Protective role of 20-hydroxyecdysone against lead stress in Chlorella vulgaris cultures

Treatment of cultured C. vulgaris cells with 10(-6)-10(-4) M lead decreased their growth and chemical composition during the first 48 h of cultivation. However, at concentrations above 10(-4) M, lead is cytotoxic to Chlorella vulgaris cells, resulting in cellular fragmentation and lysis. In contrast, at concentrations below 10(-6) M lead had no influence on the growth and metabolism of C. vulgaris cells. 20-Hydroxyecdysone (20E) (10(-10)-10(-8) M) increased growth and chemical composition of C. vulgaris cells over a concentration range. Levels per cell of chlorophylls, protein, sugars are all increased by 20E treatment, when compared to non-treated control cells. However, the cultures treated with 20E and lead show a lower stimulation than the cultures treated with 20E alone. The effects of 20E mixed with lead on the growth and the level of cellular lead, chlorophyll, sugar and protein in C. vulgaris are also reported. The decreased growth and composition of C. vulgaris cells treated with lead was restored by the 20E. Application of 20E to C. vulgaris cultures reduced the impact of lead stress on growth, prevented chlorophyll, sugar and protein loss and increased phytochelatins synthesis. Furthermore, 20E did not restore toxic effect of lead on C. vulgaris cells. The combined treatment with lead and 20E appeared to have a stimulatory effect on the above parameters during the 48 h of cultivation, as compared to the control. 20E reduced the toxicity of lead and the growth recovered to the level of cells treated with 20E alone. Concentration-dependent stimulation was observed with increasing concentration of 20E and decreasing concentration of lead.     

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.     

Characteristics of arylsulfatase in Russian sturgeon (Acipenser gueldenstaedti) semen

Spermatozoa of sturgeons (Acipenseriformes), unlike teleosts, possess an acrosome. This paper provides data concerning biochemical characteristics of arylsulfatase (AS), an acrosomal enzyme, found in Russian sturgeon spermatozoa and seminal plasma. The enzymes were purified by a four-step procedure, using n-butanol extraction, ion-exchange chromatography repeated twice and gel filtration. High purity of our enzymes was confirmed by silver staining electrophoresis and an immunological experiment. Kinetic parameters indicated that the purified enzymes belong to arylsulfatase type A. Similarity of the seminal plasma arylsulfatase to the spermatozoan enzyme showed us that arylsulfatase from seminal plasma might originate from damaged spermatozoa. The possible physiological consequences of the presence of arylsulfatase in Russian sturgeon semen are discussed.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Major histocompatibility complex and mate choice in sand lizards

In mice and man, females prefer males with a major histocompatibility complex (MHC) genotype different to their own. We tested whether this phenomenon also occurs in the Swedish sand lizard (Lacerta agilis). Females in a laboratory experiment preferred to associate with odour samples obtained from more distantly related males at the MHC class 1 loci. Data on free-ranging lizards suggest that associations between males and females are nonrandom with respect to MHC genotype. However, male spatial distribution and mobility during the mating season suggest that the non-random pairing process in the wild may also be driven by corresponding genetic benefits to males pairing with less related females.     

GDP-mannose 3',5'-epimerase forms GDP-L-gulose,a putative intermediate for the de novo biosynthesis of vitamin C in plants

Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of L-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3',5'-epimerase catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3',5'-epimerase of Arabidopsis thaliana. GDP and GDP-D-glucose are potent competitive inhibitors of the enzyme, whereas GDP-L-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P)+. A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3',5'-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-l-galactose, a novel intermediate: GDP-L-gulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous L-gulose and L-gulono-1,4-lactone serve as direct precursors of l-ascorbic acid in plant cells. We propose an L-gulose pathway for the de novo biosynthesis of vitamin C in plants.     

Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.