Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

Change in membrane fatty acid compositions and cold-induced responses in chickpea

Plant cells often increase cold tolerance by reprogramming their genes expression which results in adjusted metabolic alternations, a process enhanced under cold acclimation (CA) phase. In present study, we assessed the changes of membrane fatty acid compositions and defense machine (like antioxidative enzymes) along with damage indexes like electrolyte leakage index (ELI) and malondialdehyde (MDA) during CA, cold stress (CS) and recovery (R) phases in chickpea (Cicer arietinum L.). Results showed an increase in unsaturated fatty acids ratio compare to saturated ones which is a sign of cold tolerance especially after CA phase. Antioxidant enzymes had an important role during CA and R phases while CS affected their activity which can be a sign for associating other metabolites or enzymes activities to create cold tolerance in plants. To investigation of enzymes assay under experimental treatments, the expression pattern of some enzymes including superoxide dismutase (sod), catalase (cat) and lipoxygenase (lox) was studied using quantitative real time PCR. LOX activity has shown a bilateral behavior: a positive relation with membrane damage index in CA and an interesting link with double bond index (DBI) in CS indicating probably its role in secondary metabolites like jasmonic acid signaling pathway. It was suggested that increased DBI and low LOX activity under CS could be a reason for plant cold tolerance.     

Towards β-globin gene-targeting with integrase-defective lentiviral vectors

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.     

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.     

The Effects of Oral Consumption of Selenium Nanoparticles on Chemotactic and Respiratory Burst Activities of Neutrophils in Comparison with Sodium Selenite in Sheep

The present study was designed to compare the effects of nano-selenium and of sodium selenite on the chemotactic and respiratory burst activities of neutrophils in sheep. Fifteen sheep were randomly divided into three groups. Groups 1 and 2 received selenium nanoparticles (1 mg/kg) or sodium selenite (1 mg/kg) orally, respectively, for ten consecutive days, and the third group was considered as the control. To determine the chemotactic and respiratory burst activities of the neutrophils, the leading front assay and the NBT test were used on heparinized blood samples that were collected at different intervals (days 0, 10th, 20th, and 30th). The results obtained showed that the chemotactic activities in groups 1 and 2 increased significantly on the 10th, 20th, and 30th day, compared to day 0, and on the 20th day in comparison with the 10th day, while in group 2, there was a significant decrease on the 30th day compared to the 20th day. The chemotactic activities in group 1 were significantly higher than in group 2 on the 10th day and in the control group on the 10th, 20th, and 30th day, but the chemotactic activities in group 2 were significantly higher than those in the control group only on the 20th day. On the 30th day into the experiment, the respiratory bursts in groups 1 and 2 were significantly stronger in comparison with those at day 0. Overall, nano-selenium increased the chemotactic and respiratory burst activities more significantly than sodium selenite, which is suggestive of a stronger stimulatory effect of the Se nanoparticles on intracellular activities.     

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of F?rster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding.     

Investigations on possible roles of C-terminal propeptide of a Ca-independent α-amylase from bacillus

Previously, an extracellular α-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [BKAΔ(N44) and BKAΔ(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The Km and V(max) values for BKAΔ(N44) were lower than BKAΔ(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.     

DNA binding studies of 3, 5, 6-trichloro-2-pyridinol pesticide metabolite

3, 5, 6-Trichloro-2-pyridinol (TCP) is a stable metabolite of two major pesticides, Chlopyrifos insecticide and Triclopyr herbicide, which are widely used in the world. The potential health hazard associated with TCP is identified due to its high affinity to the DNA molecule. Therefore, in this study, the interaction of native calf thymus DNA with TCP has been investigated using spectrophotometric, circular dichroism (CD), spectrofluorometric, viscometric and voltametric techniques. It was found that TCP molecules could interact with DNA via a groove-binding mode, as evidenced by hyperchromism, with no red shift in the UV absorption band of TCP, no changes in K(b) values in the presence of salt, no significant changes in the specific viscosity and CD spectra of DNA, and a decrease in peak currents with no shift in the voltamogram. In addition, TCP is able to release Hoechst 33258, a strong groove binder, in the DNA solutions. The results are indicative of the groove-binding mode of TCP to DNA.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.