Biomedical subjects

T Shibasaki

Publications and source records attributed to T Shibasaki.

At least 19 recordsLinked to original sources

PubMed2006

Interleukin-18 mRNA expression in the rat pituitary gland.

The present study investigated the expression of IL-18 mRNA under several stimuli, and molecular structures of IL-18 mRNA of the rat pituitary. Real-time PCR demonstrated that IL-18 mRNA, highly expressed in anterior pituitary, significantly increased following stress and adrenalectomy. In situ hybridization combined with immunohistochemistry demonstrated that corticotrope cells expressed IL-18 mRNA. RACE and sequence analysis demonstrated that pituitary IL-18 mRNA possesses five new exons at the upstream of exon 1 and between exon 1 and exon 2, indicating the preferential usage of promoter 1. The present study suggests that IL-18 in the corticotrope cells may play some roles in stress responses.

Adrenalectomy↗

PubMed2006

A Turkish case with molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MIM 252150) is a rare progressive neurodegenerative disorder with about 100 cases reported worldwide. We have identified a male with molybdenum cofactor deficiency and analyzed the molybdenum cofactor synthesis (MOCS)1 gene, MOCS2 gene, MOCS3 gene and GEPH gene. We homozygously identified the CGA insertion after A666 of the MOCS1 gene which produces arginine insertion at codon 222 of MOCS1A. The parents, his brother and his sister who did not have any symptoms were heterozygous for the same mutation. This region was highly conserved in various species. The N-terminal part of MOCS1 a protein is suggested to form the central core of the protein and be composed of an incomplete [(alpha/beta)6] triosephosphate isomerase (TIM) barrel with a lateral opening that is covered by the C-terminal part of the protein. The insertion is located in the loop connecting the fifth beta strand to the sixth alpha helices of the TIM barrel structure. This arginine insertion would induce the conformation change and the lack of the activity.

Arginine↗

PubMed2005

Distribution of urocortin 2 in various tissues of the rat.

Urocortin (Ucn) 2 is a new member of the corticotrophin-releasing hormone (CRH) neuropeptide family that is expressed in the central nervous system and peripheral tissues. However, the expression levels of Ucn 2 in various tissues of the rat remains unclear. Thus, the aim of the present study was to characterise the expression of Ucn 2 in the various tissues of the rat. Reverse transcriptase-polymerase chain reaction analysis demonstrated that Ucn 2 mRNA is expressed in the hypothalamus, pituitary, adrenal, stomach, skin, ovary, uterus and skeletal muscle. Histologically, Ucn 2 mRNA and Ucn 2-like immunoreactivity (LI) were demonstrated in both the anterior and intermediate lobes of the pituitary, but not detected in the posterior lobe. Furthermore, all Ucn 2-positive cells in the anterior and intermediate lobes were also positive for beta-endorphin. Ucn 2 mRNA was detected in the adrenal cortex and medulla although Ucn 2-LI was only found in the adrenal medulla. High-performance liquid chromatography analysis of hypothalamic, pituitary, and adrenal extracts showed that the main Ucn 2-LI peak occurred at the same molecular size as that of synthetic Ucn 2. These results suggest that Ucn 2 is synthesised in various tissues, including the anterior and intermediate lobes of the pituitary and the adrenal.

Adrenal Glands↗

PubMed2004

Sexual differentiation of the effects of emotional stress on food intake in rats.

Although gender differences in the response to stress have been reported, differences in stress-induced changes in feeding behavior have not been well studied. In this report, inhibition of food intake was compared in male and female rats following 1 h of restraint, electric footshock, or emotional stress induced by a communication box. Although the three stressors inhibited food intake in both genders, only emotional stress caused a gender difference, a greater inhibition of food intake in female rats (48%) than in male rats (22%). The inhibition of food intake by emotional stress in female rats was more prominent during proestrus than the other phases of estrous cycle. In female rats in proestrus emotional stress showed a greater inhibition of food intake than footshock and restraint. Ovariectomy reduced the inhibition of food intake by emotional stress to the same level as that in male, and replacement with estradiol restored the inhibition to the level of the normal female rats. A corticotropin-releasing factor (CRF) type 1 receptor antagonist prevented emotional stress-induced inhibition of food intake, indicating the involvement of CRF type 1 receptor in emotional stress-induced inhibition of food intake. These results suggest that female rats show a greater inhibition of food intake in response to emotional stress than male rats and that estrogen plays a role in the gender difference.

Analysis of Variance↗

PubMed2004

Interleukin-18 null mice show diminished microglial activation and reduced dopaminergic neuron loss following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment.

Recent reports have revealed an involvement of microglial cells in dopaminergic neurodegeneration. In the present study, we tested the hypothesis that interleukin-18 (IL-18) plays a role in the microglial activation. The present study investigated microglial activation and dopaminergic neurodegeneration in substantia nigra pars compacta (SNpc) following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in wild type (WT) and IL-18 knockout (KO) mice. The number of dopaminergic neuron loss in WT mice was significantly decreased 7 days after MPTP treatment compared with IL-18 KO mice. In WT mice microglial activation occurred in the SN at 1 day after MPTP treatment, progressively increased within the SNpc until 7 days post MPTP, and subsided by 14 days. In contrast, in IL-18 KO mice microglial activation occurred in the SN at 1 day post-MPTP, and decreased by 7 days, earlier than in WT mice. The lesser microglial activation and dopaminergic neurodegeneration in the SNpc following MPTP treatment in WT indicates the possibility that IL-18 may participate in microglial activation and dopaminergic neurodegeneration.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2003

Corticotropin-releasing factor as well as opioid and dopamine are involved in tail-pinch-induced food intake of rats.

Several kinds of stress such as psychological stress, restraint, and foot shock inhibit feeding behavior through corticotropin-releasing factor (CRF). In contrast, a mild tail pinch increases food intake in rats. Although dopamine and opioid are thought to be involved in tail-pinch-induced food intake, it is unknown whether CRF participates in this phenomenon. Therefore, we attempted to clarify this issue using rats. A 30-s tail pinch increased food intake in 30 min after the tail pinch, and this increase was blocked by intraperitoneal injection of CRF receptor type 1 selective antagonist. CRF increased food intake in 30 min after intracerebroventricular injection at a dose of 2 or 10 ng, and this increase was also blocked by CRF receptor type 1 antagonist. Tail-pinch- or CRF-induced food intake was blocked by naloxone, pimozide, and spiperone. These results suggest that CRF, through CRF receptor type 1 as well as opioid and dopaminergic systems, are involved in the mechanism of tail-pinch-induced food intake. The results also suggest that brain CRF has dual effects on food intake, hyperphagia and anorexia, in a stress-dependent manner.

Animals↗

PubMed2001

The effects of mitiglinide (KAD-1229), a new anti-diabetic drug, on ATP-sensitive K+ channels and insulin secretion: comparison with the sulfonylureas and nateglinide.

Mitiglinide (KAD-1229), a new anti-diabetic drug, is thought to stimulate insulin secretion by closing the ATP-sensitive K+ (K(ATP)) channels in pancreatic beta-cells. However, its selectivity for the various K(ATP) channels is not known. In this study, we examined the effects of mitiglinide on various cloned K(ATP) channels (Kir6.2/SUR1, Kir6.2/SUR2A, and Kir6.2/SUR2B) reconstituted in COS-1 cells, and compared them to another meglitinide-related compound, nateglinide. Patch-clamp analysis using inside-out recording configuration showed that mitiglinide inhibits the Kir6.2/SUR1 channel currents in a dose-dependent manner (IC50 value, 100 nM) but does not significantly inhibit either Kir6.2/SUR2A or Kir6.2/SUR2B channel currents even at high doses (more than 10 microM). Nateglinide inhibits Kir6.2/SUR1 and Kir6.2/SUR2B channels at 100 nM, and inhibits Kir6.2/SUR2A channels at high concentrations (1 microM). Binding experiments on mitiglinide, nateglinide, and repaglinide to SUR1 expressed in COS-1 cells revealed that they inhibit the binding of [3H]glibenclamide to SUR1 (IC50 values: mitiglinide, 280 nM; nateglinide, 8 microM; repaglinide, 1.6 microM), suggesting that they all share a glibenclamide binding site. The insulin responses to glucose, mitiglinide, tolbutamide, and glibenclamide in MIN6 cells after chronic mitiglinide, nateglinide, or repaglinide treatment were comparable to those after chronic tolbutamide and glibenclamide treatment. These results indicate that, similar to the sulfonylureas, mitiglinide is highly specific to the Kir6.2/SUR1 complex, i.e., the pancreatic beta-cell K(ATP) channel, and suggest that mitiglinide may be a clinically useful anti-diabetic drug.

ATP-Binding Cassette Transporters↗

PubMed2001

Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells.

A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH-DXR) inhibited glutathione S-transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH-DXR induced caspase-3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 microM GSH-DXR, GST-P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase-3 activation and DNA fragmentation were induced following the suppression of GST-P expression by treatment with GSH-DXR. When the cells were treated with 100 microM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase-3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 microM) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST-P, and consequent prevention of DXR- and GSH-DXR-induced DNA fragmentation. Overexpression of GST-pi (placental type of human GST isozymes) by transfection of GST-pi sense cDNA into AH66 cells decreased sensitivities to DXR and GSH-DXR, and the suppression of GST-P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site-directedly mutated GST-P in which the active-site residue Tyr39 was replaced with His (W39H) or the substrate-binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST-P in AH66 cells treated with GSH-DXR must play an important role in the induction of apoptosis.

Animals↗

PubMed2001

Critical role of cAMP-GEFII--Rim2 complex in incretin-potentiated insulin secretion.

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic beta-cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII--Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic beta-cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretin-potentiated insulin secretion approximately 50%, while a combination of antisense ODNs and H-89 inhibited the secretion approximately 80-90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII--Rim2 pathway is critical in the potentiation of insulin secretion by incretins.

Animals↗

PubMed2001

Effect of cadmium on gap junctional intercellular communication in primary cultures of rat renal proximal tubular cells.

Cadmium mainly accumulates in the kidney and causes renal injury. To clarify the mechanism of Cd nephrotoxicity, we investigated the effects of this element on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Sixty minutes after exposure to 100 microM Cd, dye coupling experiments showed that gap junctional intercellular communication (GJIC) was significantly inhibited. This inhibition occurred before the appearance of cytotoxicity. Intracellular calcium concentrations [Ca2+]i, which modulate the function of gap junctions, gradually increased after exposure to Cd and reached a maximum after 60 minutes. These results suggest that the inhibition of GJIC as a result of Cd exposure is related to an increase in [Ca2+]i, and that GJIC inhibition may be an indicator of nephrotoxicity.

Acetylglucosaminidase↗

PubMed2001

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.

Animals↗

PubMed2001

Fibroblast growth factor inhibits locomotor activity as well as feeding behavior of rats.

The effects of acute and chronic intracerebroventricular (i.c.v.) administration of basic fibroblast growth factor (bFGF) on behavior were examined in free-feeding rats. An i.c.v. injection of bFGF induced behavioral changes, such as an increase in resting and decreases in grooming, moving, and food intake at a dose of 20 or 50 ng. These effects appeared at 4-5 h and lasted at least 11 h after the injection. These changes, as well as inhibition of body weight gain, were also found during a 6-day period of chronic i.c.v. infusion of bFGF at a dose of 20 ng/h. These results indicate that bFGF as both bolus i.c.v. injection and chronic i.c.v. infusion inhibits not only feeding behavior but also locomotor activity in rats. It is suggested that the inhibitory effect of bFGF on food intake may be in part ascribed to the suppression of behavior by bFGF.

Animals↗

PubMed2001

Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats.

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.

Animals↗

PubMed2001

Characterization of the gene EPAC2: structure, chromosomal localization, tissue expression, and identification of the liver-specific isoform.

The liver-specific protein cAMP-GEFII (also known as Epac2) belongs to a family of cyclic adenosine monophosphate (cAMP) binding proteins having guanine nucleotide exchange factor (GEF) activity (the cAMP-GEF family). Here we clone the gene EPAC2, encoding cAMP-GEFII, from a human liver cDNA library. Human EPAC2 has at least 31 exons and is mapped to human chromosome 2q31. Analyses by primer extension, reverse transcriptase-polymerase chain reaction, and in situ hybridization revealed the presence of three transcription start sites of liver-specific Epac2: two major sites located in exon 10 and a minor site in intron 9. The same translation start site is used in all three transcripts. Liver-specific cAMP-GEFII protein, which lacks the first cAMP-binding domain and the Dishevelled/Egl-10/Pleckstrin domain, was detected at 79 kDa by immunoblot analysis, confirming the presence of the short form of cAMP-GEFII in the liver. Liver-specific cAMP-GEFII also has GEF activity toward Rap1. These results demonstrate the presence of liver-specific cAMP-GEFII. Together with the previous finding that cAMP-GEFII is responsible for cAMP-dependent exocytosis in secretory cells, our study suggests that cAMP-GEFII may have a distinct role in liver.

Amino Acid Sequence↗

PubMed2001

The expression of thyrotrophin-releasing hormone receptor 1 messenger ribonucleic acid in human pituitary adenomas.

OBJECTIVE: Thyrotrophin-releasing hormone (TRH) paradoxically induces the release of growth hormone (GH) when injected intravenously into acromegalic patients, although the mechanism of this action is unknown at present. Several research groups have reported that the level of TRH receptor-1 (TRHR-1) mRNA expression is variable in pituitary adenomas, and does not correlate with the degree of paradoxical GH response to TRH administration in a limited number of acromegalic patients. We aimed to compare the expression levels of TRHR-1 mRNA among various types of pituitary adenoma and to clarify whether these levels correlate with the degree of pituitary hormone response to TRH. PATIENTS: Pituitary adenoma tissue was obtained by surgery from 14 patients with acromegaly, four with prolactinomas, nine with nonfunctioning adenomas and one with a TSH-producing adenoma. METHODS: The level of human TRHR-1 mRNA expression in each adenoma was quantified using the competitive reverse transcription polymerase chain reaction (RT-PCR) METHOD: For amplification of a TRHR-1 cDNA fragment, a sense primer was designed according to the sequence in exon 2 and an antisense primer designed according to the sequence located at the region in exon 3 that does not encode for the alternative splicing-generated short form of TRHR-1 mRNA. RESULTS: TRHR-1 mRNA was detected in all pituitary adenomas examined and did not correlate with their size. The mean level of TRHR-1 mRNA expression was significantly lower in GH-producing adenomas than in prolactinomas and nonfunctioning adenomas (1.4 +/- 0.4 x 10(-2) attomol/microg total RNA, 10.7 3.4 x 10(-2) attomol/microg total RNA, and 7.2 +/- 3.3 x 10(-2) attomol/g total RNA, respectively). The ratio of plasma peak GH induced by TRH administration to the basal level of plasma GH in the patients with acromegaly correlated positively with the level of TRHR-1 mRNA expression in their GH-producing adenomas (r = 0.620, P = 0.0179). The responsiveness of plasma PRL and gonadotrophin to TRH in the patients with prolactinoma and nonfunctioning pituitary adenoma did not significantly correlate with the levels of TRHR-1 mRNA expression in their pituitary adenomas, respectively. CONCLUSIONS: The findings of the present study suggest that the level of TRHR-1 mRNA expression varies among different types of pituitary adenoma. Furthermore, in acromegaly, the responsiveness of plasma GH to TRH administration appears to at least partially depend on the level of TRHR-1 mRNA expression in the GH-producing pituitary adenoma.

Acromegaly↗

PubMed2001

Ghrelin and growth hormone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adenomas.

OBJECTIVE: The level of growth hormone (GH), growth hormone secretogogue (GHS) and GHS receptor (GHSR) messenger ribonucleic acid (mRNA) expression has been reported as being higher in GH-producing pituitary adenomas than in other types of pituitary adenomas. Recently, ghrelin, an endogenous ligand specific for GHSR, was isolated. Therefore, we attempted to clarify whether ghrelin mRNA is expressed in various types of human pituitary adenoma by competitive reverse transcription-polymerase chain reaction (RT-PCR). We also examined the relationship between the levels of ghrelin or GHSR mRNA and hormonal and tumour characteristics in patients with pituitary adenomas. PATIENTS: Pituitary adenoma tissue was obtained at surgery from 13 patients with acromegaly, 4 with prolactinomas, 5 with gonadotrophin (Gn)-producing adenomas, 4 with non-functioning adenomas, 2 with ACTH-producing adenomas and 2 with TSH-producing adenomas. METHODS: The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. RESULTS: Ghrelin mRNA was detected in all pituitary adenoma tissues examined, with the highest mean level detected in non-functioning adenomas, a moderate level in GH-producing adenomas and Gn-producing adenomas, and the lowest level in prolactinomas. The level of ghrelin mRNA expression in GH-producing adenomas correlated negatively with the size of the adenoma (n = 13) (r = - 0.756, P = 0.0028). Furthermore, the mean level of ghrelin mRNA expression in high-grade (III and IV of Hardy classification) GH-producing adenomas was significantly lower than that in low-grade (I and II) GH-producing adenomas (P = 0.0016). GHSR mRNA was also detected in all pituitary adenomas with the highest mean level in GH-producing adenomas, a moderate level in nonfunctioning adenoma, and the lowest level in prolactinoma and Gn-producing adenomas. CONCLUSIONS: Ghrelin mRNA, in addition to GHSR mRNA, is expressed in various types of pituitary adenoma with different levels of expression in each type. Our findings suggest that ghrelin produced in pituitary adenoma may play some role in the mechanism underlying the development of adenoma cells through autocrine and/or paracrine pathways.

Adenoma↗

PubMed2001

Nicotine infusion alters leptin and uncoupling protein 1 mRNA expression in adipose tissues of rats.

We attempted to clarify whether leptin and uncoupling protein 1 (UCP1) are involved in the action of nicotine on the energy balance. Male Wistar rats were infused subcutaneously with nicotine (12 mg x kg(-1) x day(-1)) for 4 or 14 days. At the end of the 4-day period, the plasma concentrations of leptin of the nicotine-treated and pair-fed rats were lower than those of the freely fed rats, although the levels of leptin mRNA expression in various white adipose tissues did not differ among the three groups. At the end of the 14-day nicotine infusion period, plasma concentrations of leptin were higher, and leptin mRNA expression in the omentum and epididymal and retroperitoneal adipose tissues was stronger in the nicotine-treated rats than in the pair-fed and freely fed rats. UCP1 mRNA expression in the brown adipose tissue of nicotine-treated was stronger than that of the pair-fed rats. These results suggest that continuous nicotine infusion differentially affects the synthesis and secretion of leptin according to the duration of infusion and stimulates UCP1 mRNA expression, probably in a manner independent of leptin.

Adipose Tissue↗

PubMed2001

Visual disturbance due to carotid artery thrombosis in a patient with familial hypercholesterolemia; response to surgical thrombotectomy.

A 48 years-old Japanese man suffered from marked xanthomas on ankles, knees, hand fingers, and foot joints due to insufficient control of serum hypercholesterolemia despite low density lipoprotein (LDL-C) absorptive therapy followed by treatment with potent anti-hypercholesterolemic agents. He had undergone surgical resection of xanthoma on the knee, foot and hand finger joints. Treatment with simvastatin returned the serum total cholesterol levels to nearly normal levels, followed by marked fluctuations. He subsequently experienced transient right-visual disturbance, and roentogenographic examination was performed. The patient was diagnosed as right-common carotid artery thrombosis. After the thrombotectomy of the right-common carotid artery, his visual power was markedly improved.

Carotid Artery Diseases↗