Evaluation of the leptin receptor in human spermatozoa

Background  

Leptin, a 167 amino acid peptide hormone, profoundly effects reproduction exerting its biological effects via interaction with the leptin receptor (ObR) which is widely expressed on peripheral tissues. In this study, we have attempted to assess leptin receptor expression in the spermatozoa of fertile males and those diagnosed with male factor infertility; both at the mRNA or protein levels.     

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.     

Biomimetic oxidation of Hantzsch 1,4-dihydropyridines with tetra-n-butylammonium periodate catalyzed by tetraphenylporphyrinatomanganese(III) chloride [Mn(TPP)Cl

Efficient oxidation of Hantzsch 1,4-dihydropyridines to their corresponding pyridine derivatives with (Bu(4)N)IO(4) catalyzed by tetraphenylporphyrinatomanganese(III) chloride [Mn(TPP)Cl] is reported. This catalytic system shows high efficiency in the oxidation of 1,4-dihydropyridines at room temperature in the presence of imidazole.     

The influence of peroxisome proliferator-activated receptor γ(1) during differentiation of mouse embryonic stem cells to neural cells

Peroxisome proliferator activated receptor γ, belongs to PPARs, which exerts various metabolic functions including differentiation process. To testify the importance of PPARγ in neural differentiation of mouse embryonic stem cells (mESCs), its expression level was assessed. Data revealed an elevation in expression level of PPARγ when neural precursors (NPs) are formed upon retinoic acid treatment. Thus, involvement of PPARγ in two stages of neural differentiation of mESCs, during and post-NPs formation was examined by application of its agonist and antagonist. Our results indicated that PPARγ inactivation via treatment with GW9662 during NPs formation, reduced expression of neural precursor and neural (neuronal and astrocytes) markers. However, PPARγ inactivation by antagonist treatment post-NPs formation stage only decreased the expression of mature astrocyte marker (Gfap) suggesting that inactivation of PPARγ by antagonist decreased astrocyte differentiation. Here, we have demonstrated the stage dependent role of PPARγ modulation on neural differentiation of mESCs by retinoic acid treatment for the first time.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

Comparison of Glutathione S-transferase Activity and Concentration in Aflatoxin-Producing and their Non-Toxigenic Counterpart Isolates

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 mumol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 mumol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.     

The origin of reactive oxygen species in mouse embryos cultured in vitro.

The increase in production of reactive oxygen species such as H2O2 at the G2/M phase of the second cell cycle may be related to the in vitro block to development of mouse 2-cell embryos. The occurrence of the H2O2 rise is independent of the activation of the embryonic genome and of passage through the S, G2 and M phases of the first cell cycle and G1 and M phases of the second cell cycle, but does require the activation of the unfertilized oocyte. The H2O2 is produced via dismutation of superoxide by the enzyme superoxide dismutase. Production of superoxide via mitochondrial, NADPH-oxidase and xanthine/xanthine oxidase systems has been investigated. The evidence suggests that superoxide, and thereby H2O2, is produced by the xanthine/xanthine oxidase system, but an involvement of the other superoxide generating systems has not been excluded. The relation between H2O2 and development in vitro is discussed.     

Neuronal apoptosis inhibitory protein,NAIP, is an inhibitor of procaspase-9

Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.