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Biomedical subjects

H Ishida

Publications and source records attributed to H Ishida.

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

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase.

The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur.

Amino Acid Sequence↗
PubMed1992

Bone marrow transplantation as a strategy for treatment of non-insulin-dependent diabetes mellitus in KK-Ay mice.

The effects of allogeneic bone marrow transplantation (BMT) on non-insulin-dependent diabetes mellitus (NIDDM) were examined using KK-Ay mice. KK-Ay mice reconstituted with KK-Ay bone marrow cells showed glycosuria, hyperinsulinemia, and hyperlipidemia. However, KK-Ay mice (H-2b) that had been lethally irradiated (9.0 Gy) and then reconstituted with T cell-depleted bone marrow cells from normal BALB/c mice (H-2d) showed negative urine sugar with decreases in serum insulin and lipid levels 4 mo after BMT. Morphological recovery of islets and glomeruli was also noted after allogeneic BMT. These findings suggest that BMT can be used to treat not only a certain type of NIDDM but also its complications such as hyperlipidemia and diabetic nephropathy.

Animals↗
PubMed1992

Group II phospholipase A2 induced by interleukin-1 beta in cultured rat gingival fibroblasts.

Previously, we reported the presence of group II-like phospholipase A2 activity in the soluble fraction of rat gingiva. In the present study, we found that treatment of rat gingival cells with human recombinant interleukin-1 beta resulted in dose-dependent stimulation of intracellular and extracellular phospholipase A2 activity. Antisera against group II phospholipase A2 totally blocked the interleukin-1 beta-induced phospholipase A2 activity, but antisera against group I phospholipase A2 did not. Moreover, immunoblot analysis showed that the induced phospholipase A2 was group II phospholipase A2. These findings suggest that the induced enzyme belongs to the group II phospholipase A2 family of proinflammatory enzymes.

Animals↗
PubMed1992

Continuous anti-interleukin 10 antibody administration depletes mice of Ly-1 B cells but not conventional B cells.

Ly-1 B cells have the distinctive property of continuous self-replenishment and, as we have shown previously, can be further distinguished from conventional B cells on the basis of greatly elevated constitutive and inducible production of the recently described cytokine interleukin 10 (IL-10). To test the possibility that IL-10 acts as either an autocrine or paracrine growth factor for Ly-1 B cells, we treated mice continuously from birth to 8 wk of age with a monoclonal rat IgM antibody that specifically neutralizes mouse IL-10. Mice treated in this way lacked peritoneal-resident Ly-1 B cells, contained greatly reduced serum immunoglobulin M levels, and were unable to generate significant in vivo antibody responses to intraperitoneal injections of alpha 1,3-dextran or phosphorylcholine, antigens for which specific B cells reside in the Ly-1 B cell subset. In contrast, conventional splenic B cells of anti-IL-10-treated mice were normal with respect to total numbers, phenotype, and in vitro responsiveness to B cell mitogens and the thymus-dependent antigen trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The mechanism of Ly-1 B cell depletion appeared to be related to elevation of endogenous interferon gamma (IFN-gamma) levels in anti-IL-10-treated mice, since coadministration of neutralizing anti-IFN-gamma antibodies substantially restored the number of peritoneal-resident Ly-1 B cells in these mice. These results implicate IL-10 as a regulator of Ly-1 B cell development, and identify a procedure to specifically deplete Ly-1 B cells, thereby allowing further evaluation of the role of these cells in the immune system.

Animals↗
PubMed1992

Synthesis of an S-(alpha-sialosyl)-(2----9)-O-(alpha-sialosyl)-(2----3')-beta-lactos ylceramide.

A ganglioside GD3 analog has been synthesized having an N-acetylneuraminic acid (Neu5Ac) residue alpha-thioglycosidically linked to C-9 of the Neu5Ac residue in the ganglioside GM3 structure. Glycosylation of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-beta-D-galactopyranosyl)-(1----4)-2,6-di-O-benzoyl-beta-D -glucopyranoside with methyl (methyl 5-acetamido-4,7,8-tri-O-acetyl-9-bromo-3,5,9-trideoxy-D-glycero-alpha-D- galacto-2-nonulopyranosid)onate, which was prepared from methyl (methyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2- nonulopyranosid)onate by 8,9-O-isopropylidenation, O-acetylation, hydrolysis of the isopropylidene group, selective bromination, and O-acetylation, using dimethyl(methylthio)sulfonium triflate (DMTST) as a promoter, gave the alpha-sialosyl-(2----3')-lactoside 8. Coupling of the O-acetyl derivative of 8 with the sodium salt of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha-D- galacto-2-nonulopyranosonate gave an alpha-thioglycosidically linked tetrasaccharide. This was converted, via selective removal of the 2-(trimethylsilyl)ethyl group, trichloroacetimidation, and glycosidation with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol into a ganglioside precursor. Finally the precursor on selective reduction of the azide group, coupling with octadecanoic acid, O-deacylation, and de-esterification gave the analog, S-(N-acetyl-alpha-neuraminosyl)-(2----9)-O-(N-acetyl-9-thio-alpha- neuraminosyl)-(2----3')-beta-lactosylceramide.

Antigens, CD↗
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

Glucagon, insulin and somatostatin secretion in response to sympathetic neural activation in streptozotocin-induced diabetic rats. A study with the isolated perfused rat pancreas in vitro.

Changes in glucagon, insulin and somatostatin secretion induced by electrical splanchnic nerve stimulation were examined in rats treated with streptozotocin as neonates and as adults. In order to study the direct neural effects we used the isolated perfused rat pancreas with intact left splanchnic nerve in vitro. In normal rats splanchnic nerve stimulation causes significant decreases in insulin (30-40%) and somatostatin (30-50%) secretion at both 16.7 mmol/l and 1 mmol/l glucose concentrations. In the neonatal streptozotocin-diabetic rat splanchnic nerve stimulation at 16.7 mmol/l glucose decreased insulin secretion (14%) further than in the control rats (30%), however, somatostatin secretion did not decrease to the same extent. Similar results were also observed at the low (1 mmol/l) glucose concentration. On the other hand, percent decreases of insulin and somatostatin secretion induced by splanchnic nerve stimulation in the streptozocin-diabetic rats were similar to the values observed in the normal control rats. The glucagon secretion in response to splanchnic nerve stimulation at 16.7 mmol/l glucose from pancreatic Alpha cells in both types of induced diabetes is exaggerated, and the degree of exaggeration seems to parallel the severity of the hyperglycaemia. However, the splanchnic nerve stimulation-induced glucagon secretion at 1 mmol/l glucose was impaired in the streptozotocin-diabetic rats, but not in the neonatal streptozotocin-diabetic rats. These data suggest that the sensitivity of diabetic Alpha and Delta cells to sympathetic neural activation are blunted, whereas the sensitivity of Beta cells is enhanced in the diabetic animal model.

Animals↗
PubMed1992

Diurnal variation of amylase secretion is coupled with alterations of beta-adrenoceptors in the rat parotid gland.

Diurnal changes in the neurotransmitter receptors are important for studying the receptor function in neurophysiology. The purpose of this study is to gain an insight into the regulatory mechanisms of the diurnal variation of amylase secretion. Rat salivary amylase levels showed a diurnal variation with two peaks, a marked peak at 13 h and a lesser peak at 21 h. This increase in salivary amylase levels was completely inhibited by pretreatment of rats with the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. Amylase level in parotid tissue homogenate also showed a diurnal change, but there was only one peak, at 13 h. The number of maximal binding sites (Bmax) for [3H]dihydroalprenolol (DHA) in parotid membranes showed a diurnal variation with two marked peaks at 13 and 21 h, but the affinity of parotid beta-adrenoceptors for agonists or antagonists did not show any diurnal changes. Phosphorylation of nuclear non-histone proteins in the rat parotid gland showed diurnal variation with two marked peaks at 13 and 21 h. These results indicate that a diurnal variation in the number of rat parotid beta-adrenoceptors, which is presumably regulated by gene expression, is coupled with a change in salivary amylase secretion.

Amylases↗
PubMed1992

Dimensional accuracy of castable apatite ceramic crowns: the influence of heat treatment on dimensional changes and distortion of crowns.

Castable apatite ceramic crowns are subjected to heat treatment during crystallizing and during coloring after casting. The dimensional accuracy of the heat-treated crowns was investigated with respect to its influence on dimensional changes in castable apatite ceramic crowns and on distortion of the interior of the crowns, using two investment techniques and a three-coordinate measuring machine. Dimensional changes were not influenced by the number of coloring firings. Distortion of the interior of crowns was slightly outward at the gingival plane. Shrinkage because of crystallizing was approximately 20 microns and the shrinkage of the gingival plane was equal to that of the occlusal plane.

Apatites↗
PubMed1992

Establishment of a multi-dose study of chondroitin sulfate iron colloid for evaluation of the reticuloendothelial system function.

The assay system of chondroitin sulfate iron colloid (CSFe) was established to evaluate the reticuloendothelial system (RES) function in individual rabbits. In the multi-dose study of CSFe, CSFe was repeatedly administered to each individual rabbit with increasing doses (0.6, 1.2, 2.4, 6.0 mg/kg) at set intervals. Blood samples were serially collected after injection of CSFe and the concentration of CSFe in serum was directly measured as an iron concentration by modifying the previously described assay method [1] to minimize the sample volume. The clearance rate of CSFe at each injected dose was computed by the least-squares method and the double-reciprocal plotting of the doses against the phagocytic velocities by the Lineweaver-Burk method was obtained in each rabbit. The maximum phagocytic velocity (Vmax) and the CSFe concentration producing 1/2 Vmax (Kp) obtained in ten rabbits were 0.129 +/- 0.025 mg/kg per min and 0.417 +/- 0.121 mg/kg (mean +/- S.D.), respectively. The results obtained from this multi-dose study were comparable to our previous results obtained from the mean values of five groups given different doses [1]. The clearance rates of CSFe (0.6, 1.2, 6 mg/kg) decreased after the co-injection of 80 mg/kg of carbon colloid. The calculated Vmax and Kp in 29 rabbits were 0.125 mg/kg per min and 1.167 mg/kg. The Kp was apparently greater than that of the control (Vmax = 0.128 mg/kg per min, Kp = 0.421 mg/kg). Carbon colloid (80 mg/kg) was injected to six rabbits after the completion of the first multi-dose study of CSFe and then the second multi-dose study of CSFe was repeated after 24 h. No differences were found in Vmax and Kp between the two studies as were in the control group (10 rabbits) where saline was injected instead of carbon colloid. These results indicated that carbon colloid (80 mg/kg) gives a competitive and reversible inhibition on the RES. This multi-dose study of CSFe may be applicable for a bed-side analysis of the RES function in a patient.

Animals↗
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

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

Local chromatin changes accompany the expression of the calbindin-D28K gene: tissue specificity and effect of vitamin D activation.

The high affinity calcium-binding protein calbindin-D28K is one of the known proteins transcriptionally up-regulated by the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. This regulation is tissue specific, since in the absence of 1,25-(OH)2D3, the expression of calbindin-D28K is virtually abolished in intestine, whereas it is decreased, but clearly detectable, in kidney, and it remains present at its highest level in cerebellum. Several studies have shown that there is a strong correlation between an increase in the sensitivity to nuclease digestion of a given gene locus and its potential for transcription. Furthermore, hypersensitive sites have often been mapped to regions of DNA including or surrounding sequences known to be important for the regulation of gene transcription. In this study we have scanned the 5'-end and flanking DNA of the calbindin-D28K gene for the presence of DNase-I-hypersensitive (DH) sites in order to localize possible regulatory regions involved in the tissue-specific and hormone-dependent regulation of this gene. We have found that in tissues where calbindin is not expressed, such as liver, no DH sites could be detected. In cerebellum, the same set of DH sites was observed in the presence or absence of 1,25-(OH)2D3 treatment, reflecting the vitamin D-independent expression of the calbindin gene in this tissue. A more complex pattern of DH sites was found in intestine, independently of the vitamin D status of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Enhanced myocardial adenylate cyclase activity in spontaneously hypertensive rats.

This study was designed to clarify the state of beta-adrenergic signal transduction and the disordered level of its transduction in hypertensive hearts, using myocardium from spontaneously hypertensive rats (SHR) as a generic model of essential hypertension. Beta-adrenergic receptor binding sites and dissociation constants in the extracted membranes of adult (70-100 days of age) SHR heart were not significantly different from those of Wistar-Kyoto (WKY) rats, the non-hypertensive control. The adenylate cyclase activities stimulated by isoproterenol with GTP, NaF and forskolin were significantly higher in SHR compared to those in WKY. To determine whether differences in signal transduction are natural or are a result of hypertension, we evaluated chronotropic responses in cultured cells of fetal hearts which had not been exposed to hypertension. Fetal cardiac muscle cells of SHR were more sensitive than WKY to isoproterenol stimulation over a wide concentration range. However, there were no statistically significant differences between these two strains with respect to the density of binding sites. These results suggest that in the transduction of adrenergic signals, alterations distal to the beta-receptors are present in the adult hearts of hypertensive rats, and, that the adrenergic signal transduction is already exaggerated in the pre-hypertensive fetal stage.

Adenylyl Cyclases↗