin-1 (IL-1 alpha, beta) and tumor necrosis factor (TNF alpha)].

It seems to be generally agreed that periodontal disease is a local manifestation of a systemic immune response. Interleukin-1 (IL-1), which has multiple biologic activities, is detected in the gingival sulcus fluid of periodontitis sites. Recent investigations have revealed that IL-1 and tumor necrosis factor (TNF) are analogous to osteoclast activating factor and promote bone resorption. These findings have suggested the possibility that IL-1 and TNF may play a significant role in the initiation and development of periodontal disease. However, it remains to be determined whether these cytokines influence periodontal tissue breakdown in periodontitis. To elucidate the mechanisms of tissue breakdown in periodontitis, we examined cytokine production by human periodontitis gingival tissue. Twelve periodontitis patients were included in this study. Control subjects with healthy periodontium consisted of nine individuals. Gingival samples were biopsied from inflamed or healthy gingival tissues. Biopsy specimens were dissected into fragments 3 mm in diameter and plated onto 24 well culture plates with RPMI 1640 medium. IL-1 activity was measured by a growth inhibition assay using melanoma cell line A 375. An enzyme-linked immunosorbent assay (ELIZA) was used for measuring levels of human IL-1 alpha, IL-1 beta. TNF alpha activity was measured by a growth inhibition assay using cell line LM2D6. IL-1 activity was detected in significantly (p less than 0.001) higher levels in culture supernatants from gingival tissues in periodontitis (48.0 +/- 23.3 units/ml) than in control tissues (2.3 +/- 0.6 units/ml), however, levels of IL-1 activity were not associated with periodontal pocket depth or extent of alveolar bone resorption in periodontitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Resorption↗

PubMed1989

[Pathophysiological analysis of rapidly progressive periodontitis].

Pathophysiological features were studied on 7 patients with rapidly progressive periodontitis but without any evidence of systemic disease, to analyse the clinical pathogenesis. The patients consisted of 5 females, 2 males, between the ages of 32 and 42 years. All patients had severe and rapid alveolar bone destruction on the basis of radiographic measurement. Abnormal serum levels of IgG and IgM were detected in some patients. Higher IgG level was found in 4 patients and higher IgM level was found in 2 patients. The proportion of lymphocyte subsets was calculated in mononuclear cells from peripheral blood of patients. Higher OKT4/OKT8 ratio was found in all patients. The percentage of OKT4 positive cells in 2 patients was higher than that in normal subjects while the percentage of OKT8 positive cells in 4 patients was lower than that in the healthy controls. Microorganisms from periodontal pockets were examined in 5 patients. Bacteriodes was isolated in all 5 patients and Haemophilus actinomycetemcomitans in 2 patients.

Adult↗