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Biomedical subjects

Isao Tanaka

Publications and source records attributed to Isao Tanaka.

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

Metformin causes reduction of food intake and body weight gain and improvement of glucose intolerance in combination with dipeptidyl peptidase IV inhibitor in Zucker fa/fa rats.

An incretin hormone, glucagon-like peptide-1 (GLP-1), has been shown to lower plasma glucose via glucose-dependent insulin secretion and to reduce appetite. We previously found that the biguanide metformin, an antidiabetic agent, causes a significant increase of plasma active GLP-1 level in the presence of dipeptidyl peptidase IV (DPPIV) inhibitor in normal rats. This finding suggested that the combination treatment might produce a greater antidiabetic and anorectic effect, based on enhanced GLP-1 action. In this study, we assessed the effects of subchronic treatment with metformin and a DPPIV inhibitor, valine-pyrrolidide (val-pyr), on glycemic control, food intake, and weight gain using Zucker fa/fa rats, a model of obesity and impaired glucose tolerance. The combination treatment caused a significant increase of GLP-1 level in Zucker fa/fa rats. In a subchronic study, val-pyr, metformin, or both compounds were administered orally b.i.d. for 14 days. The combination treatment significantly decreased food intake and body weight gain, although neither metformin nor val-pyr treatment alone had any effect. In an oral glucose tolerance test on day 1, the coadministration caused a greater improvement of glucose tolerance and a prominent increase of plasma active GLP-1 without marked insulin secretion. The 14-day combination treatment produced a potent reduction of fasting blood glucose and plasma insulin levels. These results demonstrate that the combination therapy of metformin with DPPIV inhibitor leads to reduced food intake and body weight gain, most likely through the significant increase of plasma GLP-1 level. The combination therapy seems to be a good candidate for treatment of type 2 diabetes with obesity.

Animals↗
PubMed2004

The combination of metformin and a dipeptidyl peptidase IV inhibitor prevents 5-fluorouracil-induced reduction of small intestine weight.

Glucagon-like peptide 2 (GLP-2), which has intestinotrophic effects, is secreted from L-cells in the intestine in response to nutrient ingestion and is degraded by dipeptidyl peptidase IV (DPPIV). In this report, we show that biguanides promote GLP-2 release. Plasma GLP-2 levels were significantly increased by 1.4- to 1.6-fold in fasted F344 rats 1 h after oral meformin (300 mg/kg), phenformin (30 and 100 mg/kg) and buformin (100 mg/kg) treatment. In addition, metformin administration (300 mg/kg, p.o.) significantly elevated plasma GLP-2 in fasted CD-1 mice by about 2.0-fold 1 and 3 h after the treatment. Metformin and/or valine-pyrrolidide, a DPPIV inhibitor, was orally given (300 and 30 mg/kg, respectively, p.o., b.i.d., 3 days) to BALB/c mice treated with 5-fluorouracil (5-FU; 60 mg/kg, s.i.d.), which induces gastrointestinal damage leading to a reduction of small intestine wet weight. Metformin and valine-pyrrolidide co-administration prevented the 5-FU-induced reduction of wet weight of the small intestine, whereas metformin or valine-pyrrolidide alone had no effect. These results suggest that GLP-2 is co-secreted with GLP-1 flollowing biguanide stimulation, and that the combination of metformin with a DPPIV inhibitor might a useful oral treatment for gastrointestinal damage, based on GLP-2 actions.

Animals↗
PubMed2004

Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.

Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos.

Amino Acid Sequence↗
PubMed2004

Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA.

CutA is a small protein that appears to be involved in the mechanism of divalent metal cation tolerance in microorganisms. Here we report the crystal structure of Pyrococcus horikoshii CutA (PhoCutA), with and without Cu(2+), and its metal-binding properties. Crystallographic analyses revealed that PhoCutA forms a stable trimeric structure with intertwined antiparallel beta-strands. The crystal structure of the Cu(2+)-PhoCutA complex shows that the Cu(2+) is located at a trimer-trimer interface and is recognized by the side chains of one Asp(48) from each trimer. In an in vitro experiment, PhoCutA bound to several heavy metals, most of which led to reversible aggregation of the protein; i.e. the aggregates could be completely solubilized by addition of ethylenediamine tetraacetic acid (EDTA) or dialysis against metal free buffer. Substitution of Asp(48) with Ala led to a decrease in the amount of aggregates, suggesting the significant contribution of Asp(48) to the reversible aggregation. To the best of our knowledge, this is the first report which provides the structural evidence for heavy metal-induced multimerization of a protein.

Amino Acid Sequence↗
PubMed2004

Crystal structure of archaeal ribonuclease P protein Ph1771p from Pyrococcus horikoshii OT3: an archaeal homolog of eukaryotic ribonuclease P protein Rpp29.

Ribonuclease P (RNase P) is the endonuclease responsible for the removal of 5' leader sequences from tRNA precursors. The crystal structure of an archaeal RNase P protein, Ph1771p (residues 36-127) from hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined at 2.0 A resolution by X-ray crystallography. The structure is composed of four helices (alpha1-alpha4) and a six-stranded antiparallel beta-sheet (beta1-beta6) with a protruding beta-strand (beta7) at the C-terminal region. The strand beta7 forms an antiparallel beta-sheet by interacting with strand beta4 in a symmetry-related molecule, suggesting that strands beta4 and beta7 could be involved in protein-protein interactions with other RNase P proteins. Structural comparison showed that the beta-barrel structure of Ph1771p has a topological resemblance to those of Staphylococcus aureus translational regulator Hfq and Haloarcula marismortui ribosomal protein L21E, suggesting that these RNA binding proteins have a common ancestor and then diverged to specifically bind to their cognate RNAs. The structure analysis as well as structural comparison suggested two possible RNA binding sites in Ph1771p, one being a concave surface formed by terminal alpha-helices (alpha1-alpha4) and beta-strand beta6, where positively charged residues are clustered. A second possible RNA binding site is at a loop region connecting strands beta2 and beta3, where conserved hydrophilic residues are exposed to the solvent and interact specifically with sulfate ion. These two potential sites for RNA binding are located in close proximity. The crystal structure of Ph1771p provides insight into the structure and function relationships of archaeal and eukaryotic RNase P.

Amino Acid Sequence↗
PubMed2003

Flash-cooling of macromolecular crystals in a capillary to overcome increased mosaicity.

This paper describes the usefulness of flash-cooling in a capillary (FCC) for X-ray diffraction data collection from macromolecular crystals. FCC may be applied when conventional cooling using a cryoloop fails. This technique cools crystals in a capillary instead of in a cryoloop and thus cools crystals more slowly than conventional cooling. Measurements of cooling rates have shown that the time taken to reach the final temperature is two to eight times longer using FCC than using a cryoloop, depending on the volume of cryoprotectant solution around the crystal. Using this cooling technique, the crystal structures of isocitrate dehydrogenase and protein L-isoaspartate O-methyltransferase have been solved at 1.95 and at 2.50 A resolution, respectively. Both crystals could not be cooled by the conventional method using a cryoloop. Moreover, diffraction data from crystals of the hypothetical proteins PH-A and PH-B were also collected successfully using the FCC method. These results show that some crystals, especially larger ones, need to be cooled slowly.

Crystallography, X-Ray↗
PubMed2003

High-density electron anions in a nanoporous single crystal: [Ca24Al28O64]4+(4e-).

We removed approximately 100% of clathrated oxygen ions from the crystallographic cages in a single crystal of 12CaO.7Al2O3, leading to the formation of high-density (approximately 2 x 10(21) cm-3) electrons highly localized in the cages. The resulting electron forms a structure that we interpret as an F+ center and migrates throughout the crystal by hopping to a neighboring cage with conductivity approximately 100 siemens per centimeter, demonstrating that the encaged electron behaves as an anion. The electron anions couple antiferromagnetically with each other, forming a diamagnetic pair or singlet bipolaron. The resulting [Ca24Al28O64]4+(4e-) may be regarded as a thermally and chemically stable single crystalline "electride."

Journal Article↗
PubMed2003

Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction.

The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1). covalent-bonded external aldimine with the coenzyme in the K51T form and 2). the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.

Binding Sites↗
PubMed2003

Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition, catalysis, and evolution.

NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the beta-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of beta-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.

Amino Acid Sequence↗
PubMed2003

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity.

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

Crystallography, X-Ray↗
PubMed2003

Insight into a natural Diels-Alder reaction from the structure of macrophomate synthase.

The Diels-Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions. It has been applied to the synthesis of complex pharmaceutical and biologically active compounds. Although evidence on natural Diels-Alderases has been accumulated in the biosynthesis of secondary metabolites, there has been no report on the structural details of the natural Diels-Alderases. The function and catalytic mechanism of the natural Diels-Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels-Alder adducts. Here we present the 1.70 A resolution crystal structure of the natural Diels-Alderase, fungal macrophomate synthase (MPS), in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.

Alcohols↗
PubMed2003

Functional characterization of the adenosine receptor contributing to glycogenolysis and gluconeogenesis in rat hepatocytes.

The adenosine receptor subtype mediating glucose production by glycogenolysis and gluconeogenesis was studied in primary cultured rat hepatocytes. Adenosine and adenosine agonists caused cyclic AMP accumulation in rat hepatocytes. The order of potency was 5'-N-ethylcarboxamidoadenosine (NECA)>R(-)-N(6)-(2-phenylisopropyl)adenosine (RPIA)>adenosine>2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Furthermore, adenosine agonists stimulated glycogenolysis and gluconeogenesis. The order of potency was NECA>RPIA>CGS21680. The rank order of potency is typical for adenosine A(2B) receptors. Glycogenolysis stimulated by NECA was fully inhibited by nonselective adenosine antagonists, 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943). However, the adenosine A(2A) receptor-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC), and the adenosine A(1) receptor-selective antagonist, (+)-(R)-[(E)-3-(2-phenylpyrazolo[1,5-alpha]pyridin-3-yl)acryloyl]-2-piperidine ethanol (FK453), had a low inhibitory potency. A strong correlation was found between the inhibitory effect of adenosine antagonists on NECA-induced glucose production and that on intracellular cyclic AMP generation in rat hepatocytes. Our results suggest that adenosine stimulates cyclic AMP formation and regulates glycogenolysis and gluconeogenesis, most likely through the adenosine A(2B) receptor subtype in rat hepatocytes.

Animals↗
PubMed2003

Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations. Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system. For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value. The value of epsilon;(c) increased with an increase in HU concentration. Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex. The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations. Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU.

Bacterial Proteins↗
PubMed2003

Theoretical prediction of ELNES/XANES and chemical bondings of AlN polytypes.

The first principles calculations of ELNES/XANES of AlN polytypes were carried out by the first-principles OLCAO method using large supercells composed of more than 100 atoms. It can quantitatively reproduce the experimental spectra from wurtzite AlN using a 108-atoms supercell. ELNES from rock-salt and zinc-blend AlN were predicted by using 128 atoms supercells. The spectral features of rock-salt phase are different from other phases, whereas that of zinc-blend phase have numerous similarities with that from wurtzite AlN. Characteristic differences between the wurtzite and zinc-blend phases are predicted to appear at the first peak of Al L(2,3) and K edges. The first peak of zinc-blend AlN is broader than that of wurtzite AlN. The same tendency was found in the case of SiC. In order to elucidate the cause of the broadness at the first peak, partial density of states and chemical bondings were investigated. The theoretical analysis revealed that the broadness of the first peak is related to the covalency of the compounds. This result suggests that the spectral features at the first peak of L(2,3) and K edges contain information about the covalency at the illuminated area.

Journal Article↗
PubMed2003

Identification of ultradilute dopants in ceramics.

The properties of ceramic materials are strongly influenced by the presence of ultradilute impurities (dopants). Near-edge X-ray absorption fine structure (NEXAFS) measurements using third-generation synchotron sources can be used to identify ultradilute dopants, provided that a good theoretical tool is available to interpret the spectra. Here, we use NEXAFS analysis and first-principles calculations to study the local environments of Ga dopants at levels of 10 p.p.m in otherwise high-purity MgO. This analysis suggests that the extra charge associated with substitutional Ga on a Mg site is compensated by the formation of a Mg vacancy. This defect model is then confirmed by positron lifetime measurements and plane-wave pseudopotential calculations. This powerful combination of techniques should provide a general method of identifying the defect states of ultradilute dopants in ceramics.

Ceramics↗
PubMed2003

Enteroinsular axis of db/db mice and efficacy of dipeptidyl peptidase IV inhibition.

In type 2 diabetic patients, the administration of glucagon-like peptide-1 (GLP-1), known as an incretin, exerts antidiabetic effects. However, GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. DPPIV inhibition is thought to be a rational strategy to treat type 2 diabetes. In this study, using C57BLKS/J-db/db (db/db) mice as a model of type 2 diabetes, we examined the effect of acute DPPIV inhibition on glucose tolerance at the early and later stages of diabetes, determining plasma active GLP-1 and insulin levels. In addition, we investigated changes of plasma DPPIV activity. Compared with normal C57BL6/J (B6) and db/+ mice, significantly increased plasma DPPIV activities were observed in db/db mice. Expression of the proglucagon gene encoding GLP-1 was significantly upregulated in the colon of db/db mice. The administration of valine-pyrrolidide, a DPPIV inhibitor, resulted in potentiated insulin secretion mediated by increased endogenous GLP-1 action, leading to improved glucose tolerance in db/db mice at 6 weeks of age. However, although acute DPPIV inhibition with valine-pyrrolidide resulted in higher plasma active GLP-1 and insulin levels in db/db mice at 23 weeks of age, it did not improve glucose tolerance. The function of the enteroinsular axis is preserved in both stage of diabetes and the DPPIV inhibitor potentiated it, but the progression of insulin resistance appeared to block the improvement of glucose tolerance through DPPIV inhibition. Our results suggest that DPPIV inhibition is a suitable approach for treatment of impaired glucose tolerance (IGT), and type 2 diabetes in the early stage.

Actins↗