Viruses and prions of Saccharomyces cerevisiae

Cahpter one.-- PMC4141569Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for blocking the propagation of the viruses and prions, and proteins involved in the expression of viral components. Here, we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast. © 2013 Elsevier Inc.R. W. was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases. T. F. and R. E. were supported by Grant BFU2010-15768 from the Spanish Ministry of Education and Science.Peer Reviewe

Conformational diversity of dynactin

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm subcomplex remain elusive. Here, we report the flexible nature and diverse conformations of dynactin sidearm observed by electron microscopy. Using nanogold labeling and deletion mutant analysis, we determined the domain organization of the largest subunit p150 and discovered that its coiled-coil (CC1), dynein-binding domain, adopted either a folded or an extended form. Furthermore, the entire sidearm exhibited several characteristic forms, and the equilibrium among them depended on salt concentrations. These conformational diversities of the dynactin complex provide clues to understanding how it binds to microtubules and regulates dynein

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum. Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives

Tyr-Pro-Trp-Gly-NH_2(Tyr-W-MIF-1)analogであるTyr-D-Pro-Trp-Gly-NH_2の抗侵害作用におけるμオピオイド受容体の関与について

We have previously reported that Tyr-D-Pro-Trp-Gly-NH_2 (D-Pro^2-Tyr-W-MIF-1) given spinally produces clearly a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 without affecting endomorphins- and[D-Ala^2, NMePhe^4, Gly(ol)^5]-enkephalin (DAMGO)-induced antinociception, and D-Pro^2-Tyr-W-MIF-1 at any doses used (0.025-1.2 nmol) does not show any antinociception or hyperalgesic effect by itself. In the present study, we found that D-Pro^2-Tyr-W-MIF-1 given supraspinally produced the antinociception, which is mediated by stimulation of μ-opioid receptors. D-Pro^2-Tyr-W-MIF-1 (0.5-16 nmol) given intracerebroventricularly (i.c.v.) produced an apparent dose-dependent antinociception. However, at the three highest doses (4, 8 or 16 nmol), there was a ceiling effect (about 30% MPE) where the increase in dose did not lead to a greater effect. The antinociception induced by D-Pro^2-Tyr-W-MIF-1 at a dose of 4 nmol was blocked by i.c.v. co-administration with the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH_2 (CTOP), but not by i.c.v. pretreatment with the μ_1-opioid receptor antagonist naloxonazine, the κ-opioid receptor antagonist, nor-binaltorphimine, or the δ-opioid receptor antagonist naltrindole. In contrast, the antinociception induced by DAMGO and Tyr-W-MIF-1 was blocked by i.c.v. co-administration with CTOP or by i.c.v. pretreatment with higher doses of naloxonazine, but not by pretreatment with nor-binaltorphimine or naltrindole. We propose that the antinociception induced by D-Pro^2-Tyr-W-MIF-1 and Tyr-W-MIF-1 is mediated by the stimulation of different subtypes of μ_2-opioid receptors

〔報 文〕シイタケ子実体のスーパーオキシドジスムターゼの精製と諸性質

Crude extract of Lentinus edodes(L. edodes)contained more than three kinds of superoxide dismutases(SODs)which were distinguished electrophoretically. All of these were found to be insensitive to H2O2 and cyanide. A centrifugal study of the crude extract in 0.25M sucrose solution revealed that most of the SOD activities are located in the cytoplasmic fraction. We purified one of these superoxide dismutases from pilei of L. edodes to homogeneity by ammonium sulfate fractionation, DE-32 ion-exchange, Sephadex G-100 gel filtration and Butyl-toyopearl hydrophobic chromatographies. The purified enzyme showed that a single protein band coincided with the single SOD activity band in native polyacrylamide gel electrophoresis(PAGE). The purified SOD showed a single band in PAGE in the presence of sodium dodecyl sulfate(SDS-PAGE)and its subunit molecular weight was estimated to 23±0.5kDa. The subunit molecular weight of SOD was also estimated by LC-MS analysis to be 22,184Da. Using the sedimentation equilibrium centrifugation method the molecular mass of native SOD was estimated to be 84,240Da. These observations suggest that the enzyme is a tetramer composed of subunits of equal size. Metal analysis of the native enzymes revealed 0.64g-atoms Mn per mole subunit in the preparations whose specific activity were 3500U/mg. Direct analysis of N-terminal amino acid of this enzyme by Edman degradation using a protein sequencer cannot detect any amino acid residue, but amino acid residue of N-terminal appeared as serine residue after the treatment of the enzyme with tetrafluoroacetic acid in a vapor phase at 60℃ for 10min. Therefore, the N-terminal amino acid was modified with acetyl group. The modification of N-terminal amino acid is the first example of Mn-SOD. Some of other physiological and biochemical properties of the enzyme were also investigated

Endothelial Dysfunction, Increased Arterial Stiffness, and Cardiovascular Risk Prediction in Patients With Coronary Artery Disease: FMD‐J (Flow‐Mediated Dilation Japan) Study A

BackgroundThe usefulness of vascular function tests for management of patients with a history of coronary artery disease is not fully known.Methods and ResultsWe measured flow‐mediated vasodilation (FMD) and brachial–ankle pulse wave velocity (baPWV) in 462 patients with coronary artery disease for assessment of the predictive value of FMD and baPWV for future cardiovascular events in a prospective multicenter observational study. The first primary outcome was coronary events, and the second primary outcome was a composite of coronary events, stroke, heart failure, and sudden death. During a median follow‐up period of 49.2 months, the first primary outcome occurred in 56 patients and the second primary outcome occurred in 66 patients. FMD above the cutoff value of 7.1%, derived from receiver‐operator curve analyses for the first and second primary outcomes, was significantly associated with lower risk of the first (hazard ratio, 0.27; 95% confidence interval, 0.06–0.74; P=0.008) and second (hazard ratio, 0.32; 95% confidence interval, 0.09–0.79; P=0.01) primary outcomes. baPWV above the cutoff value of 1731 cm/s was significantly associated with higher risk of the first (hazard ratio, 1.86; 95% confidence interval, 1.01–3.44; P=0.04) and second (hazard ratio, 2.19; 95% confidence interval, 1.23–3.90; P=0.008) primary outcomes. Among 4 groups stratified according to the combination of cutoff values of FMD and baPWV, stepwise increases in the calculated risk ratio for the first and second primary outcomes were observed.ConclusionsIn patients with coronary artery disease, both FMD and baPWV were significant predictors of cardiovascular events. The combination of FMD and baPWV provided further cardiovascular risk stratification

FMD, PWV, and Cardiovascular Events

Background The usefulness of vascular function tests for management of patients with a history of coronary artery disease is not fully known. Methods and Results We measured flow‐mediated vasodilation (FMD) and brachial–ankle pulse wave velocity (baPWV) in 462 patients with coronary artery disease for assessment of the predictive value of FMD and baPWV for future cardiovascular events in a prospective multicenter observational study. The first primary outcome was coronary events, and the second primary outcome was a composite of coronary events, stroke, heart failure, and sudden death. During a median follow‐up period of 49.2 months, the first primary outcome occurred in 56 patients and the second primary outcome occurred in 66 patients. FMD above the cutoff value of 7.1%, derived from receiver‐operator curve analyses for the first and second primary outcomes, was significantly associated with lower risk of the first (hazard ratio, 0.27; 95% confidence interval, 0.06–0.74; P=0.008) and second (hazard ratio, 0.32; 95% confidence interval, 0.09–0.79; P=0.01) primary outcomes. baPWV above the cutoff value of 1731 cm/s was significantly associated with higher risk of the first (hazard ratio, 1.86; 95% confidence interval, 1.01–3.44; P=0.04) and second (hazard ratio, 2.19; 95% confidence interval, 1.23–3.90; P=0.008) primary outcomes. Among 4 groups stratified according to the combination of cutoff values of FMD and baPWV, stepwise increases in the calculated risk ratio for the first and second primary outcomes were observed. Conclusions In patients with coronary artery disease, both FMD and baPWV were significant predictors of cardiovascular events. The combination of FMD and baPWV provided further cardiovascular risk stratification

Resting complexes of the persistent yeast 20S RNA Narnavirus consist solely of the 20S RNA viral genome and its RNA polymerase p91

The positive strand 20S RNA narnavirus persistently infects Saccharomyces cerevisiae. The 20S RNA genome has a single gene that encodes the RNA-dependent RNA polymerase (p91). 20S RNA forms ribonucleoprotein resting complexes (RNPs) with p91 and resides in the cytoplasm. Here we found no host proteins stoichiometrically associated with the RNP by pull-down experiments. Furthermore, 20S RNA, when expressed from a vector in Escherichia coli, formed RNPs with p91 in the absence of yeast proteins. This interaction required the 3' cis signal for complex formation. Moreover, when 23S RNA, the genome of another narnavirus, was expressed in E. coli, it also formed RNPs with its RNA polymerase p104. Finally, when both RNAs were expressed in the same E. coli cell, they formed RNPs only with their cognate RNA polymerases. These results altogether indicate that narnaviruses RNPs consist of only the viral genomes and their cognate RNA polymerases. Because the copy number of the RNPs can be induced almost equivalent to those of rRNAs in some yeast strains, the absence of host proteins may alleviate the burden on the host by not sequestering proteins into the RNPs. It may also contribute to the persistent infection of narnaviruses by decreasing their visibilityThis work was supported by Grant BFU2010-15768 from The Spanish Ministry of Education and Science. We wish to thank ‘Fundación Ramón Areces’ for financial support of our Institute.Peer Reviewe