Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data

Journal of Plant Research - A group of temperate grassland plant species termed the “Mansen elements” occurs in Japan and is widely distributed in the grasslands of continental East...     

Changes in contractile properties and neuromuscular propagation evaluated by simultaneous mechanomyogram and electromyogram during experimentally induced hypothermia.

The present study examined, whether or not mechanomyogram (MMG) amplitude and frequency component could reflect the contractile properties of the triceps surae muscles, composed of relatively slow soleus (SOL) and fast medial gastrocnemius (MG), during experimentally induced hypothermia condition. In eight male subjects, lying in prone position, supramaximal single twitch and repetitive electrical stimulations at 10 Hz were applied at the intramuscular temperatures of control (34 degrees C), 15, 20, and 25 degrees C, respectively. The hypothermia induced substantial reduction in muscle contractile properties, e.g. prolonged twitch contraction and half relaxation times, resulted in a highly significant reduction in the fluctuation of force signal during the repetitive stimulations. These changes were almost mirrored by the similar and significant reductions in the MMG amplitude in both SOL and MG. Power spectrum analysis revealed that peak frequency components of MMG and fluctuation of force were almost matched with the applied stimulation frequencies, independent of the temperature condition. These results strongly suggest that MMG analysis could be employed to study muscle contractile properties varying across different physiological conditions.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Cloning of cDNA for Vitellogenin of the Parasitoid Wasp, Pimpla nipponica (Hymenoptera: Apocrita: Ichneumonidae): Vitellogenin Primary Structure and Evolutionary Considerations

The cDNA for vitellogenin (Vg) of the parasitoid wasp Pimpla nipponica (Hymenoptera: Apocrita) was cloned and sequenced.¹ The deduced amino acid sequence with 1807 residues was obtained. The N-terminal 20 amino acids chemically determined for vitellin (Vn) agreed completely with the deduced 20 amino acids that follow the 16 amino acid residues for putative signal peptide. The cDNA clone for the Vg of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta), previously obtained and partially sequenced, was also completely sequenced and the amino acid sequence deduced. Amino acid sequences were compared between these two species and also with known Vg sequences from other insects. Common to all these insects is the presence of two long regions with relatively well-conserved amino acid sequences, one near the N-terminal extending 267–282 residues (including two cysteines at conserved locations), and the other starting at position 450 to 655 and extending 279–283 residues, and of a region at the C-terminal extending some 200 residues (about 250 in Aedes aegypti due to the presence of a serine-rich stretch) with 10 cysteines at conserved locations. A molecular phylogenetic tree was constructed.