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Y Tokuyama

Publications and source records attributed to Y Tokuyama.

At least 37 records · Page 2Linked to original sources
PubMed1996

Retinoids as Ig Isotype-Switch Modulators

The role of retinoids was analyzed in directing isotype switching to IgA and IgG1 (IgE) by LPS-stimulated murine μ(+)B-cells in the presence of two Th2-type cytokines, IL-4 and IL-5. All trans retinoic acid (RA) enhanced the production of IgA at high concentrations (10-100 nM) in the presence of IL-5. Addition of IL-4 to the system modulated the IgA response in a dose-dependent manner. Namely, IL-4 inhibited the response at concentrations higher than 250 usolidusml, but showed slight enhancement at lower concentrations (130 usolidusml). IL-4 alone, which is considered to be an IgE isotype-switch inducer, strongly enhanced the IgG1 and IgE responses. Addition of IL-5 to the system showed a synergistic effect which could be attenuated by addition of low concentrations of RA (about 1 nM). Thus, the presence of switch modulators such as IL-4 and IL-5, their concentration ratios, and concentrations of retinoids are crucial factors in initiating and directing isotype switching to IgA and IgG1 (IgE).

Journal Article↗
PubMed1996

Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

Animals↗
PubMed1996

Rat inwardly rectifying potassium channel Kir6.2: cloning electrophysiological characterization, and decreased expression in pancreatic islets of male Zucker diabetic fatty rats.

The ATP-sensitive potassium channel of insulin-secreting pancreatic beta-cells is a complex of Kir6.2, a member of the inwardly rectifying potassium channel superfamily, and the sulfonylurea receptor. We have isolated cDNA clones encoding rat Kir6.2. Co-expression of rat Kir6.2 and sulfonylurea receptor in human embryonic kidney cells generated a potassium current with the properties of the beta-cell ATP-sensitive potassium channel. A quantitative reverse transcriptase-polymerase chain reaction assay indicated that Kir6.2 and sulfonylurea receptor mRNAs were abundantly expressed in rat islets and that expression of Kir6.2 mRNA was reduced by >70% in islets from Zucker diabetic fatty male rats, whereas there was no significant change in sulfonylurea receptor mRNA levels. Thus, decreased expression of Kir6.2 could contribute to the beta-cell dysfunction which characterizes diabetes mellitus in this animal model.

Adenosine Triphosphate↗
PubMed1996

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Amino Acid Sequence↗
PubMed1996

Differential effects of overexpressed glucokinase and hexokinase I in isolated islets. Evidence for functional segregation of the high and low Km enzymes.

Glucose-stimulated insulin secretion is believed to require metabolism of the sugar via a high Km pathway in which glucokinase (hexokinase IV) is rate-limiting. In this study, we have used recombinant adenoviruses to overexpress the liver and islet isoforms of glucokinase as well as low Km hexokinase I in isolated rat islets of Langerhans. Glucose phosphorylating activity increased by up to 20-fold in extracts from islets treated with adenoviruses containing the cDNAs encoding either tissue isoform of glucokinase, but such cells exhibited no increase in 2- or 5-[3H]glucose usage, lactate production, glycogen content, or glucose oxidation. Furthermore, glucokinase overexpression enhanced insulin secretion in response to stimulatory glucose or glucose plus arginine by only 36-53% relative to control islets. In contrast to the minimal effects of overexpressed glucokinases, overexpression of hexokinase I caused a 2.5-4-fold enhancement in all metabolic parameters except glycogen content when measured at a basal glucose concentration (3 mM). Based on measurement of glucose phosphorylation in intact cells, overexpressed glucokinase is clearly active in a non-islet cell line (CV-1) but not within islet cells. That this result cannot be ascribed to the levels of glucokinase regulatory protein in islets is shown by direct measurement of its activity and mRNA. These data provide evidence for functional partitioning of glucokinase and hexokinase and suggest that overexpressed glucokinase must interact with factors found in limiting concentration in the islet cell in order to become activated and engage in productive metabolic signaling.

Animals↗
PubMed1996

Islet amyloid polypeptide/amylin contents in pancreas change with increasing age in genetically obese and diabetic mice.

To search for a possible relationship between islet amyloid polypeptide (IAPP)/amylin and the pathophysiology of non-insulin-dependent (type 2) diabetes mellitus (NIDDM), we examined the changes in IAPP contents in the pancreata of genetically obese and diabetic mice (C57BL/6J ob/ob and C57BL/KsJ db/db mice). In the male ob/ob mice, IAPP and insulin contents began to increase at 16 weeks and continued to increase. In the male db/db mice, IAPP content began to increase at 8 weeks of age and insulin content at 4 weeks. Both contents continued to increase until 16 weeks, but drastically decreased at 24 weeks. Immunohistochemical studies using anti-IAPP8-17 antibody showed the increase of islet cell mass and the heterogeneous immunoreactivity for IAPP in islet cells in the ob/ob mice at 24 weeks of age. In the db/db mice at the same age, the immunoreactivity was heterogeneous and weak in many islet cells. These results suggest that genetic factors that are important in the manifestation of NIDDM influence the capacity of beta-cells to synthesize and secrete IAPP, and that IAPP synthesis and secretion change in the course of the disease.

Aging↗
PubMed1996

Increased expression of endothelin B receptor mRNA following subarachnoid hemorrhage in monkeys.

These studies tested the hypothesis that the cerebral vasospasm that follows subarachnoid hemorrhage (SAH) is due to alterations in endothelin (ET) and ET receptor expression. Eight monkeys underwent cerebral angiography and induction of SAH. Angiography was repeated 7 days later to confirm the presence of cerebral vasospasm, and animals were killed. RNA was isolated from right (vasospastic) and left (control) side middle cerebral arteries and surrounding cerebral cortex. The levels of prepro (PP) ET-1 (ppET-1) and ppET-3 and ETA and ETB receptor MRNAs were determined using a quantitative reverse transcriptase polymerase chain reaction-based assay. ET-1 peptide was also measured in CSF at baseline and after 7 days. Specific agonist binding to ETA and ETB receptors in both middle cerebral arteries and in surrounding brain cortex was measured in three animals by autoradiographic binding assays. Levels of ETB receptor mRNA were 3.4 +/- 2.2-fold higher in the right than in the left cerebral arteries (p < 0.01). There were no significant differences in the levels of ppET-1, ppET-3, or ETA receptor mRNA in cerebral arteries. ET-1 peptide was not elevated in CSF. Levels of ETA and ETB receptor mRNAs were 2.6 +/ 1.1- and 2.1 +/ 1.3-fold higher, respectively, in the right than in the left cerebral cortex, while the level of ppET-3 mRNA was 2.1 +/- 1.0-fold lower. There were no differences in ppET-1 mRNA levels between right and left cerebral cortex. Binding to ETA and ETB receptors in cerebral arteries and cortex did not differ significantly between right and left sides. These results do not support the hypothesis that overexpression of ET-1 is principal cause of vasospasm, but rather they suggest that SAH causes complex changes in the ET system that together are responsible for the cellular response to SAH.

Animals↗
PubMed1996

Changes in endothelial nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys.

OBJECTIVE: We attempt to determine whether changes in messenger ribonucleic acid (mRNA) for nitric oxide synthase (NOS) and soluble guanylate cyclase, enzymes that mediate endothelium-dependent vasodilation in cerebral arteries, occur after subarachnoid hemorrhage (SAH) in monkeys. METHODS: Baseline cerebral angiograms were obtained, and right-sided SAH was induced by microsurgically placing autologous blood clot against the right anterior circle of Willis in seven monkeys. Seven days later, angiographic studies were repeated and the animals were killed. Right (vasospastic) and left (control) middle cerebral arteries and underlying cortex were removed. The competitive reverse transcriptase polymerase chain reaction was used to quantify mRNA for soluble guanylate cyclase and two isoforms of constitutive NOS in these tissues. RESULTS: Comparison of angiograms at baseline and after 7 days showed a 41 +/- 7% (mean +/- standard error of the mean, P < 0.05, Wilcoxon test) decrease in diameter of the right middle cerebral artery. After the animals were killed, comparison of right and left middle cerebral arteries showed a 56 +/- 11% decrease (P < 0.005, paired t test) in endothelial NOS mRNA. There was a 142 +/- 39% (P < 0.05) increase in right cortex endothelial NOS mRNA compared to the left cortex. There were no significant differences between right and left sides in mRNAs for soluble guanylate cyclase or brain NOS. CONCLUSION: Decreased endothelial NOS mRNA in cerebral arteries 7 days after SAH may be caused by endothelial cell damage and could contribute to vasospasm after SAH. Increased endothelial NOS in brain tissue may reflect a compensatory vasodilator mechanism of the brain against the cerebral ischemia associated with vasospasm and SAH.

Animals↗
PubMed1996

NIDDM is associated with loss of pancreatic beta-cell L-type Ca2+ channel activity.

Development of non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in glucose-stimulated insulin secretion. We have investigated Zucker diabetic fatty rats (ZDF), an animal model of NIDDM, and found that, compared with control islets, the expression of mRNA encoding C- and D-isoforms of alpha 1-subunits of beta-cell L-type voltage-dependent Ca2+ channels (VDCC) was significantly reduced in islets isolated from ZDF rats. This correlated with a substantial reduction of L-type Ca2+ currents (ICa) in ZDF beta-cells. Intracellular Ca2+ concentration responses in ZDF islets after glucose, KCI, or BAY K 8644 stimulation were markedly attenuated, whereas responses evoked by carbachol were unimpaired, consistent with a specific decrease in ICa in the diabetic islets. This reduction was accompanied by loss of pulsatile insulin secretion from ZDF islets treated with oscillatory increases of external glucose concentration. Our findings suggest that the attenuation of ICa in diabetic islets may contribute to the abnormal glucose-dependent insulin secretory responses associated with NIDDM and indicate that this defect is caused by decreased expression of genes encoding beta-cell VDCC alpha 1-subunits.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Cloning of rat KATP-2 channel and decreased expression in pancreatic islets of male Zucker diabetic fatty rats.

ATP-regulated potassium channels play a key role in regulating insulin secretion. We have isolated cDNA clones from a RINm5F insulinoma cell cDNA library that encode a protein, KATP-2, whose sequence shows 72% identity with the rat heart potassium channel KATP. RNA blotting showed that KATP-2 mRNA was present at high levels in brain and undetectable in heart, spleen, lung, liver, skeletal muscle, kidney and testis. A quantitative RT-PCR assay indicated that there were 1.85 +/- 0.32 x 10(5) molecules of KATP-2 mRNA per microgram of total RNA in pancreatic islets from nondiabetic rats. The levels of KATP-2 mRNA were reduced by 34% in islets from diabetic Zucker diabetic fatty male rats, a model of non-insulin dependent diabetes mellitus, compared to their lean nondiabetic littermates (p < 0.05), suggesting that decreased expression of KATP-2 may contribute to beta-cell dysfunction in this animal model.

Adenosine Triphosphate↗
PubMed1995

Cloning of rat and mouse P2Y purinoceptors.

The cellular responses to ATP are mediated by specific high-affinity receptors designated as P2 purinoceptors, five subclasses of which have been defined pharmacologically-P2X, P2Y, P2U, P2T, and P2Z. A cDNA clone encoding a rat P2Y purinoceptor was isolated from an insulinoma cDNA library. The 373-amino acid rat P2Y purinoceptor sequence has 85.7% and 37.8% identity with chicken P2Y and rat P2U purinoceptors, respectively. The sequence of the mouse P2Y purinoceptor was also determined and there was 97.1% amino acid identity with the corresponding rat sequence. RNA blotting studies showed that rat P2Y purinoceptor mRNA was expressed at variable levels in many tissues including heart, brain, spleen, lung, liver, skeletal muscle and kidney, although it was not detected in testis. The cloned rat P2Y purinoceptor was expressed in Xenopus laevis oocytes and possessed the properties expected for this receptor subtype.

Adenosine Triphosphate↗
PubMed1995

Retinoids inhibit IL-4-dependent IgE and IgG1 production by LPS-stimulated murine splenic B cells.

The effects of retinoids (all-trans-retinoic acid (RA) and vitamin A) and hydrocortisone (HC) on the IL-4-dependent IgE and IgG1 response by mouse whole spleen cells and splenic B cells were studied. Whole spleen cells were cultured for 8 days in the presence of LPS and IL-4 and the supernatants were assayed for IgG1 and IgE. The levels of the two classes were enhanced about twofold by the addition of HC at 10 nM. The addition of retinoids to the above culture system within 1 day from the start strongly inhibited both IgG1 and IgE responses. The concentration inhibiting both of the reactions by 50% was about 3-10 nM for RA and 300 nM-1 microM for vitamin A. In the case of splenic B cells, HC also showed an enhancing effect on the IgG1 and IgE production at extremely low concentrations (20 pM-2 nM), but at higher concentrations (above 10 nM) it was inhibitory. IL-4-dependent IgG1 and IgE production of LPS-stimulated splenic B cells was also inhibited by retinoids. The concentration giving 50% inhibition was 0.2 nM for RA and 150 nM for vitamin A. Exogenous TGF-beta 1 also inhibited the IL-4-dependent IgG1 and IgE production by splenic B cells and the inhibition was reverted in the presence of the specific neutralizing antibody to TGF-beta 1. However, the inhibition by 1 nM RA could not be abolished by the excess antibodies to TGF-beta 1 and TGF-beta 2.

Animals↗
PubMed1995

Endogenous cytokine expression profiles in retinoic acid-induced IgA production by LPS-stimulated murine splenocytes.

All-trans-retinoic acid (RA) enhances IgA production by LPS-stimulated murine splenocytes. After stimulation by RA and LPS, or by LPS alone, total RNA was extracted from cultured cells on Days 1 to 4, and the kinetics of expression of various cytokine mRNAs were analyzed by the RT-PCR method. RA induced the expression of IL-5 and TGF-beta 2 mRNAs in the LPS-stimulated cells. In addition, the expression of IL-6 and IL-2 mRNAs was more intensive in RA-stimulated cells than in unstimulated cells. TGF-beta 1 and TGF-beta 3 mRNAs were constitutively expressed in both culture groups. RA enhanced IgA production by LPS-stimulated spleen cells but not that by LPS-stimulated mu(+) naive splenic B-cells. For RA-induced IgA production, the B-cells required T-cells or the culture supernatant from RA-stimulated T-cells. Furthermore, exogenous IL-5 replaced the T-cell requirement, at least in part, in RA-induced IgA production by LPS-stimulated B-cells. This reaction was partially inhibited by anti-TGF-beta-neutralizing antibodies. These findings suggest that RA induces IgA production by (IL-5 + LPS)-stimulated B-cells in TGF-beta-independent and TGF-beta-dependent manners.

Adjuvants, Immunologic↗
PubMed1995

A long-term follow-up study in direct cerebral aneurysm surgery.

A long-term follow-up study covering between one and twenty-two years after surgery was carried out on 177 patients with direct operations for cerebral aneurysms. Rebleeding was detected in five out of 18 patients with incomplete operations; one patient within 6 months and four patients within 5 to 8 years after surgery. The rebleeding rate of incompletely operated patients in the first decade was similar to that of untreated patients with ruptured aneurysms. This suggests that wrapping, coating and incomplete clipping procedures provide some protection against rebleeding in the first 6 months but little in the period extending beyond 5 years after surgery. Showing two types of newly-formed aneurysms after complete surgery and enlarged residual aneurysms after incomplete surgery, the possibility of rebleeding from these aneurysms is discussed in the present report.

Cerebral Angiography↗
PubMed1995

Magnesium supplementation reduces development of diabetes in a rat model of spontaneous NIDDM.

We examined the effects of a magnesium-supplemented (Mg-S) diet in the male obese Zucker diabetic fatty rat, a model of non-insulin-dependent diabetes mellitus (NIDDM). Obese rats were maintained on either a control (0.20% Mg) or magnesium-supplemented (Mg-S; 1% Mg) diet for 6 wk beginning at 6 wk of age. The rats maintained on the Mg-S diet had markedly lower fasting and fed-state blood glucose concentrations and an improved glucose disposal. By 12 wk of age, all of the eight animals on the control diet became diabetic, whereas diabetes developed in only one of eight animals on the Mg-S diet. Insulin and C-peptide concentrations, in addition to pancreatic GLUT-2 and insulin mRNA expression, were higher in the male obese Mg-S rats than in their control-fed counterparts. A subgroup of rats on the control diet with established diabetes was switched to a Mg-S diet for an additional 4 wk. The Mg-S diet did not reverse diabetes once already established. These data indicate that an increased dietary Mg intake in male obese rats prevents deterioration of glucose tolerance, thus delaying the development of spontaneous NIDDM.

Aging↗
PubMed1995

Formation of islet amyloid fibrils in beta-secretory granules of transgenic mice expressing human islet amyloid polypeptide/amylin.

To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we developed transgenic mice using a human IAPP cDNA connected to an insulin promoter. Ribonucleic acid blotting and immunohistochemistry revealed the expression of the transgene in the pancreatic beta cells. Immunogold electron microscopy showed that beta-secretory granules contained the human C-terminal flanking peptide of the IAPP precursor. Reverse-phase HPLC demonstrated human and mouse IAPP amide in the pancreas. Electron microscopy showed the accumulation of fibril-like material in a considerable number of beta-secretory granules. These results suggest that in transgenic mice, the human IAPP precursor is expressed in beta cells and becomes normally sorted into beta-secretory granules in which normal conversion to mature human IAPP takes place. The human IAPP molecules, because of their amyloidogenesis, aggregate into amyloid fibrils in secretory granules. Glucose tolerance was normal at 7 months old and islet amyloid was not observed. A longer time may be required for islet amyloid deposits and hyperglycemia to develop in mice. Our working hypothesis is that in human NIDDM, IAPP aggregates into amyloid fibrils in beta-secretory granules, and that the fibrils are released into the extracellular space and islet amyloid deposits become substantial with time.

Amyloid↗
PubMed1995

Evolution of beta-cell dysfunction in the male Zucker diabetic fatty rat.

The molecular basis for the beta-cell dysfunction that characterizes non-insulin-dependent diabetes mellitus (NIDDM) is unknown. The Zucker diabetic fatty (ZDF) male rat is a rodent model of NIDDM with a predictable progression from the prediabetic to the diabetic state. We are using this model to study beta-cell function during the development of diabetes with the goal of identifying genes that play a key role in regulating insulin secretion and, thus, may be potential targets for therapeutic intervention aimed at preserving or improving beta-cell function. As a first step, we have characterized morphology, insulin secretion, and pattern of gene expression in islets from prediabetic and diabetic ZDF rats. The development of diabetes was associated with changes in islet morphology, and the islets of diabetic animals were markedly hypertrophic with multiple irregular projections into the surrounding exocrine pancreas. In addition, there were multiple defects in the normal pattern of insulin secretion. The islets of prediabetic ZDF rats secreted significantly more insulin at each glucose concentration tested and showed a leftward shift in the dose-response curve relating glucose concentration and insulin secretion. Islets of prediabetic animals also demonstrated defects in the normal oscillatory pattern of insulin secretion, indicating the presence of impairment of the normal feedback control between glucose and insulin secretion. The islets from diabetic animals showed further impairment in the ability to respond to a glucose stimulus. Changes in gene expression were also evident in islets from prediabetic and diabetic ZDF rats compared with age-matched control animals. In prediabetic animals, there was no change in insulin mRNA levels. However, there was a significant 30-70% reduction in the levels of a large number of other islet mRNAs including glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca(2+)-ATPase, and transcription factor Islet-1 mRNAs. In addition, there was a 40-50% increase in the levels of glucose-6-phosphatase and 12-lipoxygenase mRNAs. There were further changes in gene expression in the islets from diabetic ZDF rats, including a decrease in insulin mRNA levels that was associated with reduced islet insulin levels. Our results indicate that multiple defects in beta-cell function can be detected in islets of prediabetic animals well before the development of hyperglycemia and suggest that changes in the normal pattern of gene expression contribute to the development of beta-cell dysfunction.

Animals↗