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Biomedical subjects

N Ohta

Publications and source records attributed to N Ohta.

At least 73 records · Page 4Linked to original sources
PubMed1999

A novel bacterial tyrosine kinase essential for cell division and differentiation.

Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The Caulobacter crescentus divL gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550 in vitro, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription in vitro of the cell cycle-regulated fliF promoter. Suppressor mutations in ctrA bypass the conditional cell division phenotype of cold-sensitive divL mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.

Amino Acid Sequence↗
PubMed1999

Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins.

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.

Amino Acid Sequence↗
PubMed1999

CD80 (B7-1) and CD86 (B7-2) antigens on house dust mite-specific T cells in atopic disease function through T-T cell interactions.

BACKGROUND: CD80 (B7-1) and CD86 (B7-2) play an important role in antigen presentation to effector cells. Recent studies have demonstrated that these costimulatory molecules are also expressed on activated T cells. However, the functional role of CD80 and CD86 expressed on allergen-specific T cells in atopic diseases has not yet been clarified. OBJECTIVE: We sought to determine the functional role of CD80 and CD86 expressed on allergen-specific T cells in atopic diseases. METHODS: We assayed the expression of CD80 and CD86 on allergen-specific T-cell lines from patients with perennial allergic rhinitis stimulated by Dermatophagoides farinae-crude (Der f-c) antigen, 1 of the major allergens causing house dust mite allergy. T-cell proliferation induced by Der f-c-specific T-T cell interactions was measured, and the role of CD80 and CD86 in this proliferation was examined. In addition, we compared the proportion of CD45RO+CD86(+) T cells in primary culture of PBMCs stimulated by Der f-c antigen between patients with perennial allergic rhinitis and control subjects. RESULTS: On T-cell activation, CD86 antigen was upregulated earlier than CD80. Both CD80 and CD86 expressed on Der f-c-specific T cells could provide costimulatory signals to induce allergen-specific T-cell proliferation that was partially inhibitable by both anti-CD80 and anti-CD86 mAbs. The proportion of CD45RO+CD86(+) T cells in primary culture from atopic patients was significantly higher than that from control subjects. CONCLUSION: These results suggest that costimulatory molecules, such as CD80 and CD86, expressed on allergen-specific T cells may be involved in the amplification of allergen-specific immune responses through T-T cell interactions in atopic diseases.

Adult↗
PubMed1999

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.

Animals↗
PubMed1999

Immune response against protozoal and nematodal infection in mice with underlying Schistosoma mansoni infection.

Schistosoma mansoni infection induces T helper (Th) 2-dominant immune response in mice not only to S. mansoni itself but also to other coexisting antigens. In the present study, we challenged S. mansoni-infected mice with the intestinal nematode, Strongyloides venezuelensis, and the intracellular protozoa, Leishmania major to see whether such Th2-dominant immune responses alter susceptibility of the host to other concomitant parasitic infections. The recovery of S. venezuelensis adult worms from the small intestine was significantly decreased by S. mansoni infection, and the protection to S. venezuelensis appeared to act on migrating larvae. Antibodies elicited by S. mansoni infection showed cross-binding to third-stage larvae antigen of S. venezuelensis. On the other hand, S. mansoni infection did not affect the outcome of L. major infection in both susceptible BALB/c and resistant C57BL/6 mice. Popliteal lymph node cells of BALB/c mice expressed mRNA for interleukin (IL)-10 rather than IL-4, regardless of S. mansoni infection, and those of C57BL/6 mice expressed IFN-gamma mRNA upon L. major antigen stimulation, even in S. mansoni-infected mice. Our findings suggest that Th2-dominant immune response induced by S. mansoni protects mice from intestinal helminthic infections, whereas they do not always modulate protozoal infections.

Animals↗
PubMed1999

Two ftsH-family genes encoded in the nuclear and chloroplast genomes of the primitive red alga Cyanidioschyzon merolae.

The red algal chloroplast genome encodes an essential prokaryotic cell division gene, ftsH, which has never been found in the mitochondrial genome of any organism. To compare the conserved prokaryote-derived mechanism for mitochondrial division with that of chloroplasts, we cloned chloroplast- and nuclear-encoded ftsH genes from the primitive red alga Cyanidioschyzon merolae. The deduced amino-acid sequence of chloroplast ftsH (ftsHcp) consists of 603 amino acids and shows the highest similarity with algal-chloroplast and cyanobacterial FtsH. On the other hand, the nuclear-encoded ftsH (ftsH2) encodes a protein of 920 amino acids and has the highest similarity with two yeast mitochondrial FtsHs, Rca1p and Afg3p. Furthermore, the amino-terminal extension of FtsH2 appears to be an amphipathic alpha-helix, a characteristic mitochondrial targeting signal, suggesting that FtsH2 is a mitochondrial protein. Southern hybridization revealed that ftsH2 is a single gene located on chromosome III of the 17 C. merolae chromosomes. The level of expression of the 3.0 and 4.0 kb transcripts of this gene decreased in concert during the organelle division phase of a synchronized culture, indicating a cell-cycle-dependent manner of ftsH2 transcription, while northern hybridization did not detect ftsHcp transcripts. Nevertheless, reverse transcription-PCR and immunoblotting demonstrated for the first time that chloroplast-encoded ftsH is transcriptionally and translationally active. Overproduction of FtsHcp and FtsH2 in Escherichia coli disrupted cytokinesis and produced filamentous cells, but had no effect on the replication, segregation, or distribution of their nucleoids, as also occurs in ftsH-deficient E. coli. These observations suggest the possible involvement of both C. merolae FtsHs in organelle division.

ATP-Dependent Proteases↗
PubMed1999

Medium-chain triglycerides modulate ileitis induced by trinitrobenzene sulfonic acid.

BACKGROUND: It is important to develop an appropriate animal model for further investigation into inflammatory bowel disease (IBD). We therefore investigated a trinitrobenzene sulfonic acid (TNBS) ileitis model. Dietary fat in Crohn's disease is still a controversial risk factor for IBD. We therefore also studied the effects of medium-chain triglycerides (MCT) and long-chain triglycerides (LCT) on TNBS ileitis. METHODS AND RESULTS: An intraileal injection of TNBS induced ulceration and inflammation with thickening of the intestinal wall, which were characterized histologically by infiltration of polymorphic nuclear leucocytes and by granuloma formation. The mucosal damage score and serum sialic acid levels reached their highest 7 days after the TNBS injection and then gradually decreased. The mucosal damage series in the MCT group was significantly lower than in the LCT group, and levels of tumour necrosis factor-alpha (TNF-alpha) and leukotriene B4 (LTB4) tended to be lower in the MCT group. CONCLUSIONS: These results suggested that TNBS enteritis might be useful as an IBD animal model and that MCT modulates intestinal inflammation and is less damaging than LCT.

Analysis of Variance↗
PubMed1999

Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in-vitro fertilization programmes.

CD44 is a polymorphic and polyfunctional transmembrane glycoprotein widely expressed in many types of cells. Here, the expression of this protein on human membrana granulosa was studied by two techniques. Using confocal laser scanning microscopy (CLSM) with the mouse monoclonal antibody to human CD44 (clone G44-26), cells immunoreactive for CD44 were observed in both cumulus and mural granulosa cell masses. On the other hand, using monoclonal antibody to human CD44v9, goat polyclonal antibody to human CD44v3-10 and the clone G44-26, no immunoreactivity for CD44v9 and/or CD44v3-10 was observed in either cell group by flow cytometry. In the flow cytometric analysis of 32 patients, the incidence of CD44 expression in cumulus cells (62.6+/-1.3%) was significantly higher than that in mural granulosa cells (38.5+/-3.2%) (P<0.0001). In the comparison of CD44 expression by flow cytometry according to the maturation of each cumulus-oocyte complex, the incidence of CD44 expression of cumulus cells was significantly higher in the mature group than in the immature group (P<0.05). In a flow cytometric analysis, patients with endometriosis showed a significantly lower incidence of CD44 expression in cumulus cells compared to the infertility of unknown origin group (P<0.05), and compared to both the male infertility group and the unknown origin group in mural granulosa cells (P<0.01). These findings suggest that the standard form of CD44 is expressed in human membrana granulosa with polarity and may play an important role in oocyte maturation.

Adult↗
PubMed1999

Merits of soft nitriding scalers.

BACKGROUND: It has been demonstrated that nitriding modifies the physical characteristics of metals. The purpose of this study was to evaluate the changes induced by 3 levels of inexpensive soft nitriding treatments on commercial sickle scalers. METHODS: Taglite scalers (NT) were soft nitrided for 30 (SN30), 60 (SN60), or 90 (SN90) minutes. The cumulated scaled material was weighed every 10 strokes x 10 and thereafter every 1,000 to 8,000 strokes by an automatic scaling apparatus against epoxy resin. Weight differences were used to indicate abrasion resistance; the relative efficiency (RE) was calculated as the ratio of scaled amount at a given number of strokes (SN/NT). The hardness and the tensile strengths were determined for each soft nitriding treatment level. The nitrided layer thickness of representative SN scalers was observed by electron probe x-ray microanalysis. RESULTS: The SN60 was not significantly different from SN30 or SN90, but the SN90 was more efficient than SN30 for the first 100 strokes (P<0.01). The RE of all SN scalers was significantly greater (2.3 to 2.7 times) than the NT scalers from the beginning of the study and throughout all time periods (SN90 > SN60 > SN30); it increased further during the first 100 strokes (9.7 to 15.5 times), indicating the NT scalers wore out faster than SN scalers. The untreated scalers' performance decreased to 10% of baseline after 100 strokes; but even after 1,000 strokes, the SN60 and SN90 performed better than new untreated scalers. Thereafter, all scalers' performance, including SN scalers, decreased. While the NT blades ceased to cut measurable amounts after 7,000 to 8,000 strokes, all SN scalers continued to cut. Although SN90 scalers had the thickest soft layer and were the hardest (P<0.01), the SN60 had the highest tensile strength (P <0.01), suggesting that it might be the safest in practice. CONCLUSIONS: Sixty minutes of soft nitriding treatment of commercially available taglite scalers seem to be the optimal treatment duration to increase their durability, on the order of 100 to 1,000 times, without jeopardizing safety for clinical use.

Dental Alloys↗
PubMed1999

Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells.

PURPOSE: Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated. METHODS: Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis. RESULTS: Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells. CONCLUSIONS: Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells.

Aldehyde Reductase↗
PubMed1998

Structure and organization of the mitochondrial genome of the unicellular red alga Cyanidioschyzon merolae deduced from the complete nucleotide sequence.

The complete nucleotide sequence of the mitochondrial genome of a very primitive unicellular red alga, Cyanidioschyzon merolae , has been determined. The mitochondrial genome of C.merolae contains 34 genes for proteins including unidentified open reading frames (ORFs) (three subunits of cytochrome c oxidase, apocytochrome b protein, three subunits of F1F0-ATPase, seven subunits of NADH ubiquinone oxidoreductase, three subunits of succinate dehydrogenase, four proteins implicated in c-type cytochrome biogenesis, 11 ribosomal subunits and two unidentified open reading frames), three genes for rRNAs and 25 genes for tRNAs. The G+C content of this mitochondrial genome is 27.2%. The genes are encoded on both strands. The genome size is comparatively small for a plant mitochondrial genome (32 211 bp). The mitochondrial genome resembles those of plants in its gene content because it contains several ribosomal protein genes and ORFs shared by other plant mitochondrial genomes. In contrast, it resembles those of animals in the genome organization, because it has very short intergenic regions and no introns. The gene set in this mitochondrial genome is a subset of that of Reclinomonas americana , an amoeboid protozoan. The results suggest that plant mitochondria originate from the same ancestor as other mitochondria and that most genes were lost from the mitochondrial genome at a fairly early stage of the evolution of the plants.

Base Sequence↗
PubMed1998

p53 gene mutations in rectal cancer associated with schistosomiasis japonica in Chinese patients.

Mutations in p53 tumor suppressor gene were examined in 44 Chinese patients with rectal cancer, including 22 cases with advanced schistosomiasis japonica and 22 cases without schistosomiasis. In schistosomal rectal cancer (SRC), 13 mutations were found in 10 cases, which included 11 base-pair substitutions and two deletions. Of 11 base substitutions, nine were transitions and two were transversions and seven of them were located at CpG dinucleotides. In non-schistosomal rectal cancer (NSRC), 13 mutations were found in nine cases, all of which were base-pair substitutions. Of 13 substitutions, 10 were transitions and three were transversions and three of them were located at CpG dinucleotides. The proportion of base-pair substitutions at CpG dinucleotides was higher in SRC patients than in NSRC patients, although this was not statistically significant (P = 0.054). Point mutation was frequent at codon 248 in SRC. A higher frequency of arginine missense mutations was observed in SRC than in NSRC. These observations suggest that the mutations in SRC are the result of genotoxic agents produced endogenously through the course of schistosomiasis japonica.

Aged↗
PubMed1998

Effect of serum fatty acid composition on coronary atherosclerosis in Japan.

The serum lipid profiles of patient's with or without significant coronary stenosis diagnosed by coronary angiography and of control subjects were compared. The level of high-density-lipoprotein cholesterol and eicosapentaenoic acid were significantly lower in the patients with significant coronary stenosis than in the control subjects. It suggests that high-density-lipoprotein cholesterol and omega3 fatty acids may have a protective effect on the progress of coronary atherosclerosis. The frequency of eating dark-meat fish was positively associated with serum eicosapentaenoic acid and docosahexaenoic acid, omega3 fatty acids, and inversely associated with serum stearic acid and linoleic acid. The frequency of eating soybean products was positively associated with serum docosahexaenoic acid and inversely associated with serum linoleic acid. It is necessary to discuss a way to popularise a diet of dark-meat fish and soybean products as a means of preventing coronary heart disease.

Aged↗
PubMed1998

The delta isoform of protein phosphatase type 1 is localized in nucleolus and dephosphorylates nucleolar phosphoproteins.

The immunolocalization and substrates of protein phosphatases present in nucleolus were investigated using Swiss 3T3 cells and Novikoff hepatoma ascites cells. The protein phosphatase activity was detected in the extract of the isolated nucleoli and its activity was inhibited by okadaic acid with IC50 value of 160 nM. Immunoblotting assay indicated that PP1c delta but not PP1c alpha, PP1c gamma 1, and PP2Ac was localized in the isolated nucleoli. Confocal microscopy showed that PP1c delta was localized in nucleoli, nuclei, and cytosol, though the intensity of fluorescence at the nucleoli was stronger than that of the cytosol or nuclei. PP1c delta was co-localized with the major nucleolar phosphoprotein B23 at nucleoli. The phosphatase was capable of dephosphorylating several proteins in the nucleolus, including B23. The Km of PP1 for the recombinant B23.1, phosphorylated by endogenous kinase(s), was 3.5 microM. These results indicate that PP1c delta is the major serine/threonine phosphatase present in nucleolus and it dephosphorylates nucleolar phosphoproteins, including B23.

3T3 Cells↗
PubMed1998

Novel structural difference between nopaline- and octopine-type trbJ genes: construction of genetic and physical map and sequencing of trb/traI and rep gene clusters of a new Ti plasmid pTi-SAKURA.

We constructed a gene library of a nopaline-type Ti plasmid (called pTi-SAKURA) which was newly isolated from Agrobacterium tumefaciens MAFF301001 of a Japanese cherry tree. The partial sequencing data, which were distributed over the entire plasmid genome, made it possible to assign typical Ti-encoded genes including trb and rep gene clusters. The trb/traI and rep gene clusters were sequenced completely. All the genes in the regions except trbJ were homologous with the corresponding genes on octopine-type Ti plasmids, based on both ORF size and sequence similarity. The trbJ on pTi-SAKURA is similar to that of an octopine-type Ti, but has an extra 282-base segment in its central domain. The above gene organization and sequences suggest a divergence of Ti plasmid during evolution in relation to Rhizobium plasmids, and is discussed in this paper.

Agrobacterium tumefaciens↗
PubMed1998

An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter.

Cell differentiation and division in Caulobacter crescentus are regulated by a signal transduction pathway mediated by the histidine kinase DivJ and the essential response regulator DivK. Here we report genetic and biochemical evidence that the DivJ and DivK proteins function to control the activity of CtrA, a response regulator required for multiple cell cycle events, including flagellum biosynthesis, DNA replication, and cell division. Temperature-sensitive sokA (suppressor of divK) alleles were isolated as extragenic suppressors of a cold-sensitive divK mutation and mapped to the C terminus of the CtrA protein. The sokA alleles also suppress the lethal phenotype of a divK gene disruption and the cold-sensitive cell division phenotype of divJ mutants. The relationship between these signal transduction components and their target was further defined by demonstrating that the purified DivJ kinase phosphorylates CtrA, as well as DivK. Our studies also showed that phospho-CtrA activates transcription in vitro from the class II flagellar genes and that their promoters are recognized by the principal C. crescentus sigma factor sigma73. We propose that an essential signal transduction pathway mediated by DivJ, DivK, and CtrA coordinates cell cycle and developmental events in C. crescentus by regulating the level of CtrA phosphorylation and transcription from sigma73-dependent class II gene promoters. Our results suggest that an unidentified phosphotransfer protein or kinase (X) is responsible for phosphoryl group transfer to CtrA in the proposed DivJ --> DivK --> X --> CtrA phosphorelay pathway.

Alleles↗
PubMed1998

Strongyloides venezuelensis: binding of orally secreted adhesion substances to sulfated carbohydrates.

Adhesion substances produced by adult worms of Strongyloides venezuelensis bound strongly to hepin-Sepharose beads after incubation at 37 degrees C for 1 h. This binding was completely inhibited by highly sulfated carbohydrates such as soluble heparin, dextran surfate, fucoidan, and pentosan polysulfate. Chondroitin sulfate E and chondroitin sulfate A inhibited to a lesser degree and chondroitin sulfate C and dextran did not inhibit significantly. Carbohydrate moieties as well as the number and position of negatively charged sulfate groups of sulfated glycans were important determinants for the interaction between sulfated carbohydrates and adhesion substances. Adhesion substances of S. venezuelensis adult worms also bound to negatively charged rat red blood cells. The binding was significantly inhibited by heparin but not by mono- or disaccharides. Thus the intraction between red cells and adhesion substances was electrostatic in nature, but did not involve lectin-sugar interactions.

Animals↗