Directed synthesis of 2',5'-oligoadenylates with increased stability towards phosphodiesterases

Phosphodiesterase stability of synthetic analogs of 2',5'-oligoadenylates, the mediators of antiviral and antiproliferative action of interferons was analysed. The analogs with a 3'-terminal acyclic nucleoside residue were prepared. These analogs were treated with NIH3T3 cell lysate, mice liver homogenate and snake venom phosphodiesterase. All analogs have demonstrated a high stability as compared with the natural 2',5'-oligoadenylate and its 3'-deoxyderivative. The possible biological activity of these stable analogs of 2',5'-oligoadenylates is discussed.     

Study of 2'-phosphodiesterase activity in cultured NIH 3T3 cells during activation of cAMP-dependent phosphorylation

A rapid and transient decrease in 2'-phosphodiesterase activity in NIH 3T3 mouse cells was observed after adrenaline addition. The decrease of activity was accompanied by an elevation of intracellular cAMP level. The 2'-phosphodiesterase activity changed similarly when cells sink deeper into the resting state. In the latter case, the fall of the enzyme activity was correlated with elevation of the activity of cAMP-dependent proteinkinase and, moreover, a considerable increase of the intracellular level of 2',5'-oligoadenylate was observed. Phosphorylation of proteins by cAMP-dependent proteinkinase in the cell lysate also produced a pronounced drop of 2'-phosphodiesterase activity. Exogenous 2',5'-oligo (A) treatment of the cells resulted in the rise of 2'-phosphodiesterase activity; actinomycin D prevented this effect. The data presented suggest the involvement of two different mechanisms in regulation of 2'-phosphodiesterase activity: cAMP-dependent phosphorylation and induction of 2'-phosphodiesterase by 2',5'-oligoadenylate.     

A strategy for construction of industrial strains of distiller's yeast

A procedure was developed for construction of industrial strains of distiller's yeast (Saccharomyces cerevisiae). It includes several steps: construction of congenic genetically marked haploid strains of opposite mating types starting from an industrial strain of hybrid nature, integrative transformation of the above haploid strains with a DNA fragment containing an expression cassette responsible for new technological facilities, and hybridization of transformants and isolation of final industrial homozygous strains under experimental conditions simulating commercial fermentation processes. This strategy permits the generation of strains that have desirable characteristics of traditional races of distiller's yeast along with new technological facilities determined by the particular expression cassette. Using this procedure, we have constructed an industrial strain with improved amylolytic activity. (c) 1995 John Wiley & Sons, Inc.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

The antidepressant-like effect of Ocimum basilicum in an animal model of depression

We investigated the efficacy of Ocimum basilicum (OB) essential oils for treating depression related behavioral, biochemical and histopathological changes caused by exposure to chronic unpredictable mild stress (CUMS) in mice and to explore the mechanism underlying the pathology. Male albino mice were divided into four groups: controls; CUMS; CUMS plus fluoxetine, the antidepressant administered for pharmacological validation of OB; and CUMS plus OB. Behavioral tests included the forced swim test (FST), elevated plus-maze (EPM) and the open ?eld test (OFT); these tests were performed at the end of the experiment. We assessed serum corticosterone level, protein, gene and immunoexpression of brain-derived neurotropic factor (BDNF) and glucocorticoid receptors (GRs) as well as immunoexpression of glial fibrillary acidic protein (GFAP), Ki67, caspase-3 in the hippocampus. CUMS caused depression in the mice as evidenced by prolonged immobility in the FST, prolonged time spent in the open arms during the EPM test and reduction of open field activity in the OFT. OB ameliorated the CUMS induced depressive status. OB significantly reduced the corticosterone level and up-regulated protein and gene expressions of BDNF and GR. OB reduced CUMS induced hippocampal neuron atrophy and apoptosis, and increased the number of the astrocytes and new nerve cells. OB significantly increased GFAP-positive cells as well as BDNF and GR immunoexpression in the hippocampus.     

Productivity of forests in the Eurosiberian boreal region and their potential to act as a carbon sink –- a synthesis

Based on review and original data, this synthesis investigates carbon pools and fluxes of Siberian and European forests (600 and 300 million ha, respectively). We examine the productivity of ecosystems, expressed as positive rate when the amount of carbon in the ecosystem increases, while (following micrometeorological convention) downward fluxes from the atmosphere to the vegetation (NEE = Net Ecosystem Exchange) are expressed as negative numbers. Productivity parameters are Net Primary Productivity (NPP=whole plant growth), Net Ecosystem Productivity (NEP = CO₂ assimilation minus ecosystem respiration), and Net Biome Productivity (NBP = NEP minus carbon losses through disturbances bypassing respiration, e.g. by fire and logging). Based on chronosequence studies and national forestry statistics we estimate a low average NPP for boreal forests in Siberia: 123 gC m^–2 y^–1. This contrasts with a similar calculation for Europe which suggests a much higher average NPP of 460 gC m^–2 y^–1 for the forests there. Despite a smaller area, European forests have a higher total NPP than Siberia (1.2–1.6 vs. 0.6–0.9 × 10¹⁵ gC region^–1 y^–1). This arises as a consequence of differences in growing season length, climate and nutrition. For a chronosequence of Pinus sylvestris stands studied in central Siberia during summer, NEE was most negative in a 67-y old stand regenerating after fire (– 192 mmol m^–2 d^–1) which is close to NEE in a cultivated forest of Germany (– 210 mmol m^–2 d^–1). Considerable net ecosystem CO₂-uptake was also measured in Siberia in 200- and 215-y old stands (NEE:174 and – 63 mmol m^–2 d^–1) while NEP of 7- and 13-y old logging areas were close to the ecosystem compensation point. Two Siberian bogs and a bog in European Russia were also significant carbon sinks (– 102 to – 104 mmol m^–2 d^–1). Integrated over a growing season (June to September) we measured a total growing season NEE of – 14 mol m^–2 summer^–1 (– 168 gC m^–2 summer^–1) in a 200-y Siberian pine stand and – 5 mol m^–2 summer^–1 (– 60 gC m^–2 summer^–1) in Siberian and European Russian bogs. By contrast, over the same period, a spruce forest in European Russia was a carbon source to the atmosphere of (NEE: + 7 mol m^–2 summer^–1 = + 84 gC m^–2 summer^–1). Two years after a windthrow in European Russia, with all trees being uplifted and few successional species, lost 16 mol C m^–2 to the atmosphere over a 3-month in summer, compared to the cumulative NEE over a growing season in a German forest of – 15.5 mol m^–2 summer^–1 (– 186 gC m^–2 summer^–1; European flux network annual averaged – 205 gC m^–2 y^–1). Differences in CO₂-exchange rates coincided with differences in the Bowen ratio, with logging areas partitioning most incoming radiation into sensible heat whereas bogs partitioned most into evaporation (latent heat). Effects of these different surface energy exchanges on local climate (convective storms and fires) and comparisons with the Canadian BOREAS experiment are discussed. Following a classification of disturbances and their effects on ecosystem carbon balances, fire and logging are discussed as the main processes causing carbon losses that bypass heterotrophic respiration in Siberia. Following two approaches, NBP was estimated to be only about 13–16 mmol m^–2 y^–1 for Siberia. It may reach 67 mmol m^–2 y^–1 in North America, and about 140–400 mmol m^–2 y^–1 in Scandinavia. We conclude that fire speeds up the carbon cycle, but that it results also in long-term carbon sequestration by charcoal formation. For at least 14 years after logging, regrowth forests remain net sources of CO₂ to the atmosphere. This has important implications regarding the effects of Siberian forest management on atmospheric concentrations. For many years after logging has taken place, regrowth forests remain weaker sinks for atmospheric CO₂ than are nearby old-growth forests.     

Inhibition of 2'-phosphodiesterase by cAMP-dependent protein kinase. Involvement of phosphorylation of protein inhibitor

2'-Phosphodiesterase from NIH 3T3 cells was purified about 530-fold. Treatment of the cell lysate with the cAMP-dependent protein kinase causing the 2'-phosphodiesterase inhibition did not result in phosphorylation of the enzyme itself. The kinase was found to phosphorylate a specific 18-kDa protein, the phosphorylated form of this protein being the inhibitor of 2'-phosphodiesterase.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.