Protective role of cytosolic NADP(+)-dependent isocitrate dehydrogenase, IDH1, in ischemic pre-conditioned kidney in mice

Ischemic pre-conditioning protects the kidney against subsequent ischemia/reperfusion (I/R). This study investigated the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDH1), a producer of NADPH, in the ischemic pre-conditioning. Mice were pre-conditioned by 30 min of renal ischemia and 8 days of reperfusion. In non-pre-conditioned mice 30 min of ischemia had significantly increased the levels of plasma creatinine, BUN, lipid peroxidation and hydrogen peroxide in kidneys, whereas in pre-conditioned mice, the ischemia did not increase them. The reductions of reduced glutathione and NADPH after I/R were greater in non-pre-conditioned mice than in pre-conditioned mice. Ischemic pre-conditioning prevented the I/R-induced decreases in IDH1 activity and expression, but not in glucose-6-phosphate dehydrogenase activity. In conclusion, protection of the kidney afforded by ischemic pre-conditioning may be associated with increased activity of IDH1 which relates to increased levels of NADPH, increased ratios of GSH/total glutathione, less oxidative stress and less kidney injury induced by subsequent I/R insult.     

A mutated PtsG, the glucose transporter, allows uptake of D-ribose.

Mutations arose from an Escherichia coli strain defective in the high (Rbs/ribose) and low (Als/allose and Xyl/xylose) affinity D-ribose transporters, which allow cells to grow on D-ribose. Genetic tagging and mapping of the mutations revealed that two loci in the E. coli linkage map are involved in creating a novel ribose transport mechanism. One mutation was found in ptsG, the glucose-specific transporter of phosphoenolpyruvate:carbohydrate phosphotransferase system and the other in mlc, recently reported to be involved in the regulation of ptsG. Five different mutations in ptsG were characterized, whose growth on D-ribose medium was about 80% that of the high affinity system (Rbs+). Two of them were found in the predicted periplasmic loops, whereas three others are in the transmembrane region. Ribose uptakes in the mutants, competitively inhibited by D-glucose, D-xylose, or D-allose, were much lower than that of the high affinity transporter but higher than those of the Als and Xyl systems. Further analyses of the mutants revealed that the rbsK (ribokinase) and rbsD (function unknown) genes are involved in the ribose transport through PtsG, indicating that the phosphorylation of ribose is not mediated by PtsG and that some unknown metabolic function mediated by RbsD is required. It was also found that D-xylose, another sugar not involved in phosphorylation, was efficiently transported through the wild-type or mutant PtsG in mlc-negative background. The efficiencies of xylose and glucose transports are variable in the PtsG mutants, depending on their locations, either in the periplasm or in the membrane. In an extreme case of the transmembrane change (I283T), xylose transport is virtually abolished, indicating that the residue is directly involved in determining sugar specificity. We propose that there are at least two domains for substrate specificity in PtsG with slightly altered recognition properties.     

Development of species-specific quantitative real-time PCR primers for detecting anginosus group streptococci based on the rpoB

In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene for detecting anginosus group streptococci (AGS), Streptococcus anginosus, S. constellatus, and S. intermedius. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 76 strains regarding 44 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of these species-specific qPCR primers was 40 fg at below a cycle threshold (C_T) value of 35. These results suggest that AGS species-specific qPCR primers are suitable for applications in epidemiological studies associated with infectious diseases related to AGS.     

Crystal structures of aprotinin and its complex with sucrose octasulfate reveal multiple modes of interactions with implications for heparin binding

The crystal structures of aprotinin and its complex with sucrose octasulfate (SOS), a polysulfated heparin analog, were determined at 1.7-2.6 Å resolutions. Aprotinin is monomeric in solution, which associates into a decamer at high salt concentrations. Sulfate ions serve to neutralize the basic amino acid residues of aprotinin to stabilize the decameric aprotinin. Whereas SOS interacts with heparin binding proteins at 1:1 molar ratio, SOS was surprisingly found to induce strong agglutination of aprotinins. Five molecules of aprotinin interact with one molecule of the sulfated sugar, which is stabilized by electrostatic interactions between the positively charged residues of aprotinin and sulfate groups of SOS. The multiple binding modes of SOS with five individual aprotinin molecules may represent the diverse patterns of potential heparin binding to aprotinin, reflecting the interactions of densely packed protein molecules along the heparin polymer.     

Senescent phenotype can be reversed by reduction of caveolin status

Hyporesponsiveness to growth factors is one of the fundamental characteristics of senescent cells. We previously reported that the up-regulation of caveolin attenuates the growth factor response and the subsequent downstream signal cascades in senescent human diploid fibroblasts. Therefore, in the present experiment, we investigated the modulation of caveolin status in senescent cells to determine the effect of caveolin on mitogenic signaling efficiency and cell cycling. We reduced the level of caveolin-1 in senescent human diploid fibroblasts using its antisense oligonucleotides and small interfering RNA, and this resulted in the restoration of normal growth factor responses such as the increased phosphorylation of Erk, the nuclear translocation of p-Erk, and the subsequent activation of p-Elk upon epidermal growth factor stimulation. Moreover, DNA synthesis and the re-entry of senescent cells into cell cycle were resumed upon epidermal growth factor stimulation concomitantly with decreases in p53 and p21. Taken together, we conclude that the loss of mitogenic signaling in senescent cells is strongly related to their elevated levels of caveolin-1 and that the functional recovery of senescent cells at least in the terms of growth factor responsiveness and cell cycle entry might be achieved simply by lowering the caveolin level.     

Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals

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Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H₂O₂ level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.     

Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects

The 15,360-bp long complete mitogenome of Caligula boisduvalii possesses a gene arrangement and content identical to other completely sequenced lepidopteran mitogenomes, but different from the common arrangement found in most insect order, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA Ile. The 330-bp A+T-rich region is apparently capable of forming a stem-and-loop structure, which harbors the conserved flanking sequences at both ends. Dissimilar to what has been seen in other sequenced lepidopteran insects, the initiation codon for C. boisduvalii COI appears to be TTG, which is a rare, but apparently possible initiation codon. The ATP8, ATP6, ND4L, and ND6 genes, which neighbor another PCG at their 3' end, all harbored potential sequences for the formation of a hairpin structure. This is suggestive of the importance of such structures for the precise cleavage of the mRNA of mature PCGs. Phylogenetic analyses of available sequenced species of Bombycoidea, Pyraloidea, and Tortricidea supported the morphology-based current hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (Antheraea pernyi and C. boisduvalii) formed a reciprocal monophyletic group.