Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.     

Genetic heterogeneity within individual bovine rotavirus isolates

The genomic RNA patterns of six different bovine rotavirus isolates were analyzed on high-percentage polyacrylamide gels (12.5, 13.6, and 17.5%). In contrast to the RNA patterns exhibited by conventional gel systems, those on the high-percentage gels showed an improvement in segment resolution which consequently aided in the detection of extensive band splitting in these patterns. The ability to clone out various electrophoretically distinct virus subpopulations from each of the six isolates provided an explanation for the band splitting detected by the high-resolution gels. The significance of the coexistence of genetically distinct rotavirus populations within a single host is discussed.     

RNase E, an RNA processing enzyme from Escherichia coli.

An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Laboratory evaluation of a carabid larva,Chlaenius bioculatus [Col.: Carabidae] as a predator of lepidopterous pests

Food preference as well as feeding efficiency studies of the carabid predator revealed that the grub of theChlaenius bioculatus Chaud preferred mostPolytella gloriosae F. larvae in both 2nd and 3rd instar.Tarache tropica Guénée was preferred least by the grub predator in both instar.T. tropica was found toxic and grubs of the predator died after consumption.

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Résumé Les préférences alimentaires, de même que l'efficacité prédatrice deChlaenius bioculatus Chaud, montrent que ce carabide prédateur préfère le plus les larves dePolytella gloriosae F. du 2^e et 3^e stade.Tarache tropica Guénée est le moins attaqué aux deux stades.T. tropica est toxique et les larves du prédateur meurent après sa consommation.

     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2

In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.     

Paclobutrazol Arrests Vegetative Growth and Unveils Unexpressed Yield Potential of Jatropha curcas

Although the process for making EN 14214 grade Jatropha methyl ester (biodiesel) capable of running unmodified diesel engines in neat form has been demonstrated, getting higher seed yield from Jatropha shrubs in wastelands is critical to the success of Jatropha biodiesel. But, low productivity is inherent to many Jatropha curcas germplasms and raising large-scale plantations using such untested planting material can lead to wasteful expenditures. Unreliable and poor flowering and fruiting are important factors responsible for low productivity in the species. Although much is known about growth retardants applied to field and horticultural crops, their role in improving the seed productivity of Jatropha has never been explored. Here we report for the first time that paclobutrazol could be an extremely useful chemical, whose dose and time of application, if optimized, can significantly reduce unwanted vegetative growth, with concomitant improvement in yield and seed oil content of Jatropha. In the year following application of paclobutrazol, an unexpected increase in seed yield, as high as 1127% relative to controls, was obtained from one such unproductive Jatropha germplasm. We hypothesize that low seed production in this species may be a result of excess vegetative growth caused by an unfavorable endogenous hormonal configuration which competes with growth and development of flower, fruit, or seed. This undesired physiological state can be reversed by paclobutrazol application to achieve maximum oil yield from this energy shrub that holds great promise in the future.