Biomedical subjects

I Tanaka

Publications and source records attributed to I Tanaka.

At least 37 records · Page 2Linked to original sources

PubMed2001

Altered expression of HES-1, BETA2/NeuroD, and PDX-1 is involved in impaired insulin synthesis induced by glucocorticoids in HIT-T15 cells.

Expression of the insulin gene is highly specific to pancreatic beta cells and is upregulated mainly by PDX-1 and BETA2/NeuroD depending on the extracellular glucose concentration. However, its downregulation has not been well studied. Reporter gene analyses using pancreatic HIT-T15 cells revealed that the glucose-dependent insulin promoter activity was blocked by glucocorticoids, dexamethasone (DEX) and hydrocortisone, in a dose-dependent manner. After the addition of DEX (20 nM) to HIT-T15 cells, a decrease of insulin mRNA was observed at 12-24 h, followed by a decline of insulin protein at 48 h. Expressions of PDX-1 and BETA2/NeuroD decreased within 2 h. HES-1, a potent negative regulator of bHLH-type transcription factors, was found to be expressed in HIT-T15 cells, and its expression was increased 6 h after the addition of DEX. Overexpression of HES-1 suppressed the insulin promoter activity in a dose-dependent manner. These results suggest that glucocorticoids impair insulin synthesis in HIT-T15 cells by decreasing PDX-1 and BETA2/NeuroD and that enhancement of HES-1 expression is involved in this regulation.

Animals↗

PubMed2001

Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Animals↗

PubMed2001

Direct observation of deuterium migration in crystalline-state reaction by single-crystal neutron diffraction. III. Photoracemization of 1-cyanoethyl cobaloxime complexes.

The H atoms bonded to the chiral C atoms (stereogenic center) of the 1-cyanoethyl groups in two cobalt complexes, [(R)-1-cyanoethyl]bis(dimethylglyoximato)(pyridine)cobalt(III) (2) and [(R,S)-1-cyanoethyl]bis(dimethylglyoximato)(piperidine)cobalt(III) (3), were replaced with D atoms, such as Co--C*D(CH(3))CN. The crystals of the two cobalt complexes were irradiated with a xenon lamp for 72 h and 27 d, respectively. The unit-cell dimensions were gradually changed with retention of the single-crystal form. The crystal structures after irradiation were determined by neutron diffraction. In each crystal the chiral 1-cyanoethyl group of one of the two crystallographically independent molecules was partly inverted to the opposite configuration, whereas that of the other molecule kept the original configuration. The C*--D bond in the inverted group was completely conserved in the process of the inversion of the chiral alkyl group. This suggests that the inversion of the chiral 1-cyanoethyl group proceeds with the rotation of the cyanoethyl radical after the Co--C bond cleavage by photo-irradiation so that the opposite side of the radical faces the Co atom. This is followed by recombination of the Co--C bond to form the inverted 1-cyanoethyl group.

Journal Article↗

PubMed2001

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the human calcium-binding protein MRP14 (S100A9).

MRP14 is a protein that is specifically expressed in myeloid and epithelial cells during the stages of acute or chronic inflammatory states such as rheumatoid arthritis or sarcoidosis. MRP14 has EF-hand motifs as Ca(2+)-binding sites and belongs to the S100 family of proteins. This paper deals with the sample preparation (cloning, overexpression and purification), crystallization and preliminary crystallographic analysis of recombinant human MRP14. Crystals of MRP14 were obtained by the hanging-drop vapour-diffusion method. MRP14 crystals belong to space group P2(1), with unit-cell parameters a = 57.59, b = 178.44, c = 61.23 A, beta = 113.17 degrees, and diffract to 2.1 A resolution.

Amino Acid Sequence↗

PubMed2001

Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats.

Glucagon-like peptide-1 (GLP-1) is an incretin, which induces glucose-dependent insulin secretion. GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. We investigated whether DPPIV-deficient F344/DuCrj rats show improved glucose tolerance when compared with DPPIV-positive F344/Jcl rats. Oral glucose tolerance test indicated improved glucose tolerance in F344/DuCrj rats, but blood glucose levels of the two strains were almost the same 120 min after the glucose bolus. Valine-pyrrolidide, a DPPIV inhibitor, had no effect on the glucose tolerance of F344/DuCrj rats, but improved that of F344/Jcl rats. Enhanced insulin secretion and high plasma active GLP-1 levels were detected in an intraduodenal glucose tolerance test. Glucose tolerance is improved in DPPIV-deficient F344/DuCrj rats via enhanced insulin release mediated by high active GLP-1 levels. Our results suggest that DPPIV inhibition is a rational strategy to treat diabetic patients by improving glucose tolerance with low risk of hypoglycemia.

Animals↗

PubMed2001

The MinD protein from the hyperthermophilic archaeon Pyrococcus horikoshii: crystallization and preliminary X-ray analysis.

MinD is one of the proteins regulating cell division. MinD from Escherichia coli has been designated as a type of motor protein which has an ATPase activity. This paper deals with the first crystallization and preliminary crystallographic analysis of recombinant MinD from Pyrococcus horikoshii (molecular weight 26.3 kDa) expressed in E. coli. Crystals of MinD were obtained by the hanging-drop vapour-diffusion method. MinD crystals belong to space group P2(1)3, with unit-cell parameters a = b = c = 98.5 A, and diffract to 3.0 A resolution. The asymmetric units each contain one molecule of MinD, giving a crystal volume per protein mass (V(M)) of 3.0 A(3) Da(-1) and a solvent content of 59.0%.

Adenosine Triphosphatases↗

PubMed2001

Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav 2.2) of N-type calcium channels.

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the alpha(1B) subunit (Ca(v) 2.2). The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Animals↗

PubMed2001

Morphology of pulmonary rapidly adapting receptor relay neurons in the rat.

The term rapidly adapting pulmonary stretch receptor (RAR) refers to one of the major pulmonary sensory receptors that responds to inflation and deflation of the lungs as well as to irritant stimuli with rapidly adapting irregular discharges. The functional role and central pathways are largely unknown. The aim of this study was to elucidate morphological characteristics of second-order neurons (RAR cells) activated by vagal afferent fibers originating from RARs. A mixture of horseradish peroxidase (HRP) and Neurobiotin was injected intracellularly into physiologically identified RAR cells in Nembutal-anesthetized, immobilized, and artificially ventilated Wister rats. Direct visualization of individual RAR cells (n = 12), including their somata, dendritic arborizations, and fine axonal branches with terminal boutons, was possible for the first time. Their somata were located in the commissural or medial subdivision of the nucleus of the solitary tract, caudal to the level of the area postrema. The RAR cells had, in addition to dendrites extending into the NTS area, one or two long dendrites extending laterally and/or ventrolaterally into the medullary reticular formation. The stem axons issuing from the RAR cells first coursed ventrolaterally toward the reticular formation in the vicinity of the ambiguus nucleus and then bifurcated into ascending and descending axons: three RAR cells possessed only ascending axons. Some of the ascending axons could be traced as far as the level of the facial nucleus and some of the descending axons beyond the spinomedullary junction. These ascending and/or descending axons gave off extensive axon collaterals distributing boutons within and in the vicinity of the ambiguus nucleus. These results, showing an anatomical substrate for the network implicated in RAR-evoked reflexes, provide useful clues for study of the RAR system.

Afferent Pathways↗

PubMed2001

Enhanced expression of the early retrotransposon in C3H mouse-derived myeloid leukemia cells.

Cells of acute myeloid leukemia (AML) from C3H/He mice express an increased amount of RNA for an endogenous retrovirus-like retrotransposon, intracisternal A-particle element. We analyzed the transcription of other mouse retrotransposons in C3H-derived tumor cells and found that all the AML lines from different mice overexpress early-transposon (ETn) RNA. In contrast, only faint levels of ETn were detected in the cells from other tumors, including hepatoma and lymphoma. The polyadenylation sites of the ETn RNA in the AML cells varied. We also determined the binding site for the nuclear extract of the AML cells in the long terminal repeat sequence of ETn. The overexpression of ETn as a common phenotype of AML cells suggests that myeloid cells with this phenotype are the origin of all the AML cells or that the phenotype is acquired during leukemogenesis.

Animals↗

PubMed2001

Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C-mediated proteolysis.

Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with K(d) values of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation.

Antigen-Antibody Complex↗

PubMed2001

2-Alkynyl-8-aryl-9-methyladenines as novel adenosine receptor antagonists: their synthesis and structure-activity relationships toward hepatic glucose production induced via agonism of the A(2B) receptor.

Novel adenosine antagonists, 2-alkynyl-8-aryl-9-methyladenine derivatives, were synthesized as candidate hypoglycemic agents. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. In general, aromatic moieties at the 8-position and alkynyl groups at the 2-position had significantly increased activity compared to unsubstituted compounds. The preferred substituents at the 8-position of adenine were the 2-furyl and 3-fluorophenyl groups. In modifying the alkynyl side chain, change of the ring size, cleavage of the ring, and removal of the hydroxyl group were well tolerated. The order of the stimulatory effects of adenosine agonists on rat hepatocytes was NECA > CPA > CGS21680, which is consistent with involvement of the A(2B) receptor. In Chinese hamster ovary cells stably transfected with human A(2B) receptor cDNA, one of the compounds potent in hepatocytes, 15o (IC(50) = 0.42 microM), antagonized NECA-induced stimulation of cyclic AMP production (IC(50) = 0.063 microM). This inhibitory effect was much more potent than those of FK453, KF17837, and L249313 which have been reported to be respectively A(1), A(2A), and A(3) selective antagonists. These findings agree very well with the result that, compared to 15o, these selective antagonists for each receptor subtype showed only marginal effects in rat hepatocytes. These results suggest that adenosine agonist-induced glucose production in rat hepatocytes is mediated through the A(2B) receptor. Furthermore, 15o showed hypoglycemic activity in an animal model of noninsulin-dependent diabetes mellitus, the KK-A(y) mice. It is possible that inhibition of hepatic glucose production via the A(2B) receptor could be at least one of the mechanisms by which 15o exerts its in vivo effects. Further elaboration of this group of compounds may afford novel antidiabetic agents.

Adenine↗

PubMed2001

Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity.

The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.

Amino Acid Sequence↗

PubMed2001

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma.

Genomic imprinting is a parental origin-specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

Adenocarcinoma↗

PubMed2001

Role of adrenomedullin and its receptor system in renal pathophysiology.

Adrenomedullin (AM), a potent vasorelaxing, natriuretic and cell growth-modulating peptide, is thought to act as an autocrine/paracrine regulator in renal glomeruli and tubules. AM receptors comprise the calcitonin receptor-like receptor (CRLR) and a family of receptor-activity-modifying proteins (RAMPs 1-3); however, the pathophysiological role of AM and its receptor system in the kidney remains to be clarified. We examined the regulation of their expression in a rat model of renal injury and found that RAMP1, RAMP2 and CRLR expressions were markedly upregulated upon induction of fibrosis during obstructive nephropathy. Since AM exerts potent antiproliferative effects in various cell types, upregulation of the AM receptor system may play important roles in modulating the progression of renal diseases.

Adrenomedullin↗

PubMed2001

Electron-energy-loss near edge structures of six-fold-coordinated Zn in MgO.

Electron-energy-loss near edge structures (ELNES) at the Zn-L(2,3) edge and the O-K edge have been measured for 10 mol% ZnO-doped MgO, and were compared with spectra from reference materials. In order to interpret the spectra, first principles molecular orbital calculations were made using model clusters composed of 125 and 153 atoms. Photoabsorption cross sections (PACS) were computed at the Slater's transition state in which a half-filled core hole was included in the self-consistent calculations. The difference in the coordination numbers of Zn was found well distinguishable by the Zn-L(2,3)-edge ELNES. The experimental spectra in the first 25 eV were well reproduced by the theoretical PACS. In this energy region, the Zn-L(2,3)-edge ELNES from four-fold coordinated Zn showed four sets of peaks, whereas the six-fold coordinated Zn exhibits three sets of peaks. The origin of these peaks can be explained by the point symmetry within the first coordination unit. A small shift toward the lower energy side was observed in the O-K edge ELNES of the ZnO-doped MgO as compared with pure MgO. This can be ascribed to the lower energy of the Zn-4s orbital as compared with the Mg-3s orbital, which is the common mechanism to the difference in the band gap between MgO and ZnO.

Journal Article↗

PubMed2001

2-Alkynyl-8-aryladenines possessing an amide moiety: their synthesis and structure-activity relationships of effects on hepatic glucose production induced via agonism of the A(2B) adenosine receptor.

A series of 2-alkynyl-8-aryladenine derivatives bearing an amide moiety at the 9-position of adenine was synthesized. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. The m-primary benzamide derivative 15f was the most potent compound (IC(50)=0.017 microM), being 15-fold more active than the corresponding 9-methyl derivative (1). Compound 15f showed 72- and 5.2-fold selectivity for human A(2B) receptor versus human A(1) and A(2A) receptors, respectively. Structure-activity relationship (SAR) studies of the synthesized compounds indicated that a three-carbon linker, fixed in the form of a benzene ring, between the adenine core and the amide moiety is important for both A(2B) antagonistic activity and selectivity. The IC(50) values in rat hepatocyte glucose assay correlated well with the IC(50) values in cAMP assay using Chinese hamster ovary cells stably transfected with human A(2B) receptors (r(2)=0.94). The A(1) and A(2A) affinities showed no correlation with the potency to inhibit NECA-induced glucose production. These results strongly support our previous conclusion that adenosine agonist-induced hepatic glucose production in rat hepatocytes is mediated through the A(2B) receptor.

Adenine↗

PubMed2001

The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP.

BACKGROUND: In Escherichia coli, the cell division site is determined by the cooperative activity of min operon products MinC, MinD, and MinE. MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the supressor of MinC. MinD plays a multifunctional role. It is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE. MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed by fluorescent microscopy. RESULTS: The three-dimensional structure of the MinD-2 from Pyrococcus horikoshii OT3 (PH0612) has been determined at 2.3 A resolution by X-ray crystallography using the Se-Met MAD method. The molecule consists of a beta sheet with 7 parallel and 1 antiparallel strands and 11 peripheral alpha helices. It contains the classical mononucleotide binding loop with bound ADP and magnesium ion, which is consistent with the suggested ATPase activity. CONCLUSIONS: Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the P loop-containing nucleotide triphosphate hydrolase superfamily of proteins. Unlike nitrogenase or other member proteins that normally work as a dimer, MinD was present as a monomer in the crystal. Both the 31P NMR and Malachite Green method exhibited relatively low levels of ATPase activity. These facts suggest that MinD may work as a molecular switch in the multiprotein complex in bacterial cell division.

Adenosine Diphosphate↗

PubMed2001

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Amino Acid Sequence↗