Search PubMed⌕ Search

Biomedical subjects

T Nagata

Publications and source records attributed to T Nagata.

At least 361 records · Page 20Linked to original sources
PubMed1993

Structure and expression of the mRNA encoding urinary stone protein (osteopontin).

The chemical nature of urinary stone protein is poorly understood. We have sequenced a cDNA of urinary calcium oxalate stone protein extracted with EDTA. cDNA sequences showed complete identity between urinary stone protein and human osteopontin. Osteopontin protein was detected by staining with Stains-All, which specifically stains phosphoproteins, and by digestion with the highly specific protease thrombin, demonstrating that urinary calcium oxalate stones consist of osteopontin protein. We used a technique of in situ hybridization to detect osteopontin mRNA in the kidney. In control rats, distal tubular cells were sporadically positive, and proximal tubular cells and glomeruli were negative for osteopontin mRNA. A rat model of stone formation was induced with glyoxylic acid. In stone-forming rats, staining of distal tubular cells was remarkably increased, but proximal tubular cells and glomeruli were still negative. Immunostaining for the osteopontin protein also revealed that epithelial cells of distal tubules were weakly positive in control rats and significantly increased in stone-forming rats, although proximal tubular cells and glomeruli were negative. Northern blot analysis showed a significant increase of osteopontin mRNA in stone-forming rats in proportion to the dosage and the duration of the stone-inducing drugs. These results show that osteopontin in the kidney is presumably involved in urinary stone formation as the stone matrix.

Animals↗
PubMed1993

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study.

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.

Aging↗
PubMed1993

Primary colonic epithelial cell culture of the rabbit producing prostaglandins.

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E2 production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35% collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10% fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE2 and PGF2 alpha were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF1 alpha, thromboxane B2, PGF2 alpha, PGE2, and PGD2 by cultured cells. Quantitatively, 117 +/- 7 ng/mg-protein/hour PGE2 and 7.4 +/- 0.7 ng/mg-protein/hour PGF2 alpha were produced. While hydrocortisone (10(-4) - 10(-2) M) did not show a significant effect on PGE2 production, indomethacin (10(-8) - 10(-6) M), and 5-aminosalicylic acid (2X10(-4)-5X10(-3) M) inhibited PGE2 production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE2 synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

De novo synthesis of phospholipase A2 and prostacyclin production by proliferating rat smooth muscle cells.

We investigated the role of phospholipase A2 (PLA2) in cell cycle-dependent alterations of endogenous prostacyclin (PGI2) synthesis in aortic smooth muscle cells in culture (VSMC) from Wistar Kyoto rats. Randomly cycling VSMC generated more PGI2 than the stationary cells. Cell cycle analysis showed that PGI2 production capacity was increased from the G0/G1 through the early DNA synthetic (S) phases. Enzyme analysis revealed that, although there were different mechanisms underlying this increase in the PGI2 production during the G0/G1, the peak at 4 hours coincided with a sharp increase in PLA2 activity. This increase in PLA2 activity was preceded by an increased expression of functional PLA2 messenger RNA, and protein synthesis inhibition prevented most of the increase in PGI2 production at 4 hours. These data indicate that endogenous PGI2 generation is mainly increased during the G0/G1 period and that this event is secondary to de novo synthesis of PLA2 and probably, at least in part, to cyclooxygenase induction. This mechanism provides a negative feedback regulating VSMC proliferation.

Animals↗
PubMed1993

Possible radical scavenging properties of cicletanine and renal protection in Dahl salt sensitive rats.

Much interest in cicletanine, a novel antihypertensive drug, has grown because it uniquely stimulates prostacyclin (PGI2) production and may, thereby, provide further cardiovascular protection. We postulated that cicletanine may be an antioxidant, and assessed its ability to protect the kidney in Dahl salt-sensitive (Dahl S) rats on a high salt diet. Cicletanine eradicated in vitro a stable radical, DPPH, and decreased the lipid peroxidation both in rat brain homogenate and in a xanthine-xanthine oxidase (X-XOD) superoxide generating system. Furthermore, cicletanine attenuated the inhibition of PGI2 synthase activity by 15HPETE. However, cicletanine did not exhibit superoxide dismutase-like activity in X-XOD system, suggesting that it behaves primarily as a hydroxy radical scavenger. A 6 week cicletanine treatment reduced blood pressure in Dahl S rats fed a high salt diet, and ameliorated functional and morphological injury to the kidney. This attenuation of glomerular sclerosis correlated with the attenuation of lipid peroxidation in the kidney homogenate. These data indicate that cicletanine is an antioxidant that protects the kidney from salt-induced hypertension in Dahl salt-sensitive strain rats.

Animals↗
PubMed1993

CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection.

CD69, an 'activation marker' that is rapidly induced on mature T cells after stimulation through the T cell antigen receptor (TCR) was found to be expressed on approximately 10% of normal thymocytes. All of these CD69+ thymocytes express alpha beta TCR, and they include both TCRlowCD4+CD8+ and TCRhighCD4+CD8- or CD4-CD8+ thymocytes. The CD69+ cells can be further segregated into heat-stable antigen (HSA)+TCRlow, HSA+TCRhigh and HSA-TCRhigh thymocyte populations. None of CD69+ cells express the mature T cell marker Qa-2. Thus CD69+ cells present in vivo appear phenotypically to represent transitional cell populations between immature TCRlowHSA+Qa-2-double-positive cells and mature TCRhighHSA-QA-2+ single-positive cells. In addition, TCR engagement by MHC molecules is required for CD69 expression in the thymus. Taken together, the CD69+ thymocytes appear to represent the cells auditioning in positive selection process or they are the cells that have been positively selected recently. Analysis of a TCR transgenic mouse model revealed an increased number of CD69+ thymocytes in a positively selecting thymus, whereas no CD69+ transgenic TCR+ thymocytes were observed in the non-selecting thymus. Based on the results of this study, we suggest that the surface expression of CD69 serves as a useful marker to identify and trace those thymocytes that are engaged in the TCR-mediated positive selection process in the thymus.

Animals↗
PubMed1993

Inhibition of protein synthesis and antiproliferative effect of the angiotensin converting enzyme inhibitor trandolaprilat in rat vascular smooth muscle cells.

OBJECTIVE: To investigate the effect of the angiotensin converting enzyme (ACE) inhibitor trandolaprilat on vascular smooth muscle cell growth, and to analyse its mechanism of action. DESIGN: Aortic vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats were cultured, and cell proliferation was analysed using a cell synchrony technique. METHODS: Proliferative activity was assessed by [3H]-thymidine uptake and doubling time. Protein synthesis was assessed by [3H]-leucine incorporation. Actin formation was measured using sodium dodecylsulphate-polyacrylamide slab gel electrophoresis and a densitometric assay. The effect of trandolaprilat on translational protein synthesis was also examined using the cell-free protein synthesis system of reticulocyte lysate and messenger RNA from VSMC. RESULTS: Trandolaprilat decreased [3H]-thymidine uptake and increased the doubling time of randomly cycling VSMC. The cell synchrony study revealed that this antiproliferative effect was due to increased transition time from S to G2-M. Decreased cell cycle progression during G2-M was reflected by inhibition of cellular protein synthesis during this period. Cellular protein in randomly cycling VSMC was also decreased by trandolaprilat. This decreased protein synthesis was probably produced by inhibition of RNA translation. CONCLUSIONS: The ACE inhibitor trandolaprilat reduces VSMC proliferation by lengthening the G2-M phase of the cell cycle, and produces a decrease in cellular protein content. This effect is probably mediated by inhibition of protein synthesis at the translational level.

Actins↗
PubMed1993

Suppression of anti-tumor CD4+ T cell responsiveness in the tumor-bearing state and its recovery in in vitro culture free of tumor burden.

We investigated whether the responsiveness of anti-tumor CD4+ T cells suppressed in the tumor-bearing state is reversed in conditions free of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1-3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and IL-4 upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4+ T cells and antigen-presenting cells (APC) that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. However, spleen cells from late (8-10 wk) tumor-bearing stages produced reduced levels of lymphokine production despite the presence of comparable proportions of CD4+ T cells. Because APC in these cell populations exhibited enhanced capacities to present tumor antigens, reduced responsiveness was ascribed to the dysfunction of CD4+ T cells themselves. When spleen cells from early tumor-bearing mice were preincubated for 1-2 days and recultured in fresh medium, the magnitude of lymphokine production by these cells was not changed. In contrast, the same protocol of preincubation and reculture for cells from late tumor-bearing mice resulted in the recovery of anti-tumor lymphokine-producing capacity. The recovered capacity was comparable to or slightly higher than that expressed by cells from early tumor-bearing stages. Since the CD4+ T cell content did not significantly differ before and after preincubation, enhanced lymphokine production was due to the recovered responsiveness of anti-tumor CD4+ helper T cells. The recovery of anti-tumor responsiveness was also induced in vivo by tumor removal at the late tumor-bearing stage: spleen cells from mice 2-4 wk after tumor resection efficiently produced IL-2 and IL-4. These results indicate that the immunodysfunction of anti-tumor CD4+ T cells induced in the tumor-bearing state is reversible because release from tumor burden either by preincubation in vitro or by tumor removal in vivo results in almost complete recovery of the potent anti-tumor responsiveness initially expressed.

Animals↗
PubMed1993

Transforming growth factor-beta (TGF-beta)-mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-beta and a progressive increase in TGF-beta susceptibility of anti-tumor CD4+ T cell function.

The present study deals with the effect of transforming growth factor-beta (TGF-beta) on anti-tumor immune responsiveness at various stages of the tumor-bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1-3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and macrophage-activating factor (MAF)/interferon-gamma (IFN-gamma) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4+ T cells and antigen-presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The IL-2-producing capacity of CD4+ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor-bearing stages. In contrast, the capacity of CD4+ T cells to produce MAF/IFN-gamma was not affected but was maintained at high levels even late in the tumor-bearing state. The addition of recombinant TGF-beta (rTGF-beta) to cultures of spleen cells from various tumor-bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF-beta-induced suppression varied depending on which tumor-bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1-3 wk) tumor-bearing stages but increased in cells from donor mice at later tumor-bearing stages. Thus, spleen cells from late tumor-bearing stages with weak but significant IL-2-producing and considerable MAF/IFN-gamma producing capacities failed to produce these lymphokines when rTGF-beta was present in cultures. A progressive increase in the TGF-beta susceptibility was also observed for IL-4-producing Th2 as well as IL-2/MAF-producing Th1 cells. In addition, increased levels of TGF-beta were detected in plasma from tumor-bearing mice at late stages. Taken together, these results indicate that tumor-bearing mice exhibit enhanced production of TGF-beta as well as a progressive increase in the susceptibility of anti-tumor CD4+ T cells to TGF-beta-induced suppressive mechanisms.

Animals↗
PubMed1993

Purification and Characterization of Carbaryl Hydrolase from Blastobacter sp. Strain M501.

A bacterium capable of hydrolyzing carbaryl (1-naphthyl-N-methylcarbamate) was isolated from a soil enrichment. This bacterium was characterized taxonomically as a Blastobacter sp. and designated strain M501. A carbaryl hydrolase present in this strain was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, gel filtration, and hydroxylapatite chromatographies. The native enzyme had a molecular mass of 166,000 Da and was composed of two subunits with molecular masses of 84,000 Da. The optimum pH and temperature of the enzyme activity were 9.0 and 45 degrees C, respectively. The enzyme was not stable at temperatures above 40 degrees C. The purified enzyme hydrolyzed seven N-methylcarbamate insecticides and also exhibited activity against 1-naphthyl acetate and 4-nitrophenyl acetate.

Journal Article↗
PubMed1993

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.

Animals↗
PubMed1993

Light and electron microscopic radioautographic studies on the RNA synthesis of peri-implanting pregnant mouse uterus during activation of receptivity for blastocyst implantation.

The uterine receptivity for blastocyst implantation in the rodents is activated by nidatory estradiol and is maintained for a restricted period. To establish the variation of the RNA synthesis related to the activation of the uterine receptivity for blastocyst implantation, 1 hr. pulse 3H-uridine incorporation in the mouse uterus at time 0, 3, 6, 12 and 18 hrs. after nidatory estradiol treatment was analyzed by light (LM-RAG) and electron microscopic (EM-RAG) radioautography. The qualitative and quantitative differences of 3H-uridine incorporation in the luminal epithelium and endometrial stroma of three regions of the uterus were evaluated. In the LM-RAM, both the luminal epithelium and stromal cells around the conceptus showed a high incorporation 6 hrs. after estrogen treatment. After the peak of incorporation, the RNA synthesis both of the epithelial and stromal cells of antimesometrial side of the implantation site (AI) decreased abruptly and the grain count values become lower than seen in the other two sites. Both the epithelial and stromal cells of the IN sites showed a gradual but low increase of 3H-uridine incorporation through the time analyzed. The cells of the MI sites also showed a peak of incorporation 6 hrs. after estradiol treatment, but less intense than the AI sites. The EM-RAM showed the silver grains distributed over the cell structures related to the mRNA and rRNA metabolism both on the epithelial and stromal cells. The labeling were also seen on the trophoblastic cells, endothelial cells, smooth muscle cells of the myometrium and mesothelial cells. These results proved the strong influences of the estrogen on the pregnant uterus and showed the topological differences on the responses of the endometrium according to their relation to the implanting blastocyst, and suggest a time restricted responses of the endometrium for activation of uterine receptivity for blastocyst implantation.

Animals↗
PubMed1993

Proliferative activity in the kidneys of aging mice evaluated by PCNA/cyclin immunohistochemistry.

The change of proliferative activity in mouse kidney cortex cells due to aging was studied by PCNA/cyclin immunostaining. Mouse kidney tissues of various ages: late fetal, newborn, suckling, weaning, adult and senescent were used for this experiment. Small pieces of kidney tissues were fixed in methacarn solution and embedded in paraffin. Sections were stained with PCNA/cyclin monoclonal antibody. The reaction product for PCNA/cyclin was observed mainly on nuclei. The ratio of the PCNA/cyclin positive nuclei to the total number of nuclei were calculated. PCNA/cyclin positive ratios in glomeruli and uriniferous tubules in the superficial layer were higher than those in the deep layer from late fetal period to suckling period. They decreased due to aging after birth and became to nearly zero after weaning period.

Aging↗
PubMed1993

Nifedipine-induced gingival overgrowth in the presence or absence of gingival inflammation in rats.

One adverse effect of nifedipine, a long-acting vasodilator, is gingival overgrowth. Preexisting gingival inflammation and/or dental plaque has been suggested to be responsible for the progression of this side effect, but the precise mechanism is uncertain because of a lack of suitable animal models. A study was therefore done to establish an experimental model of gingival overgrowth in rats and to investigate the possible involvement of gingival inflammation and/or dental plaque in its development. Specific pathogen-free Fischer rats (male, 14 days old) were used. Gingival inflammation and dental plaque accumulation were induced by infection with Streptococcus mutans MT8148R. The nifedipine-treated rats (experimental group) were fed a caries-inducing diet containing nifedipine either with or without infection, while the nifedipine-untreated rats (control group) were fed the same diet, similarly with or without the infection. Marked gingival overgrowth was induced in the mandibular molar region of nifedipine-treated rats regardless of S. mutans infection, although the infection resulted in a further increase in the degree of gingival overgrowth. Histological examination of the gingival overgrowth revealed the presence of redundant subepithelial connective tissue in the treated rats, and inflammatory cell infiltration was apparent only in the tissue of the S. mutans-infected rats regardless of the nifedipine administration. These findings suggest that nifedipine induces gingival overgrowth in rats either in the presence or absence of gingival inflammation and/or dental plaque, although these factors can augment the effect of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Radioautographic study on the intracellular localization of a hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat hepatocytes.

In order to demonstrate the intracellular localization of the peroxisome proliferator in the hepatocytes, we have examined the localization of silver grains due to 14C-labelled bezafibrate in cultured rat hepatocytes by means of light and electron microscopic radioautography. As it results from the difference between chemical fixation and freeze-substitution by light microscopic radioautography, more numerous silver grains, about twice, were observed in freeze-substitution specimens in comparison with chemical fixation. On light microscopic radioautograms of the epoxy resin sections during each experimental condition, about 90% of all the silver grains were localized over the cytoplasm. Then, statistical significance was evaluated on grain density in the cytoplasm. On electron microscopic radioautograms of whole mount cultured cells, silver grains were localized not on the peroxisome but on the cytoplasmic matrix specially over the endoplasmic reticulum. From these results, it is concluded that bezafibrate was localized over endoplasmic reticulum. This fact suggests that the receptor of the peroxisome proliferator should be associated with the endoplasmic reticulum or that the receptor exists on the endoplasmic reticulum. Thus, it is demonstrated that the peroxisome proliferator acts on the endoplasmic reticulum of hepatocytes to proliferate peroxisomes.

Animals↗
PubMed1993

[Paternity test by DNA fingerprinting in a sexual assault].

We have applied the method of DNA fingerprinting to resolve a paternity case. DNA samples were extracted from the blood of mother, child (4 months old fetus) and alleged father, digested with the restriction endonuclease Hinf I and Hae III, size separated by agarose gel electrophoresis, and hybridized with the multi-locus minisatellite probe B.E.S.T.-MZ1.3 digoxigenin. DNA fingerprinting patterns in the child and alleged father indicated possibly paternity. Following extraction of DNA from cord blood (infant of 26 to 39 week's gestation), the quantity of DNA isolated was determined on a spectrophotometer at 260 nm and its integrity by electrophoresis in agarose gels. We found that high-molecular-weight DNA could be recovered in large quantities from cord blood as well as from adult blood.

DNA↗