Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme

Recent studies have implied that the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid. PhIPMI is thus considered a key to understanding the fundamental metabolism of the earliest organisms. We describe for the first time the crystal structure of PhIPMI-s, which displays dual substrate specificity. The crystal structure unexpectedly shows that four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. Although the overall fold of the PhIPMI-s monomer is related closely to domain 4 of the aconitase (ACN), one alpha-helix in the ACN structure is replaced by a short loop with relatively high temperature factor values. Because this region is essential for discriminating the structurally similar substrate based on interactions with its diversified gamma-moiety, the loop structure in the PhIPMI-s must be dependent on the presence of a substrate. The flexibility of the loop region might be a structural basis for recognizing both hydrophobic and hydrophilic gamma-moieties of two distinct substrates, isopropylmalate and homocitrate.     

Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.     

Enhancement of depressed lymphokine activated killer cell activity in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) patients can be divided into two groups according to the degree of lymphokine activated killer (LAK) cell activity; a high LAK activity group (H-LAK-HCC) and a low LAK activity group (L-LAK-HCC). Interferon-gamma (IFN-gamma) production is severely defective in L-LAK-HCC but not defective in H-LAH-HCC. IFN-gamma production is suppressed with the addition of anti-Tac in dose dependent manner, though LAK activity is suppressed only in the presence of high concentration of anti-Tac. LAK activity is suppressed with the addition of anti-IFN-gamma, which is most prominent when the antibody is present during the first 12 hr of incubation. LAK generation is enhanced with the addition of recombinant IFN-gamma, which is most prominent when it is present during the first 12 hr of incubation. However, this enhancing effect is less prominent in L-LAK-HCC as compared to normals, liver cirrhosis, and H-LAK-HCC. This enhancement is regarded to depend on the presence of Leu7⁺ and Leu11⁺ subset, as this enhancement is abandoned and IFN-gamma production is inhibited when either of these subsets is deleted. These data suggest that IFN-gamma production and the participation of Leu7⁺ and Leu11⁺ subsets is important in sufficient LAK generation, and that poor IFN-gamma production and insufficient response to IFN-gamma may be the cause of severely defective LAK generation in L-LAK-HCC.     

Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice

The uncarboxylated form (ucOC), but not the γ-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide–1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Water and salt movement in soil driven by crop roots: a controlled column study

Water deficit and salt accumulation in soil presents serious problems to crop production in semi-arid regions. These problems depend on the active transpiration stream and the selective absorption of ions by crop roots. In this study, a large sized soil column system was used to examine the dynamics of water and ion transport and salt accumulation in soil layers. Special reference was placed on the effects of the active and selective absorption by roots of different crops (i.e., corn plants, sunflower plants and no plants). The column system was equipped with on-line systems for the control of groundwater level. Soil water content sensors enabled time-course evaluations of the volumetric water content and hence upward flux of the groundwater in the soils at different depths. Furthermore, the distribution and accumulation of ions in soil layers, plant organs and xylem sap were analyzed using ion chromatography. In this column experiment, diurnal and longer term changes in water movement and ion accumulation in soil, affected by root absorption characteristics of plants, were evaluated quantitatively. The results demonstrated that the column system was applicable for the quantitative analysis of the effects of root absorption by different crops on water deficit and salinization in soils.     

Occurrence of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in Canopies of a Natural Lucidophyllous Forest in Japan

A total of 39 Bacillus thuringiensis isolates were recovered from 38 leaves collected from 5- to 10-m-high canopies of 8 micro-/meso-phanerophyte species in a lucidophyllous forest of Japan. B. thuringiensis-positive leaves accounted for 1.4% of a total of 2805 leaves from 15 tree species. The frequency of the organism was 0.8% among the Bacillus cereus/B. thuringiensis group. Of 39 isolates obtained, 27 (69.2%) were allocated to 11 H serovars, and 12 isolates remained unidentified: 11 were motile but lacked reactivity to the 55 reference antisera, and 1 isolate was not flagellated. Two H serovars, kurstaki (H3abc) and tohokuensis (H17), occurred predominantly on canopy phylloplanes. Larvicidal activities against Bombyx mori and/or Aedes aegypti were associated with 49% of the canopy isolates. Strong hemolysis was induced by parasporal inclusion proteins of the two isolates of serovar israelensis (H14). Hemagglutinating (lectin) activity was associated with parasporal proteins of nine isolates. There was little correlation between insecticidal activity and lectin activity.     

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.     

CARM1 regulates proliferation of PC12 cells by methylating HuD

HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21^cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21^cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21^cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21^cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21^cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.