Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.     

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.     

Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.     

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.     

Efficacy of metronidazole as second-line drug for the treatment of Helicobacter pylori Infection in the Japanese population: a multicenter study in the Tokyo Metropolitan Area

BACKGROUND: With the increase in the frequency of clarithromycin-resistant Helicobacter pylori (H. pylori), there is rising concern about the decline of the eradication rate of this infection following treatment. The Tokyo Hp Study Group examined the eradication rate in response to a second-line regimen consisting of proton pump inhibitor (PPI), amoxicillin, and metronidazole by conducting a multicenter study in the Tokyo Metropolitan Area. MATERIALS AND METHODS: Two hundred and twenty-eight patients with H. pylori infection, in whom the first-line therapy with a PPI, amoxicillin, and clarithromycin administered for 1 week had failed to eradicate the infection, were enrolled in this study. These cases were randomly assigned to one of the two second-line regimens containing metronidazole (PPI/AM500 or PPI/AM750) administered for 1 week. 13C-urea breath test was performed as a diagnostic method test for H. pylori infection not earlier than 8 weeks after the second-line therapy. RESULTS: Intention-to-treat (ITT) and per-protocol (PP) analyses revealed an eradication rate of 87.6 and 90.6%, respectively, following PPI/AM500 treatment, and 86.9 and 88.6%, respectively, following PPI/AM750 treatment. Neither analysis revealed any significant difference in the eradication rate between PPI/AM500 and PPI/AM750 (p = .876 and .621, respectively). According to ITT and PP analyses, the eradication rates following treatment with PPI/AM500 were 85.2 and 88.5% with the use of lansoprazole, 62.5 and 62.5% with the use of omeprazole, and 93.2 and 96.5% with the use of rabeprazole, respectively. There was a significant difference in the eradication rates between PPI (omeprazole)/AM500 and PPI (rabeprazole)/AM500. In the case of PPI/AM750, the corresponding eradication rates were 84.8 and 87.0% with the use of lansoprazole, 92.9 and 92.9% with the use of omeprazole, and 92.9 and 92.9% with the use of rabeprazole, respectively. There were no significant differences in the eradication rates obtained with the use of the three PPIs. CONCLUSIONS: Both PPI/AM500 and PPI/AM750 administered for 1 week appeared to be highly effective second-line regimens for the treatment of H. pylori infection in Japanese patients. From the viewpoint of adverse events, PPI/AM500 appeared to be safe compared with PPI/AM750.     

Design,synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding to inactive kinase conformation

We synthesized a series of pyrrolo[3,2-d]pyrimidine derivatives and evaluated their application as type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase. Incorporation of a diphenylurea moiety at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker resulted in compounds that were potent inhibitors of VEGFR2 kinase. Of these derivatives, compound 20d showed the strongest inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC). The co-crystal structure of 20d and VEGFR2 revealed that 20d binds to the inactive form of VEGFR2. Further studies indicated that 20d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited PDGFR and Tie-2 kinases. Oral administration of the hydrochloride salt of 20d at 3 mg/kg/day showed potent inhibition of tumor growth in a DU145 human prostate cancer cell xenograft nude mouse model.     

Design,synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part I: Exploration of 5–6 fused rings as alternative S1 moieties

A series of cis-1,2-diaminocyclohexane derivatives were synthesized with the aim of optimizing previously disclosed factor Xa (fXa) inhibitors. The exploration of 5–6 fused rings as alternative S1 moieties resulted in two compounds which demonstrated improved solubility and reduced food effect compared to the clinical candidate, compound A. Herein, we describe the synthesis and structure–activity relationship (SAR), together with the physicochemical properties and pharmacokinetic (PK) profiles of some prospective compounds.     

Difference in seasonal activity pattern between non-native and native ants in subtropical forest of Okinawa Island,Japan

Biological invasions by non-native tramp ants are reported throughout the world, particularly in island ecosystems. In Okinawa Island, a subtropical island in south-western Japan, many tramp ants including the invasive ant, Anoplolepis gracilipes, already occur. In this study activity patterns of the ground-foraging tramp and native ants were investigated for one year by using food bait traps at the forest edges where both categories of ants coexist. On a seasonal time scale, activity patterns were different between exotic and native ants. Native ants are active and seemingly more dominant from spring to summer whereas tramp ants become more dominant from autumn to winter. These results suggest there might be temporal niche partitioning between tramp and native ants, and native ants might be able to dominate tramp ants in their suitable seasons.     

Presence of immunoreactive salusin-β in human plasma and urine

Salusin-α and salusin-β are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-β has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-β peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-β, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-β. We have characterized the molecular form of salusin-β in human plasma and urine. The assay detected immunoreactive salusin-β concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-α and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-β. Salusin-β-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean ± SD, 1.16 ± 0.84 nmol/l, n = 10). These data present the first evidence that salusin-β circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-β in humans.