Formation of novel hairpin structures by telomeric C-strand oligonucleotides.

Telomeres are specialized structures at the ends of chromosomes that are required for long term chromosome stability and replication of the chromosomal terminus. Telomeric DNA consists of simple repetitive sequences with one strand G-rich relative to the other, C-rich, strand. Evolutionary conservation of this feature of telomeric repeat sequences suggests that they have specific structural characteristics involved in telomere function. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoretic analyses showed that telomeric C-strand oligonucleotides form stable non-Watson-Crick hairpin structures containing C.C+ base pairs. Formation of such hairpins may facilitate previously reported G-strand exclusive interactions.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles.     

Potential of the neem tree (Azadirachta indica) for pest control and rural development

The neem tree (Azadirachta indica) contains promising pest-control substances found effective against many economically important pests. These materials are easy to process by village-level industries and easy to use by limited-resource farmers, thereby offering potential for crop protection and off-farm income generation. The tree’s numerous pharmacological and other complementary uses make it doubly attractive for incorporation in large-scale rural development efforts.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein

The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.     

Effects of short-term administration of sex hormones on normal and autoimmune mice

The effects of short-term administration (2 to 4 wk) of sex hormones on the immune system of normal (C57BL/6) and autoimmune (C57BL/6-lpr, C3H/lpr, B/W) strains of mice were investigated. Both estrogen (E2) and testosterone (Te) had significant effects on the numbers of T and B cells as well as on the density of cell surface antigens as demonstrated by flow cytometry. For example, Te depleted Thy-1.2+ thymocytes in normal mice and brought about a shift to lower density cells. Lyt-2+ cells appeared to be the main target cells of hormonal modulation in normal and autoimmune mice. Both sex hormones significantly depleted these cells in the thymus but had differential effects in the peripheral lymphoid organs, particularly in the spleen. In general, E2 depleted Lyt-2+ cells, whereas Te increased or maintained this subpopulation of cells in spleen and lymph nodes. Similarly, the suppressor cell activity and IL 2 production on a per cell basis in E2-treated animals was diminished, whereas Te-treated animals had normal or enhanced activity. The relevance of these findings to differential sex susceptibility in autoimmune diseases is discussed.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Asplenetin,a flavone and its glycoside from Launaea asplenifolia

A new flavone, asplenetin, has been isolated from Launea asplenifolia and characterized as 5,7,3′,4′,5′-pentahydroxy-3-(3-methylbutyl)flavone. Its glycoside, asplenetin 5-O-neohesperidoside, is also reported.     

Solvent influence on rate and mechanism of oxidation of ethylenediaminetetraacetatocobaltate(II) by periodate

The kinetics of the oxidation of Co^II(EDTA)²⁻ by IO₄⁻ were studied in various ethanol + water mixtures covering the range 7.9 to 58.0 wt% ethanol, at five different temperatures in the range 15–35 °C. The effect of solvent on the rate and mechanism of the reaction was investigated. An inner-sphere mechanism for the reaction was proposed and supported by the calculated activation parameters.