Evaluation of <Emphasis Type="Italic">GPx1</Emphasis> Pro198Leu polymorphism in idiopathic male infertility

Infertility is defined as failure to conceive a child after 1 year of unprotected regular sexual intercourse. Approximately half of all cases of infertility are caused by factors related to the male. In nearly 50% of infertile men it is not possible to determine the cause of infertility and this situation has been defined as unexplained or idiopathic. Oxidative stress plays an important role in the pathophysiology of male infertility. Oxidative stress results from an imbalance in free radicals and antioxidant defense mechanisms of the body. Genetic variations in the antioxidant gene coding for GPx enzyme may lead to decreased or impaired regulation of its enzymatic activity and alter reactive oxygen species (ROS) detoxification. We have investigated the possible association between polymorphism GPx1 Pro198Leu and idiopathic male infertility. One hundred patients with idiopathic male infertility and one hundred fifty healthy volunteers were enrolled. Genomic DNA was extracted from blood samples. Genotyping for the GPx1 Pro198Leu polymorphism was done by PCR–restriction fragment length polymorphism (RFLP) using ApaI. The genotype frequencies were 11% (Leu/Leu), 76% (Pro/Leu) and 13% (Pro/Pro) in the patient group and 8.7% (Leu/Leu), 67.3% (Pro/Leu) and 24% (Pro/Pro) in the control group. The genotype and allele frequencies of GPx1 Pro198Leu did not differ between the patient group and the control group (P = 0.09 and P = 0.1, respectively). In conclusion, there is no correlation between idiopathic male infertility and the GPx1 codon Pro198Leu polymorphism. Further studies are needed to investigate other genetic factors that influence the development of idiopathic male infertility.     

Effects of ghrelin on insulin and glucagon secretion: a study of isolated pancreatic islets and intact mice

We combined in vitro and in vivo methods to investigate the effects of ghrelin, a novel gastric hormone, on insulin and glucagon release. Studies of isolated mouse islets showed that ghrelin concentrations in the physiological range (0.5-3 nmol l(-1)) had no effect on glucose-stimulated insulin release, while low ghrelin concentrations (1-100 pmol l(-1)) inhibited and high (0.1 and 1 micromol l(-1)) stimulated. The insulin response to glucose was enhanced in the presence of a high ghrelin concentration (100 nmol l(-1)). Glucagon release was stimulated by ghrelin (0.1 pmol l(-1) to 1 micromol l(-1)); this effect was maintained in the presence of glucose (0-20 mmol l(-1)). In intact mice, basal plasma insulin was suppressed by 1 and 10 nmol kg(-1) of ghrelin, 2 and 6 min after i.v. injection. Ghrelin (0.2-10 nmol kg(-1) i.v.) suppressed also the glucose-stimulated insulin response and impaired the glucose tolerance (at a ghrelin dose of 3.3 nmol kg(-1)). Ghrelin (1 or 10 nmol kg(-1) i.v.) inhibited the insulin response to the phospholipase C stimulating agent carbachol and enhanced the insulin response to the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX) but did not affect the response to the membrane-depolarizing amino acid l-arginine. These observations suggest that the inhibitory effect of ghrelin on glucose-induced insulin release is in part exerted on phospholipase C pathways (and not on Ca(2+)entry), while the stimulatory effect of high doses of ghrelin depends on cyclic AMP. In contrast to the spectacular glucagon-releasing effect of ghrelin in vitro, ghrelin did not raise plasma glucagon. Carbachol, IBMX and l-arginine stimulated glucagon release. These responses were impaired by ghrelin, suggesting that it suppresses the various intracellular pathways (phospholipase C, cyclic AMP and Ca(2+)), that are activated by the glucagon secretagogues. Together these observations highlight (but do not explain) the different effects of ghrelin on glucagon release in vitro and in vivo. The results show that ghrelin has powerful effects on islet cells, suggesting that endogenous ghrelin may contribute to the physiological control of insulin and glucagon release. However, the narrow "window" of circulating ghrelin concentrations makes this doubtful.     

Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl⁻-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic β cells. In ClC-3^−/− mice, insulin secretion evoked by membrane depolarization (high extracellular K⁺, sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a 80% reduction in depolarization-evoked β cell exocytosis (monitored as increases in cell capacitance) in single ClC-3^−/− β cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl⁻ fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.     

Toward immunomodulation of witches broom disease of lime (WBDL) by targeting immunodominant membrane protein (IMP) of candidatus Phytoplasma aurantifolia

The witches' broom disease of lime (WBDL) caused by Candidatus Phytoplasma aurantifolia is the most devastating disease of acidian lime in southern part of Iran as it destroy thousands of trees yearly throughout these regions. Traditional methods such as eradication of infected trees and insect vector control have shown limited effect on this case. Therefore, alternative approaches such as plantibody-mediated resistance, have been considered. Throughout present study we prepared sufficient amount of antigen that is required for generation of specific monoclonal recombinant antibodies against Immunodominant membrane protein (IMP) which will be exploited for plantibody-mediated resistance approach. The gene encoding IMP protein was obtained by PCR amplification using specific primers and DNA extracted from the infected plants. Amplified fragment was then inserted into T/A cloning vector. Intact clones containing the right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. IMP encoding region having the right sequence was sub-cloned into pET28a bacterial expression vector. Large scale expression of His tagged recombinant protein was performed in the BL21-de3 strain of E. coli and purification under native conditions was carried out through immobilized metal ion affinity chromatography (IMAC) in a column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE and western blotting analyses. The results obtained indicated the successful production of about 18 mg purified recombinant IMP protein with a low level of contamination in one liter cultured medium. Finally the purified protein was dialyzed in phosphate saline buffer and applied for immunization of mice.     

Marcusenius desertus sp. nov. (Teleostei: Mormyridae), a mormyrid fish from the Namib desert

We critically compared Marcusenius specimens from the mouth of the Cunene River on the Namibia/Angola border, a harsh desert environment on the Atlantic Ocean coast virtually devoid of aerial insects with aquatic larvae which are an important food item, with Marcusenius multisquamatus Kramer & Wink, 2013 from the escarpment region of that same river, in a relatively rich and productive subtropical savannah environment. River mouth specimens were differentiated in morphology and electric organ discharges, as determined by ANOVA/MANOVA comparisons, principal component and discriminant analyses on morphological and electrophysiological characters, and genetics, including sequences of the mitochondrial cytochrome b gene, indicating reproductive isolation. Specimens from the river mouth differed from M. multisquamatus, their closest relatives, by having a shorter snout, a smaller eye diameter, and smaller nares separation. River mouth specimens were also differentiated from other, increasingly less-close relatives, such as M. altisambesi Kramer et al., 2007 from the Okavango River, Botswana, and from M. krameri Maake et al., 2014 from the Limpopo System, South Africa. We therefore designate the new species Marcusenius desertus sp. nov. for the Cunene River mouth population.     

The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.     

Ciliary Neurotrophic Factor Role in Myelin Oligodendrocyte Glycoprotein Expression in Cuprizone-Induced Multiple Sclerosis Mice

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. Myelin oligodendrocyte glycoprotein (MOG) is a minor component of the myelin sheath, but is an important autoantigen linked to the pathogenesis of MS. Ciliary neurotrophic factor (CNTF) has been shown to enhance the generation, maturation, and survival of oligodendrocytes in culture medium. The aim of this study was to demonstrate the role of CNTF on MOG expression in the cerebral cortex of Cuprizone-induced MS mice. The mice were treated by Cuprizone for five weeks in order to induce MS. The mice were then divided into 3 groups. The first group was injected subcutaneously (SC) by CNTF in the amount of 250 μg/kg BW per day. The second group (SHAM) was injected SC by normal saline and the third group was left without injection as the control group. After four weeks the mice were killed and the cerebral cortex was harvested and the expression of MOG was studied by Western blotting. The data from this study show that the MOG expression was significantly increased in the CNTF-injected group as compared to the other groups. It is concluded that CNTF increases the MOG expression and may be important in the pathophysiology of MS. It is also concluded that CNTF may play a role in the process of remyelination by inducing the MOG expression.     

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal -glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the -cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G_i-proteins, abolished the inhibitory effect of the ₂-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G_s-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G_j-proteins, but most likely exerted solely through the selective suppression of lysosomal -glucosidehydrolase activity, a step in between the proximal and the distal G_i-proteins, in glucose-induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G_s-proteins to facilitate the secretory process.