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Biomedical subjects

Gozoh Tsujimoto

Publications and source records attributed to Gozoh Tsujimoto.

At least 73 records · Page 4Linked to original sources
PubMed2004

cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period.

BACKGROUND: After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray. METHODS: Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR. RESULTS: In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs. CONCLUSIONS: A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation.

Animals↗
PubMed2004

Gene expression profile in experimental mesangial proliferative glomerulonephritis.

We analyzed gene expression in rat anti-Thy1 antibody-induced glomerulonephritis by using the cDNA microarray method. Ninety-seven genes that differed by more than 1.5-fold intensity in comparison with the controls were selected. Cluster analysis showed that the expression of genes associated with inflammation reached maximum levels at 24 h, while genes involved in the development of fibrosis increased at 7 days after injection. Microarray analysis of animal disease models may be a powerful approach for understanding the gene expression programs that underlie these disorders.

Animals↗
PubMed2004

Carvedilol selectively inhibits oscillatory intracellular calcium changes evoked by human alpha1D- and alpha1B-adrenergic receptors.

BACKGROUND: Increasing evidence from clinical trials indicates that carvedilol, an antagonist of alpha1- and beta-adrenergic receptors (ARs), provides an effective treatment for chronic heart failure, whereas nonselective alpha1-AR blockade has an adverse outcome in this disease. It is, however, not clear whether carvedilol exhibits a subtype-dependent impact on three distinct alpha1-adrenergic receptors (alpha1-ARs). METHODS AND RESULTS: We determined binding properties of human ARs for carvedilol using HEK293 human embryonic kidney cells expressing a single AR subtype. Our results showed that the affinities of alpha1D-AR and alpha1B-AR for carvedilol are higher than that of the beta1-AR subtype, a major target in heart failure treatment. The affinity rank order and pKi values of ARs for carvedilol were as follows: alpha1D-AR (8.9)>alpha1B-AR (8.6)>beta1-AR (8.4)>beta2-AR (8.0)>alpha1A-AR (7.9)?alpha2C-AR (5.9)>alpha2B-AR (5.5)>alpha2A-AR (5.3). Furthermore, temporal kinetics of intracellular calcium signaling mediated via alpha1D- and alpha1B-ARs, but not via alpha1A-AR (P<0.01), showed oscillatory patterns with frequencies ranging from 0.3 to 3 per minute in human smooth muscle and HEK293 cells, which were inhibited by the therapeutic concentrations of carvedilol (10 nM) in a subtype-dependent manner. When oscillatory alpha1B-AR and non-oscillatory alpha1A-AR were co-expressed and heteromer receptors were detected with bioluminescence resonance energy transfer and co-immunoprecipitation, carvedilol suppressed only oscillatory component of global cytosolic free calcium change. CONCLUSIONS: These results indicate that in addition to beta-ARs, receptor inhibition by carvedilol is directed to alpha1-ARs, preferably to alpha1D- and alpha1B-AR-mediated signaling events, including intracellular calcium oscillations in vascular smooth muscle.

Adrenergic Antagonists↗
PubMed2004

TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor.

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.

Animals↗
PubMed2004

Upregulation of the transcript level of GTPase activating protein KIAA0603 in T cells from patients with atopic dermatitis.

We have analyzed transcription profiles in peripheral blood CD3(+) cells from patients with allergic diseases to better understand the genes that are involved. Transcription levels of the gene KIAA0603/AS160 in CD3(+) cells from patients with atopic dermatitis (AD) were significantly higher than in normal individuals. The KIAA0603 gene encodes a 1299 amino acid protein with two phosphotyrosine interaction domains at the N-terminal region and a TBC domain at the C-terminal region. The region containing the TBC domain has a 31% homology to human rab6 GTPase activating protein (GAP). When human primary CD3(+) cells were stimulated with anti-CD3 or calcium ionophore, the KIAA0603 transcript level was upregulated. The marked upregulation of KIAA0607 was accompanied by activation induced cell death of primary CD3+ cells. KIAA0603 is likely to be a Rab GAP that participates in the regulation of activated T cells, especially helper memory T cells. Expression of KIAA0603 in T cells may be involved in pathogenesis of AD.

Base Sequence↗
PubMed2004

Characteristics of behavioral abnormalities in alpha1d-adrenoceptors deficient mice.

To investigate the functional role of alpha1d-adrenergic receptor (alpha1d-AR) in the CNS, we have generated mutant mice lacking the alpha1d-AR using a gene targeting approach and examined in detail the effects of alpha1d-AR knockout mice on motor function, sensory function, and learning and memory. alpha1d-AR knockout mice showed better motor coordination at the highest rotating speed of the rotarod performance and stronger muscle tone using the traction meter, but their locomotor activity and swimming ability in the water maze were not affected. In the water maze requiring reference memory, alpha1d-AR knockout mice showed normal spatial learning. In the Y-maze task requiring working memory or attention, alpha1d-AR knockout mice displayed an impaired spontaneous alternation performance. The alpha1d-AR knockout mice tended to display lower levels of acoustic startle responses than the wild-type group at lower pulse intensities, although the acoustic prepulse inhibition was not impaired in the alpha1d-AR knockout mice. Furthermore, the NMDA receptor antagonist, MK-801-induced deficits of acoustic prepulse inhibition were not observed in the alpha1d-AR knockout mice. These results clearly demonstrate that the alpha1d-AR receptor plays an important role in the process of auditory sensory function, attention or working memory rather than reference memory, and the sensorimotor gating deficits induced by the NMDA receptor antagonist.

Acoustic Stimulation↗
PubMed2004

Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling.

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.

Animals↗
PubMed2004

Gene expression profiling identifies platelet-derived growth factor as a diagnostic molecular marker for papillary thyroid carcinoma.

PURPOSE: Cancer diagnostics and therapeutics are often based on clinically relevant markers that are expressed specifically in a malignant tissue at levels higher than in normal tissue. We examined potential markers for papillary thyroid carcinoma (PTC) by monitoring PTC-specific gene expression using cDNA microarray. EXPERIMENTAL DESIGN: Gene expression profiles for PTC tissue, normal thyroid tissue, and healthy peripheral blood cells were compared by use of a human 4000-gene cDNA microarray. Protein expressions of the up-regulated genes in PTC were examined in thyroid tissues by immunohistochemistry. RESULTS: Sixty-four genes were overexpressed in PTC tissue relative to normal thyroid tissue and healthy peripheral blood cells. The genes that were up-regulated in PTC were involved in cell cycle regulation, DNA damage response, angiogenesis, and oncogenesis. Among these genes, basic fibroblast growth factor and platelet-derived growth factor were identified by immunochemical methods as proteins that are specifically expressed at high levels in thyroid neoplasms. Basic fibroblast growth factor, which has been identified as a biomarker for PTC, was overexpressed in 54% of PTC cases, 67% of follicular thyroid carcinomas, and 36% of benign thyroid neoplasms. Platelet-derived growth factor was overexpressed in 81% of PTC cases and 100% of follicular carcinomas, but was immunonegative in normal thyroid tissues and benign thyroid neoplasms. CONCLUSIONS: Platelet-derived growth factor may be a potential biomarker for PTC and follicular carcinoma. Expression profile analysis using a microarray followed by immunohistochemical study can be used to facilitate the development of molecular biomarkers for cancer.

Carcinoma, Papillary↗
PubMed2004

V1a receptor knockout mice exhibit impairment of spatial memory in an eight-arm radial maze.

In this study, we examined the performance of vasopressin V1a receptor (V1aR) and vasopressin V1b receptor (V1bR) knockout (KO) mice compared to wild-type (WT) mice in an eight-arm radial maze. V1aR KO mice exhibited an impairment of spatial memory in comparison to WT mice. By contrast, we did not observe any significant differences between the V1bR KO mice and the WT mice in the eight-arm radial maze. Moreover, OPC-21268, a selective V1aR antagonist, impaired spatial memory in the eight-arm radial maze in WT mice characterized by an increased number of errors. These results suggest that the V1aR controls spatial memory in mice.

Analysis of Variance↗
PubMed2004

Pregnancy-associated changes in genome-wide gene expression profiles in the liver of cow throughout pregnancy.

The objective of the present study was to fabricate and use a bovine liver complementary DNA (cDNA) microarray to profile genome-wide gene expressions in the liver of cow throughout pregnancy. A cDNA library was prepared from liver total RNA collected from cows during estrous cycle and pregnancy, and from fetuses at different stages of pregnancy. The sequenced clones were compiled and annotated by basic local alignment search tool (BLASTn) and spotted onto glass slides. The annotated liver array represented 2675 genes. Of which, 1442 were known genes while 617 sequences had matches with sequences found in expressed sequence tags databases. In addition, 616 unknown sequences were found and these sequences may possibly be identified as candidates for novel bovine genes. For gene expression profiling studies, total RNA from livers of cows slaughtered on days 19, 27-28, 49-58, 150, and 245 of pregnancy (test RNAs) was separately reverse transcribed and labeled with either cyanine 5-fluorescent dye (Cy5) or Cy3. The test samples were individually compared with liver total RNA collected from nonpregnant cycling cows (control RNA) after reverse transcription and labeling with the opposite dye following a two-color hybridization method. After scanning, image acquisition, and normalization, genes that showed either more than 1.5-fold (test/control) induction or repression were selected for further analyses. Hierarchical clustering algorithm showed a clear induction of most liver genes on days 27-28 of pregnancy. Self-organizing maps algorithm identified groups of genes whose differential expression patterns were similar across pregnancy. In conclusion, we described fabrication of a bovine liver cDNA microarray, and demonstrate, for the first time, differential expression patterns of a large number of coregulated liver genes in parallel throughout pregnancy in the bovine.

Algorithms↗
PubMed2004

Vasopressin stimulates insulin release from islet cells through V1b receptors: a combined pharmacological/knockout approach.

Vasopressin receptor subtype(s) responsible for stimulation of insulin release from pancreatic beta cells were investigated by using subtype-selective antagonists and mice that were genetically lacking either V1a or V1b receptors. Arginine vasopressin (AVP) increased insulin release from isolated mouse islet cells in a concentration-dependent manner, with a submaximal response at 100 nM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis detected V1b and oxytocin, but not V1a or V2, receptor transcripts in mouse islet cells. We characterized the recently synthesized vasopressin receptor subtype antagonists (2S)1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-difydro-1H-indole-2-carbonyl)-pyrrolidine-2-carboxamide] (SR49059), 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone (OPC-21268), and (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415) using human embryonic kidney 293 cells stably expressing the three cloned mouse vasopressin receptors (V1a, V1b, and V2). A radioligand binding study showed that SR49059 and OPC-21268 potently inhibited [3H]AVP binding to the cloned mouse V1a receptor, with Ki values of 27 and 510 nM, respectively, whereas SSR149415 potently inhibited [3H]AVP binding to the cloned mouse V1b receptor with a Ki value of 110 nM. The inhibitory effects of vasopressin antagonists on AVP-induced insulin release correlate well with the rank order of potency to inhibit [3H]AVP binding to the V1b receptor; pancreatic islet cells were significantly inhibited by SSR149415 but not by SR49059 or OPC-21268. Furthermore, the AVP effect on insulin release was entirely lost in mice lacking the V1b receptor but was preserved in mice lacking the V1a receptor. Our study, which combined pharmacological and knockout approaches, clearly demonstrates that vasopressin-stimulated insulin release from islet cells is mediated via V1b receptors.

Animals↗
PubMed2004

Expression of a human SOCS protein, HSOCP-1, in peripheral blood eosinophils from patients with atopic dermatitis.

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34(SEI-1) was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16(INK4), suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.

Apoptosis↗
PubMed2004

The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions.

The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR-/-). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR-/- mice compared with WT mice (V1bR+/+). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR-/- mice, while corticotropin-releasing hormone-stimulated ACTH release in the V1bR-/- mice was not significantly different from that in the V1bR+/+ mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR-/- mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR-/- mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions.

Adrenocorticotropic Hormone↗
PubMed2004

[Heterogeneity of G protein-coupled receptor generated by post-translational mechanisms and its clinical meanings].

G protein-coupled receptors (GPCRs) are the most famous target proteins for medicinal drugs. So far, heterogeneity of GPCRs is mainly focused on genetic variation. However, it has been reported that the structure and function of GPCRs are modified by several mechanisms after translation. RNA editing introduces the amino acid different from that encoded in genome by changing the nucleotide. Dimer formation is another example of how heterogeneity is produced. Many receptors form homo- or hetero-dimers, and obtain different function from original receptors. Receptors are regulated by several means to modulate stimulation strength. Receptor subtype is often differentially regulated by receptor kinases and/or second messenger-regulated kinases. There is a new type of receptor that shows a novel structural feature, a long amino terminal region belonging to class B seven transmembrane receptors. The physiological function of this class of receptor is assumed to play a role in cell-cell communication. This novel structural feature may directly link GPCR to the cytoskeleton. These mechanisms to produce functional and structural heterogeneity may explain how cells evoke different responses in different tissues or cells upon the same stimulation. Thus, the post-translational mechanism to produce heterogeneity provides additional flexibility when cells respond to one extracellular stimulus.

Animals↗
PubMed2003

Crystallization and preliminary X-ray diffraction studies of DNA decamer d(CCAGGCCTGG) complexed with cobalt(III)-pepleomycin.

Crystals of the self-complementary oligonucleotide d(CCAGGCCTGG) [Heinemann & Alings (1989), J. Mol. Biol. 210, 369-381] complexed with the hydroperoxide of cobalt-pepleomycin (CoPEP) were obtained by the hanging-drop vapour-diffusion method at 298 K. An X-ray diffraction data set was collected to 2.8 A at 100 K. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.52, b = 59.88, c = 72.93 A.

Antibiotics, Antineoplastic↗
PubMed2003

Responses to noxious stimuli in mice lacking alpha(1d)-adrenergic receptors.

Nociceptive behaviors were examined in the mice lacking alpha1d-adrenergic receptor (alpha1d-AR) and wild type littermates using tail-flick, hot-plate (hindpaw-licking and jumping), tail-pinch and formalin tests. The distribution of alpha1d-AR was studied using in situ hybridization in the wild type mice. Mutant mice showed longer tail-flick and hindpaw-licking latencies while their jumping latency was shorter. Mechanical and chemical nociception was not altered in alpha1d-knockout mice. In situ hybridization study revealed dense alpha1d-AR mRNA expression in the reticular thalamic nucleus, the hippocampus, the cingulate cortex and the spinal cord. These results suggest that alpha1d-AR in the spinal cord contributes to thermal pronociception; and that the jump behavior seen when escaping from heat is inhibited via the supraspinal alpha1d-AR.

Animals↗