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Biomedical subjects

T Nagata

Publications and source records attributed to T Nagata.

At least 19 recordsLinked to original sources
PubMed1992

Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar).

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.

Animals↗
PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗
PubMed1992

Retinoic acid-dependent transactivation of major histocompatibility complex class I promoters by the nuclear hormone receptor H-2RIIBP in undifferentiated embryonal carcinoma cells.

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex (MHC) class I genes. The binding occurs through the GG(T/A)CA motif present also in many other genes. The role of H-2RIIBP in developmental regulation of MHC class I genes has been studied in undifferentiated N-Tera2 embryonal carcinoma cells by transient cotransfection of an expressible H-2RIIBP plasmid and a chloramphenicol acetyltransferase reporter gene linked to the MHC class I promoter. Transfection of the expression plasmid led to production of H-2RIIBP transcripts and enhanced MHC class I promoter activity in cells that were treated with retinoic acid but not yet differentiated. Retinoic acid concentrations required for transactivation overlapped with those capable of inducing morphological differentiation and expression of endogenous MHC class I genes in these cells. This enhancement was mediated by region II, as a heterologous thymidine kinase promoter driven by region II also served as a target for H-2RIIBP transactivation. Deletion of the bulk of the DNA-binding domain or the ligand-binding domain of H-2RIIBP, but not of the N-terminal domain, abolished transactivation, indicating that the former two domains are critical for the enhancement. Moreover, H-2RIIBP transactivation exhibited a strict cell-type restriction. As observed in other cell lines, N-Tera2 cells that had undergone differentiation failed to elicit transactivation, suggesting that H-2RIIBP acts in concert with a cofactor expressed in undifferentiated N-Tera2 cells that requires retinoic acid for its function. These results suggest that H-2RIIBP can function as a developmentally specific transcription factor for MHC class I genes.

Base Sequence↗
PubMed1992

The role of 5'-methylthioadenosine on rat calvaria cell differentiation.

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.

Adenine↗
PubMed1992

parB: an auxin-regulated gene encoding glutathione S-transferase.

We have isolated an auxin-regulated cDNA, parB, from the early stage of cultured tobacco mesophyll protoplasts. The expression of parB was observed during transition from G0 to the S phase of tobacco mesophyll protoplasts cultured in vitro. The predicted amino acid sequence of parB cDNA has 213 amino acid residues with a relative molecular weight of 23,965. Nucleotide sequence analysis revealed that parB cDNA has homology to glutathione S-transferase (GST; RX:glutathione R-transferase, EC 2.5.1.18) from several sources including plant and animal cells. When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product. Accordingly a significant increase of GST activity was detected in the tobacco mesophyll protoplasts cultured in the presence of 2,4-dichlorophenoxyacetic acid. This is an example in which the function of auxin-regulated gene product is shown to be ascribed to a specific enzymatic activity. As GST, and its substrate glutathione, are shown to be related to cell proliferation as well as detoxification of xenobiotics in plant and animal cells, the role of parB is discussed in relation to the induction of proliferative activity in differentiated and nondividing mesophyll protoplasts of tobacco.

Amino Acid Sequence↗
PubMed1992

H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes.

H-2RIIBP (RXR beta) is a member of the nuclear hormone receptor superfamily that activates transcription of MHC class I genes in response to retinoic acid (RA). Using chemical cross-linking, co-immunoprecipitation, gel mobility shift and streptavidin-biotin DNA precipitation assays, we show that H-2RIIBP formed heterodimers with thyroid hormone (T3) and RA receptors (T3R alpha and RAR alpha). H-2RIIBP heterodimer formation required a conserved sub-domain of its C-terminal region, occurred independently of target DNA and was much more efficient than either T3R alpha/RAR alpha heterodimer or H-2RIIBP homodimer formation. Heterodimers displayed enhanced binding to target DNA elements and contacted DNA in a manner distinct from that of homodimers. A functional role for heterodimers in vivo was demonstrated by synergistic enhancement of MHC class I transcription following co-transfection of H-2RIIBP with T3R alpha or RAR alpha. We provide biochemical evidence that H-2RIIBP formed heterodimers with several naturally occurring nuclear proteins. The results suggest that H-2RIIBP, by virtue of its ability to heterodimerize, enhances combinatorial diversity and versatility in gene regulation mediated by nuclear hormone receptors.

Amino Acid Sequence↗
PubMed1992

Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro.

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.

Animals↗
PubMed1992

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Base Sequence↗
PubMed1992

The replication origin of proplastid DNA in cultured cells of tobacco.

When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.

Autoradiography↗
PubMed1992

Actions of parathyroid hormone on cultured human dental pulp cells.

The responsiveness of human dental pulp (HDP) cells to parathyroid hormone (PTH) was investigated by measuring their cyclic AMP (cAMP) content, DNA synthesis, alkaline phosphatase activity, collagen synthesis, and glycosaminoglycan (GAG) synthesis. PTH dose-dependently increased the intracellular cAMP 1 min after the addition of PTH. Confluent HDP cells on day 14 expressed a high level of cAMP production after addition of 3 units/ml PTH. The hormone did not affect DNA synthesis by HDP cells. Alkaline phosphatase activity was suppressed by PTH to 81% of control (p < 0.01), and addition of dibutyryl cAMP to the medium mimicked the effect of PTH (79% of control, p < 0.01). The hormone inhibited collagen synthesis (15% decrease of [3H] proline incorporation, p < 0.01), and stimulated non-collagen protein synthesis (10% increase, p < 0.05). The increase of non-collagen protein by PTH was in accordance with the enhancement of GAG synthesis (17% increase of [35S]sulfate incorporation, p < 0.01). Dibutyryl cAMP caused further increase of GAG synthesis, to 155% of control (p < 0.01). Observations of the radiolabeled proteins on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after metabolic labeling with [14C]proline and [35S]sulfate revealed a similar tendency to the quantitative determinations in which PTH inhibited collagen synthesis and stimulated GAG synthesis. These findings suggest that HDP cells have some osteoblastic characteristics in terms of PTH responsiveness, and that this culture system is a useful model for studies of human dental pulp.

Adolescent↗
PubMed1992

Determination of pyrimethamine and sulphadimethoxine residues in eggs by high performance liquid chromatography.

1. Laying hens were given food containing per kg diet: 1 mg pyrimethamine, 10 mg sulphadimethoxine (SDM) or 1 mg pyrimethamine with 10 mg SDM, for 14 days. Residues in the eggs were determined by high performance liquid chromatography. 2. Pyrimethamine was mainly found in yolk. Its concentration was slightly higher when it was given with SDM. 3. The concentrations of SDM in eggs were not different when it was given alone or in combination with pyrimethamine. 4. After withdrawal, pyrimethamine residues decreased below the detection limit (0.02 mg/kg) in the yolk after 11 to 12 d and in the albumen after 2 to 3 d. SDM in yolk fell below the detection limit (0.01 mg/kg) on day 7, whereas in albumen it was not detectable after 2 to 3 d.

Animals↗
PubMed1992

The induction of cataracts by HIV-1 in transgenic mice.

OBJECTIVE: To elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host-range barrier of this virus. RESULTS: mRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3-6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. CONCLUSIONS: Preferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Reduced bone density and major hormones regulating calcium metabolism in anorexia nervosa.

Bone density of lumbar vertebrae (L2 to L4) and the whole body in 29 patients with anorexia nervosa were measured by dual photon absorptiometry, and the results were compared with those of 10 age-matched normal controls. The patients had significantly lower bone mineral density (BMD) in L3 and L2-4 than controls. However, there was no difference in whole-body BMD. L3 and L2-4 BMD was positively correlated with body weight and was negatively correlated with duration of illness and amenorrhea. Patients who had been more active 6 months before the time of the study had significantly higher L3 BMD than the less active patients. Most patients had an abnormally low serum estrogen level, whereas the mean serum levels of thyroid hormone (T3, T4), cortisol, calcitonin, parathyroid hormone and vitamin D were within the normal range. No correlation was found between L3 or L2-4 BMD and the levels of these hormones. These results suggest that severe weight loss, low physical activity, longer duration of amenorrhea and deficiency of estrogen contribute to bone loss in patients with anorexia nervosa, whereas calcium-regulating hormones such as parathyroid hormone, calcitonin and vitamin D are unlikely to be a primary contributor to bone loss.

Adolescent↗
PubMed1992

Phospholipase A2 in rat gingival tissue.

Phospholipase A2 (PLA2) is a proinflammatory enzyme in the synovial fluids of all--and sera of some--patients with rheumatoid arthritis. Due to the similarities in pathogenesis between rheumatoid arthritis and periodontitis, we sought to study the enzymatic properties of PLA2 in periodontal tissue. In this study, we demonstrated PLA2 activity in rat gingival tissue, about 80% of which was present in the cytosolic fraction. We characterized the cytosolic PLA2 enzyme with respect to substrate specificity, sensitivity to detergent, Ca2+ ion dependency and optimum pH. We found that phosphatidylethanolamine, rather than phosphatidylcholine, was the preferred substrate, the Ca2+ ion was essential for the expression of PLA2 activity, the enzyme was active over a broad pH range, with the optimum at pH 9.0, and sodium-deoxycholate inhibited the enzyme activity strongly in a concentration-dependent manner. These results are consistent with those which have been obtained with synovial fluid PLA2 and suggest that gingival PLA2 may be involved in the pathogenic processes of gingivitis and periodontitis.

Analysis of Variance↗
PubMed1992

Regulatory effect of thromboxane A2 on proliferation of vascular smooth muscle cells from rats.

We investigated the regulatory effects of the vasoconstrictor thromboxane A2 on the proliferation of vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats using 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2. STA2 dose dependently increased incorporation of [3H]thymidine into DNA in randomly cycling VSMC and significantly shortened the doubling time. Cell cycle analysis revealed that the increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, STA2 enhanced protein synthesis in VSMC during the G2/M phase, whereas the protein synthesis was unaffected in the G0/G1 period. In fact, STA2 prompted the cells in G2/M phase to synthesize actin, a major cytoskeleton protein. Conversely, inhibition of protein synthesis by puromycin retarded the transition from S to G2/M. In addition, depolymerization of the actin molecules by cytochalasin D offset the quick progression to the G2/M phase by STA2. These data indicate that thromboxane A2 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. This enhanced progression is attributable partly to a rapid buildup of the cytoskeleton proteins during the G2/M period.

Animals↗
PubMed1992

H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements.

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.

Amino Acid Sequence↗
PubMed1992

Immunohistochemical and histopathological study of experimental rabies infection in mice.

An immunohistochemical and histopathological study using the ABC technique was carried out to examine time-sequential virus spread in the central nervous system (CNS) of mice after inoculation with the CVS strain of fixed rabies virus by different routes; intracerebral (ic), intraocular (io), intranasal (in), intramuscular (im) and subcutaneous (sc). Only the ic and io inoculations caused fatal infections, so that detailed analysis was conducted on mice inoculated by these two routes. In ic-inoculated mice, viral antigens were detected mainly in neurons in the cerebral cortex and in the pyramidal cells and granular cells of the hippocampus. After io inoculation, viral antigen was first detected in the trigeminal nerve ganglia, following which it spreads to the cerebral cortex and cerebellum. In the hippocampus only a few cells were viral antigen-positive at the early stage after io inoculation. There were no inflammatory lesions or Negri bodies in the CNS of mice infected by either route. This suggests that clinical signs such as ataxia or depression leading to death may be due to the direct effect of the virus on the functions of neural cells, but not to inflammatory reactions. The ABC method will be useful for the early diagnosis of suspected patients or animals to have the disease when conventional histopathological and immunofluorescent antibody techniques can not detect lesions or viral antigens.

Animals↗