Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Bacterial monitoring by molecular tools of a continuous stirred tank reactor treating textile wastewater

This study was performed to examine the effect of the bacterial diversity changes on the performances of a continuously stirred tank reactor (CSTR) treating textile wastewater. The molecular fingerprint established using polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) methods showed that bacterial community profiles changed simultaneously with the increase of the wastewater loading rates (WLR). For the two WLR of 0.28 g l(-1)d(-1) and 0.37 g l(-1)d(-1), the reactor maintained good performances, suggesting that the large bacterial community present a high specific activity. The increase of the WLR from 0.37 to 1.12 g l(-1)d(-1) decreased the colour and the chemical oxygen demand (COD) removal efficiencies from 90% to 55% and from 85% to 30%, respectively, explained by the decrease of the bacterial diversity and activity. The changes of the bacterial dominance had no affect on the reactor performances. However, the decrease of the bacterial diversity significantly affected the colour and the COD removal efficiencies. It should conclude that indigo dye-containing textile wastewater treatment required the concerted activity of multiple bacterial populations.     

Lipoprotein lipase deficiency is associated with elevated acylation stimulating protein plasma levels

Acylation stimulating protein (ASP, C3adesArg) is an adipose tissue derived hormone that stimulates triglyceride (TG) synthesis. ASP stimulates lipoprotein lipase (LPL) activity by relieving feedback inhibition caused by fatty acids (FA). The present study examines plasma ASP and lipids in male and female LPL-deficient subjects primarily with the P207L mutation, common in the population of Quebec, Canada. We evaluated the fasting and postprandial states of LPL heterozygotes and fasting levels in LPL homozygotes. Homozygotes displayed increased ASP (58–175% increase, P < 0.05–0.01), reduced HDL-cholesterol (64–75% decrease, P < 0.0001), and elevated levels of TG (19–38-fold, P < 0.0001) versus control (CTL) subjects. LPL heterozygotes with normal fasting TG (1.3–1.9 mmol/l) displayed increased ASP (101–137% increase, P < 0.05–0.01) and delayed TG clearance after a fatload; glucose levels remained similar to controls. Hypertriglyceridemics with no known LPL mutation also had increased ASP levels (63–192% increase, P < 0.001). High-TG LPL heterozygotes were administered a fatload before and after fibrate treatment. The treatment reduced fasting and postprandial plasma ASP, TG, and FA levels without changing insulin or glucose levels. ASP enhances adipose tissue fatty-acid trapping following a meal; however in LPL deficiency, high ASP levels are coupled with delayed lipid clearance.     

Synthesis, dihydrofolate reductase inhibition, antitumor testing, and molecular modeling study of some new 4(3H)-quinazolinone analogs

In order to produce potent new leads for anticancer drugs, a new series of quinazoline analogs was designed to resemble methotrexate (MTX, 1) structure features and fitted with functional groups believed to enhance inhibition of mammalian DHFR activity. Molecular modeling studies were used to assess the fit of these compounds within the active site of human DHFR. The synthesized compounds were evaluated for their ability to inhibit mammalian DHFR in vitro and for their antitumor activity in a standard in vitro tissue culture assay panel. Compounds 28, 30, and 31 were the most active DHFR inhibitors with IC₅₀ values of 0.5, 0.4, and 0.4 μM, respectively. The most active antitumor agents in this study were compounds 19, 31, 41, and 47 with median growth inhibitory concentrations (GI₅₀) of 20.1, 23.5, 26.7, and 9.1 μM, respectively. Of this series of compounds, only compound 31 combined antitumor potency with potent DHFR inhibition; the other active antitumor compounds (19, 41, and 47) all had DHFR IC₅₀ values above 15 μM, suggesting that they might exert their antitumor potency through some other mode of action. Alternatively, the compounds could differ significantly in uptake or concentration within mammalian cells.     

Enhancement of morphological, anatomical and physiological characteristics of seedlings of the mangrove Avicennia marina inoculated with a native phosphate-solubilizing isolate of Oceanobacillus picturae under greenhouse conditions

One hundred and twenty-nine rock phosphate (RP) solubilizing bacteria were isolated from the mangrove Avicennia marina rhizosphere sediment deficient in available phosphorus (P), in the United Arab Emirates (UAE). Among these isolates, an outstanding isolate of Oceanobacillus picturae grown in Pikovskaya’s liquid medium amended with RP, caused the highest percentage of RP solubilization (97%), the most pronounced drop in the medium pH and produced a variety of organic acids, as well as acid and alkaline phosphatases. Greenhouse experiments were carried out to investigate the effect of sediment inoculation with O. picturae (Op) with or without RP amendment on the growth, nutrient uptake, conducting tissue and photosynthetic gas exchange characteristics of mangrove seedlings. The application of Op to sediments amended with RP significantly promoted the growth of roots and shoots of seedlings compared with those grown in sediment amended with RP only. O. picturae significantly increased available sediment P, decreased sediment pH, positively enhanced nutrient uptake parameters in roots and shoots, increased stem circumference, number of xylem vessels, mean xylem diameter, and the hydraulically weighted xylem vessel diameter, compared with plants grown in non-inoculated sediment amended with only RP. Mechanistic analysis of A:Ci response curves (Assimilation versus sub-stomatal CO₂ concentration) showed that photosynthetic gas exchange characteristics were significantly enhanced by Op?+?RP treatment compared with RP alone or control treatment. The maximal Rubisco-catalyzed carboxylation velocity (V _c,max), increased by more than two fold in plants treated with RP?+?Op, over the control. Application of Op?+?RP greatly increased the maximal electron transport rate values (J _max) by 185%, over the control plants. In addition, the triose phosphate utilization rate (V _TPU) increased three fold in plants treated with RP?+?Op over the control. To the best of our knowledge, this is the first published report of Op from mangrove sediments and also the first report of this organism as a phosphate-solubilizing bacterium. These results have clearly shown that Op has the potential to be applied as an effective and economical treatment in association with RP amendment for mangrove reforestation programs in arid environments such as those found in the UAE.     

Assessment of insecticidal activity of red pigment produced by the fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Insecticidal activity of the red pigment produced by a strain of the fungus Beauveria bassiana that was locally isolated from infected whitefly, Bemisia tabaci (Genn.) was assessed. The pigment is produced extracellularly and is a water-soluble. This makes it easy and simple to be recovered from fermentation broth and used in pathogenicity experiments. When applied alone to Bm. tabaci nymphs, mortality percentages of 18% was recorded. For nymphs treated with Bv. bassiana spore suspension, mortality was 60%. The best results were obtained when red pigment was combined with fungal spores with the mortality percentage being increased to up to 92%. The highest insecticidal activity against adults emerging later on from the surviving larvae of Bm. tabaci was recorded also with treatment combining pigment and fungal spores with the longest days to pupation.     

Synthesis of novel pyrrole and pyrrolo[2,3-d]pyrimidine derivatives bearing sulfonamide moiety for evaluation as anticancer and radiosensitizing agents

Pyrroles and pyrrolo[2,3-d]pyrimidines were reported to act as potent anticancer agents, in this work, a series of novel 2-substituted-3-cyano-4-phenyl-pyrrole 5, 6, 11–18, and 5-phenyl-pyrrolo[2,3-d]pyrimidine derivatives 7–10, 19–24 bearing either sulfathiazole or sulfapyridine were synthesized. The structures of these compounds were confirmed by elemental analysis, IR, ¹H NMR and mass spectral data. All the newly synthesized compounds were evaluated for their in vitro cytotoxicity against liver and breast cancer cell line (HEPG2 and MCF7). Most of the screened compounds showed interesting cytotoxic activities compared with the used reference drug (doxorubicin). The radiosensitizing ability of some of the synthesized compounds was studied and the results showed an increase in the cell killing effect of γ-radiation after combination with the tested compounds.     

A novel chemotype of kinase inhibitors: Discovery of 3,4-ring fused 7-azaindoles and deazapurines as potent JAK2 inhibitors

Pictet–Spengler condensation of aldehydes or alpha-keto-esters with 4-(2-anilinophenyl)-7-azaindole (11) or deazapurine (12) gave high yields of the 3,4-fused cyclic compounds. SAR studies, by varying the substituted benzaldehyde components, lead to the discovery of a series of potent JAK2 kinase inhibitors.