NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Transgenic alfalfa (Medicago sativa L.) root cultures expressing sense and antisense barley (Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000-g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7–8 and a strong affinity to NADH and NADPH (K _m 3 M for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35±4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.Abbreviation Hb Hemoglobin     

Metabolic and endocrine effects of water and/or food deprivation in rats

Metabolic and endocrine effects of water and/or food deprivation in rats. We aim at studying the effect of water deprivation, food deprivation and their combination for three days on adrenal cortex, pituitary-thyroid axis and vasopressinergic system activity in rats. Corticosterone level was determined by fluorimetric method. The levels of free thyroxine (FT4) and thyroid stimulating hormone (TSH) were determined by immunoenzymatic assay and vasopressin (AVP) level was determined by radio-immunoassay. In all three groups, basal levels of plasma corticosterone were increased. A thyroid dysfunction was shown after water deprivation, food deprivation and their combination reflected by a significant decrease in FT4 levels. Paradoxically, a significant decrease in TSH level was observed in food-deprived rats and in rats subjected to simultaneous food and water deprivation, while a slight and not significant decrease in TSH level was shown in water-deprived rats. A significant increase in plasma AVP level was observed after water deprivation and simultaneous water and food deprivation, while no change was found after food deprivation. The data indicated that water deprivation, food deprivation and their combination stimulated the adrenal cortex, thereby suggesting a stress state. On the other hand, it seems that nutritional stress modifies the pituitary-thyroid axis through mechanisms different from those of osmotic stress. Moreover, it seems that food deprivation partially prevented the stimulatory effect of water deprivation on vasopressinergic system.     

Parasitoids of chickpea leafminer Liriomyza cicerina (Diptera: Agromyzidae) and their parasitism rate on chickpea fields in North Tunisia

In recent years, as a consequence of climatic changes, the leafminer Liriomyza cicerina had emerged as a serious pest in chickpea crops causing considerable damage in Tunisia. Thus, the search of control methods is necessary. The aim of this research was to monitor and identify the naturally occurring parasitoids of the chickpea leafminer Liriomyza cicerina in insecticide-free chickpea-growing areas located in North-Western Tunisia. Chickpea leaves with mines were weekly sampled from Beja and Kef sites during 2016 and 2017 and kept to observe and count emerging leafminer and parasitoid adults in the laboratory. Results revealed that L. cicerina parasitoids complex species included three hymenopteran parasitoids namely Opius monilicornis (Fisher, 1962) (Braconidae); Diglyphus isaea (Walker, 1838) (Eulophidae) and Diaulinopsis arenaria (Erdös, 1951) (Eulophidae). These parasitoids were identified and recorded for the first time in Tunisia on L. cicerina with parasitism rate varied between 13% and 52.5%. The most common species was O. monilicornis. The correlation between infestation rate on chickpea plants and number of emerged three parasitoids was calculated revealing high significant and positive correlations. The diversity index (H′) was also calculated. The three parasitoids were found in two sites Beja and Kef; while the diversity differed during the developmental stage of chickpea crops.     

Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH₂-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0–12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry.     

Structural organization of cycloartane-based saponins in the genus <Emphasis Type="Italic">Astragalus</Emphasis> (Fabaceae)

Cycloartane represents the most common aglycone in saponins of Astragalus genus. These saponins revealed high structural diversity through continuous elucidation of new molecules. Traditionally, saponins’ diversity was updated through reviews listing molecules with respect to their pharmacological activities and/or producing plant species. Beyond the long lists of reviewed molecules, this work provides statistical classification of 178 Astragalus saponins published between 1983 and 2014, leading to different molecular clusters grouping structurally close saponins and separating dissimilar ones. Clustering highlighted hierarchical factors governing structural diversification of cycloartane-based saponins in Astragalus, including rare aglycone forms, rare substitution positions and relative substitution levels of the carbon C3. Rare cycloartane forms were due to different epoxydations occurring at different degrees and positions in the lateral part of cycloartane. In common cycloartane forms, rare saponins were due to rare substituted positions consisting of atypical hydroxylations. Within the most common saponins, several classes were highlighted by the variations of C3-substitution levels in both epoxydated and not epoxydated cycloartane. Structural variations within and between saponin classes provided a strong basis for highlighting significant effects of cycloartane forms and C3-substitution levels on molecular ramification, elongation levels and substitution types (glycosylation and acetylation). Finally, these two key structural factors seemed to influence compartmental distribution of saponins in Astragalus genus.     

Influence of nitric oxide synthase inhibition on vasopressin and corticosterone secretion during water deprivation in rats

Nitric oxide (NO) is a short-lived radical that functions as a neurotransmitter in the central nervous system and plays a physiological role in the regulation of hypothalamic–pituitary–adrenal axis and vasopressinergic axis. In the present study, we aimed to investigate the interaction between the generation of NO and vasopressin (AVP) and corticosterone release after 3 days of water deprivation in rats. Animals were previously treated with intraperitoneal (i.p.) saline or l-nitro-arginine methyl ester (L-NAME) injection. l-NAME is a nonspecific inhibitor of nitric oxide synthases. In control rats given i.p. saline or l-NAME, hypothalamic, pituitary, and plasma AVP levels and plasma corticosterone did not change from baseline levels (p > 0.05). Three days of water deprivation increased significantly the corticosterone levels in plasma (p < 0.01) and AVP levels in hypothalamus and plasma (p < 0.01), but not in pituitary, which showed a significant decrease. These variations were concomitant with the elevation of nitrates/nitrates in plasma. l-NAME injection abolished significantly (p < 0.01) the elevation of plasma corticosterone and hypothalamic AVP levels induced by water deprivation. These findings showed that in water-deprived rats, nitric oxide synthase inhibition by l-NAME inhibits corticosterone and vasopressin release, suggesting a potent stimulatory role of NO.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

Occurrence and levels of pesticides in South Lebanon water

This study reviews the detection of pesticides in different surface and groundwater samples collected from South Litani region in South Lebanon during 2012. These have been analyzed using an optimized and validated solid phase extraction method followed by gas chromatography coupled with mass spectrometry. Organochlorine and organophosphate pesticides were mostly noted at levels below the recommended value for individual pesticide in water except pirimiphos-methyl that was recorded at 300.87 ng L^?1 in groundwater sample, designated for drinking water and collected in February. DDE concentration exceeded 100 ng L^?1 in both surface and groundwater in October. The reported results represent the first Lebanese statistical data illustrating the quantification of pesticides in water over a period of time. More importantly, it draws attention to the need of pesticides’ monitoring programs in the Lebanese water resources.