The cloning and characterization of a RAS gene fromSchizosaccharomyces pombe

We have cloned and determined the complete nucleotide sequence of a RAS gene from the yeast Schizosaccharomyces pombe (SP-RAS). The putative RAS protein of 214 amino acids is encoded by two noncontiguous reading frames separated by an intron of 86 bp. The SP-RAS gene product shares extensive homology with the proteins of the Saccharomyces cerevisiae (SC), Dictyostelium, Drosophila, and human RAS genes in its N-terminal region but not in its C-terminal region. The extended C-terminal regions found in the SC-RAS genes have no counterpart in the SP-RAS gene. Thus the RAS genes of these two yeasts are structurally quite distinct. The SP-RAS sequence was expressed in vivo.     

Determination of sulfite radical (SO 3 ⋅ −) reaction rate constants by means of competition kinetics

Summary The sulfite radical anion (SO ₃ ^–) was found to react rapidly with the flavonoid quercetin (k = 2.5 × 10⁸ dm³mol^–1 s^–1) and the carotenoids crocin (k = 1.0 × 10⁹ dm³mol^–1 s^1–) and crocetin (k = 1.5 × 10⁹ dm³mol^1– s^1–). The reactions can easily be monitored due to the strong absorptions of the substrates and, in the case of quercetin, the formation of a strongly absorbing transient species. Using these substances, we determined by means of competition kinetics rate constants of SO ₃ ^– reactions with nucleic acid components, polyunsaturated fatty acids, and glutathione.Abbreviations ABTS 2,2-azinobis(3-ethyl-6-benzothiazolinesulfonate) - cmc critical micellization concentration - GSH Glutathione - PUFA polyunsaturated fatty acids Preliminary results were presented at the Third Biennial Meeting of the Society for Free Radical Research in Düsseldorf in July, 1986     

Molecular Cloning and Expression of a Xylanase Gene from Bacillus polymyxa in Escherichia coli

Genomic fragments of Bacillus polymyxa derived from separate and complete digestion by EcoRI, HindIII, and BamHI were ligated into the corresponding sites of pBR322, and the resulting chimeric plasmids were transformed into Escherichia coli. Of 6,000 transformants screened, 1 (pBPX-277) produced a clear halo on Remazol brilliant blue xylan plates. The insert in the pBPX-277 recombinant, identified as an 8.0-kilobase BamHI fragment of B. polymyxa, was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 2.9-kilobase BamHI-EcoRI subfragment was found to code for xylanase activity. Xylanase activity expressed by E. coli harboring the cloned gene was located primarily in the periplasm and corresponded to one of two distinct xylanases produced by B. polymyxa. Xylanase expression by the cloned gene occurred in the absence of xylan and was reduced by glucose and xylose. Southern blot hybridization with the cloned fragment as a probe against complete genomic digests of the bacilli B. polymyxa, B. circulans, and B. subtilis revealed that the cloned xylanase gene was unique to B. polymyxa. The xylanase expressed by the cloned gene had a molecular weight of approximately 48,000 and an isoelectric point of 4.9.     

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery

IntroductionExosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current “gold standard” surrogate biomarker of myocardial damage.ConclusionsThe plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.     

NMR and molecular dynamics studies of tachykinins: conformation of substance P fragment 4-11

The conformation of the C-terminal octapeptide fragment of Substance P (SP4-11, Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with gamma-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.     

Reaction of No With O2-. Implications for the Action of Endothelium-Derived Relaxing Factor (EDRF)

Under physiological pH conditions (pH 7.2-7.4) the rate constant of the reaction NO + O₂ yielding peroxonitrite (ONOO) was determined as k = (3.7 ± 1.1) × 10⁷ M ¹ s ¹. The decay of peroxonitrite at this pH follows first order kinetics with a rate constant of 1.4 s ¹. At alkaline pH peroxonitrite is practically stable.

Possible consequences of these reactions for the biological lifetime of EDRF will be discussed.     

<Emphasis Type="Italic">Rhizoglomus venetianum</Emphasis>, a new arbuscular mycorrhizal fungal species from a heavy metal-contaminated site,downtown Venice in Italy

Rhizoglomus venetianum, a new arbuscular mycorrhizal fungal species, has been isolated and propagated from a heavy metal-contaminated site in Sacca San Biagio island, downtown Venice, Italy. Interestingly, under the high levels of heavy metals occurring in the site, the new fungus was able to grow only intraradically. In greenhouse trap and single species cultures under low heavy metal levels, the fungus produced innumerous spores, clusters, and sporocarps extraradically, which were formed terminally on subtending hyphae either singly, in small spore clusters, or, preferably, in loose to compact non-organized sporocarps up to 2500?×?2000?×?2000 μm. Spores are golden-yellow to bright yellow brown, globose to subglobose to rarely oblong, 75–145?×?72–140 μm in diameter, and have four spore wall layers. Morphologically, the new fungus is similar to R. intraradices, and phylogenetically, it forms a monophyletic clade next to R. irregulare, which generally forms irregular spores and lacks, like R. intraradices, the flexible innermost wall layer beneath the structural/persistent third wall layer. A key for the species identification is presented comprising all 18 Rhizoglomus species, so far described or newly combined.     

Molecular orbital studies on nucleoside antibiotics,VII. Conformation of 2′-amino- 2′-deoxyguanosine

Conformational properties of the nucleoside antibiotic 2-amino-2-deoxyguanosine have been investigated by the PCILO method along with those of its parent nucleoside, guanosine. This antibiotic, formed by replacement of the 2-hydroxyl group by an amino group in guanosine, shows anti-tumor activity and also inhibits RNA and protein syntheses. Both C(2)-endo and C(3)-endo sugar conformations have been considered in the computations. The results indicate striking similarity between the conformations of the antibiotic and the parent nucleoside, particularly in simulated aqueous environment. The biological implication of this result in terms of the antibiotic activity is discussed.     

The involvement of oxygen radicals during the autoxidation of adrenalin.

1. In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected. 2. The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions. 3. Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm. 4. The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds. 5. It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not produce reliable evidence for superoxide anions.     

Toxicity, uptake, and transfer efficiency of fipronil in western subterranean termite (Isoptera: Rhinotermitidae)

The potential horizontal transfer ofnonrepellent termiticides has become an important paradigm to control termites in recent years. In this study, 14C-radiolabeled fipronil was used in a series of laboratory experiments to demonstrate the extent and ability of termites to transfer lethal amounts of fipronil to unexposed nestmates. Fipronil is an active and nonrepellent termiticide against western subterranean termites, Reticulitermes hesperus Banks, on sand at relevant doses. It exhibited delayed toxicity with the lowest LD50 approximately 0.2 ng/termite expressed between day 4 and 7. Both continuous and brief exposures to fipronil-treated sand seriously impaired the termite's ability to move and respond to a dodecatrienol trail, limiting potential horizontal transfer. In tunneling studies, fipronil prevented termite tunneling at concentrations as low as 0.5 ppm and was nonrepellent even at 500 ppm. Greater than 90% mortality was recorded by day 7 with concentrations ranging from 0.5 to 500 ppm. There was a linear relationship between the time of exposure and uptake of [14C]fipronil when termites were continuously exposed to 0.5, 1.0, and 5.0 ppm for 24 h. However, uptake discontinued when the termites were immobilized. Maximum transfer of fipronil from donors to recipients occurred within the first 24 h. Fipronil was transferred by body contact and trophallaxis did not play a major role in horizontal transfer. In successive transfer studies, there was not enough fipronil on recipients for them to serve as secondary donors and kill other termites. In a linear arena study, there was an inverse relationship between the amount offipronil on dead termites and their distance from the treated zone. Maximum mortality was observed within 1.5 m from the treated zone. Results in our laboratory studies suggest that horizontal transfer was not a major factor contributing to the efficacy of fipronil in the field.