Differential expression of alpha2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats

The aim of the present study was to compare vascular dysfunction between the early (12 wk old) and later (36 wk old) stages of spontaneous diabetes in Goto-Kakizaki (GK) rats. We also evaluated the aortic expression of the alpha(2D)-adrenoceptor and endothelial nitric oxide synthase (eNOS). Vascular reactivity was assessed in thoracic aortas from age-matched control rats and 12- and 36-wk GK rats. Using RT-PCR and immunoblots, we also examined the changes in expression of the alpha(2D-)adrenoceptor and eNOS. In aortas from GK rats (vs. those from age-matched control rats): 1) the relaxation response to ACh was enhanced at 12 wk but decreased at 36 wk; 2) the relaxation response to sodium nitroprusside was decreased at both 12 and 36 wk, 3) norepinephrine (NE)-induced contractility was decreased at 12 wk but not at 36 wk, 4) the expressions of alpha(1B)- and alpha(1D)-adrenoceptors were unaffected, whereas those of alpha(2D)-adrenoceptor and eNOS mRNAs were increased at both 12 and 36 wk; and 5) NE- and ACh-stimulated NO(x) (nitrite and nitrate) levels were increased at 12 wk, although at 36 wk ACh-stimulated NO(x) was lower, whereas NE-stimulated NO(x) showed no change. These results clearly demonstrate that enhanced ACh-induced relaxation and impaired NE-induced contraction, due to NO overproduction via eNOS and increased alpha(2D)-adrenoceptor expression, occur in early-stage GK rats and that the impaired ACh-induced relaxation in later-stage GK rats is due to reductions in both NO production and NO responsiveness (but not in eNOS expression).     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p

The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.     

Intraperitoneal fluid accumulation induced by Clostridium perfringens alpha-toxin (phospholipase C)

We report that the intraperitoneal injection of Clostridium perfringens alpha-toxin into mice induces ascites. This phenomenon was monitored by measuring fluid volume and analyzing hematologic data. The mouse toxicity test provides a simple and useful model for examining C. perfringens alpha-toxin-induced vascular permeability.     

A repressor protein,PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.     

Possible factors responsible for the toxicity of Cochlodinium polykrikoides,a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.     

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.     

Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34+ megakaryocytic progenitor cells from human placental and umbilical cord blood

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34(+) cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34(+) CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.