Inhibition of protein synthesis in cortical neurons during exposure to hydrogen peroxide

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.     

Ghrelin regulates Bax and PCNA but not Bcl-2 expressions following scrotal hyperthermia in the rat

More recently, we have reported the beneficial effects of ghrelin in improvement of histopathological features of the rat testis following local heat exposure. However, the exact mechanism and the precise role of apoptosis- and proliferation-specific proteins in this regeneration process remained to be explored. Thus, thirty adult male Wistar rats were allotted for the experiment and subdivided equally into three groups: control-saline (CS), heat-saline (HS) and heat-ghrelin (HG). The scrota of HS and HG groups were immersed once in water bath at 43 °C for 15 min. HG animals received 2 nmol of ghrelin subcutaneously immediately after heating every other day until day 60 and the other groups were given physiological saline using the same method. The testes of all groups were taken after rat killing on days 30 and 60 after heat treatment for immunocytochemical detection of pro-apoptotic factor Bax, anti-apoptotic protein Bcl-2 and proliferation-associated peptide PCNA in the germ cells. Ghrelin could significantly suppress the Bax expression in spermatocytes compared to the HS group at day 30 (P < 0.05). Likewise, the mean percentages of spermatogonia containing Bax substance were lower in ghrelin-exposed animals, however the differences were not statistically significant. There were immunoreactive cells against Bcl-2 in each germ cell neither in the control nor in the heated animals of experimental groups. In contrast, the number of PCNA immunolabeling cells were higher in HG group in compared to HS or CS animals on both experimental days (P < 0.001). Down-regulation of Bax expression concurrent with overexpression of PCNA in HG group indicates the ability of ghrelin in acceleration of testicular germ cells regeneration following heat stress. These findings indicate that ghrelin may be used as a novel and efficient antioxidant agent to induce resumption of spermatogenesis upon environmental heat exposure.     

Antioxidant Defense of Betaine Against Oxidative Stress Induced by Ethanol in the Rat Testes

Oxidative stress is one of the factors associated with decline in fertility and betaine has been shown to bear antioxidant and methyl donor properties in our recent studies. Thus, we designed the present study to examine antioxidant and methyl donor abilities of betaine in oxidative stress induced by ethanol in the rat testes. The adult male Sprague-Dawley rats were divided into four experimental groups and treated daily for 2?months as follows: control, ethanol (4?g/kg, orally), betaine (1.5?% of total diet, orally), and betaine plus ethanol (betaine, 1.5?% of total diet and after 120?min, ethanol 4?g/kg). Sperm motility and concentration significantly increased in betaine group when compared to the ethanol?Ctreated rats. The main antioxidant enzyme (GPx) activity significantly increased (in order compensatory) in ethanol-treated rats when compared to betaine group while, antiperoxidative enzyme (CAT) activity significantly increased in betaine plus ethanol group as compared to ethanol-treated rats. Total homocysteine (tHcy) and TBARS concentration (as a lipid peroxidation marker) also significantly decreased in betaine and betaine plus ethanol groups as compared to ethanol-treated rats. Overall, methyl donor and antioxidant properties of betaine are promising and reduce the elevated tHcy and TBARS concentrations in betaine plus ethanol group. Therefore, betaine might be used as a potential therapy in hyperhomocysteinemia and oxidative stress induced by ethanol in alcoholism.     

Ghrelin is a Regulator of Cellular Apoptosis and Proliferation in the Rat Ovary

Apoptosis and proliferation are the common and essential events of reproductive function and development in the ovary, especially during follicular growth and atresia or luteal regression. Therefore, this study was set to investigate the influence of ghrelin treatment on apoptosis and proliferation specific indices in the rat ovary. Twenty-eight adult female Wistar rats were randomly allocated into control and treatment groups. Treatment group (n = 14) received 3 nmol of ghrelin as subcutaneous injection for 14 consecutive days or vehicle (normal saline) to the control rats. The animals from each group were equally sacrificed on days 9 and 14 after onset of ghrelin treatment and their ovaries were taken for immunohistochemical evaluation and caspase-3 assay. Accumulation of apoptosis-associated peptide Bax was significantly reduced following ghrelin treatment particularly in granulosa and luteal cells on day 14 (P < 0.01). In contrast, immunoreactivity against anti-apoptotic protein Bcl-2 was significantly elevated in ghrelin-exposed animals in granulosa, theca and luteal cells (P < 0.05). However, ghrelin administration was not able to change caspase-3 activity prominently, so that the means of enzyme activity were not statistically significant between groups (P > 0.05). Moreover, significant up-regulation of proliferation-associated peptide PCNA was also seen in the granulosa, theca and luteal cells of ghrelin-treated rats by day 14 (P < 0.05), but not on day 9. These findings indicate the first evidence of ghrelin involvement in the control of key gonadal functions, apoptosis and proliferation in the rat ovary, which is mainly mediated through decrease in Bax/Bcl-2 ratio consistent with upstream of PCNA level, however not depends on the reduction of caspase-3 activation. This may have potential implications that ghrelin can be considered as an apoptotic modulator of some ovarian disorders.     

Oleuropein prevents ethanol-induced gastric ulcers via elevation of antioxidant enzyme activities in rats

Purified oleuropein from olive leaf extract has been shown to have antioxidant effects in our recent studies. Thus, the aim of this study was to assess the antioxidant abilities of oleuropein in comparison with ranitidine in ethanol-induced gastric damages via evaluation of ulcer index inhibition, antioxidant enzyme activities, and lipid peroxidation level. Fifty-six adult male Sprague?CDawley rats were divided into seven equal groups as follows: control group, ethanol group (absolute ethanol 1?ml/rat), oleuropein group (12?mg/kg), and oleuropein (6, 12, and 18?mg/kg) plus ethanol groups, as well as ranitidine (50?mg/kg) plus ethanol group. Pretreatment with oleuropein (12 and 18?mg/kg) significantly increased the ulcer index inhibition (percent), in comparison with oleuropein (6?mg/kg). Glutathione peroxidase (GPx) activity was significantly lower in the ethanol group when compared with the other groups whereas, treatment of rats with oleuropein (12?mg/kg) significantly increased glutathione content in gastric tissue when compared with the other groups, and lipid peroxidation was significantly reduced in the oleuropein- (12 and 18?mg/kg) and ranitidine-treated animals. Superoxide dismutase (SOD) and catalase (CAT) activities were both much higher in oleuropein-treated rats than the ethanol group, and although there was a moderate increase in SOD and CAT activities in ranitidine-treated rats, the differences were not significant. These findings suggest that oleuropein has beneficial antioxidant properties against ethanol-induced gastric damages in the rat. Therefore, it seems that a combination regimen including both antioxidant and antisecretory drugs may be beneficial in prevention of ethanol-mediated gastric mucosal damages.     

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n=7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P<0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P<0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P>0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.     

The plant histaminase: a promising enzyme with antioxidant properties versus histamine release in isoprenaline-induced myocardial infarction in rats

The present study was designed to evaluate possible protective effects of purified histaminase from Lathyrus sativus L. seedling on the myocardial injuries upon isoprenaline-induced myocardial infarction in rats. In this regard, blood histamine concentration, creatine kinase-MB (CK-MB) activity, antioxidant status, and histopathological changes of the hearts were measured. A total of 40 adult male Sprague–Dawley rats were divided into five equal groups and treated in the following order: control (normal saline), isoprenaline (isoproterenol 110 mg/kg BW), Isopren.-H₁ (isoprenaline plus histaminase 80 U/kg BW), Isopren.-H₂ (isoprenaline plus histaminase 120 U/kg BW), and Isopren.-H₃ (isoprenaline plus histaminase 160 U/kg BW). Myocardial infarction was manifested by a significant elevation in the level of CK-MB and histopathological findings in isoprenaline group when compared to controls. In contrast, histaminase pretreatment at dose of 160 U/kg prevented isoprenaline-induced histamine release and significantly decreased CK-MB activity as well as histopathological changes in Isopren.-H₃ group. A significant increase in the catalase (CAT) and superoxide dismutase (SOD) activities was also observed by histaminase treatment in Isopren.-H₂ and Isopren.-H₃ groups. Although the activity of glutathione peroxidase (GPx) increased significantly to suppress oxidative stress in isoprenaline group, it was not able to prevent lipid peroxidation (as shown by TBARS concentration) in the heart of rats. In conclusion, the plant-originated histaminase presented as a promising enzyme with antioxidant properties against histamine release and myocardial infarction in rats, and it seems be a suitable therapeutic agent for future clinical trials in humans.     

Coxsackievirus Infection Induces Autophagy-Like Vesicles and Megaphagosomes in Pancreatic Acinar Cells In Vivo

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.Macroautophagy—henceforth referred to as autophagy—is an intracellular process that is important for cellular differentiation, homeostasis, and survival. Through autophagy, long-lived cytosolic proteins and organelles become encapsulated within double-membraned vesicles, called autophagosomes, which fuse with lysosomes to facilitate degradation of protein and cellular organelles and to promote nutrient recycling/regeneration. Autophagy plays a key role in the host immune response to infection by viruses, bacteria, fungi, and parasites (reviewed in references 10 and 62). Within virus-infected cells, whole virions and/or viral proteins and nucleic acids are captured inside autophagosomes and degraded (following lysosomal fusion) through the process of xenophagy. Moreover, autophagosome fusion with the endosomal/lysosomal pathway facilitates Toll-like receptor recognition of viral materials and delivers endogenous cytosolic viral proteins to the major histocompatibility complex (MHC) class II antigen presentation pathway, which in turn may help to trigger activation of innate immunity (and type I interferon production) and promote antigen presentation to virus-specific CD4⁺ T cells (reviewed in references 9, 41, 44, 47, 72, and 90). A recent study has shown that autophagy is also involved in the processing and presentation of MHC class I-restricted viral epitopes (13).Given the importance of autophagy in antiviral immunity, it is perhaps not surprising that viruses have evolved mechanisms to evade and/or subvert this pathway (reviewed in references 9, 11, 14, 35, 37, 60, 61, and 77). Several members of the herpesvirus family, most notably herpes simplex virus type 1, inhibit autophagy within an infected cell and encode proteins that block and/or target intracellular signaling pathways that regulate autophagy (reviewed in references 60 and 61). However, some viruses not only evade autophagy but also appear to take advantage of the process; several RNA viruses induce autophagy and exploit the pathway during their replication (1, 12, 15, 31, 40, 43, 76, 93, 96). Viruses belonging to the Picornaviridae family and the Nidovirales order replicate their genomes on double-membraned vesicles that resemble autophagosomes; these vesicles are notably smaller in size than cellular autophagosomes and are decorated with proteins derived from the autophagic pathway (19, 21, 31, 37, 67, 68, 71, 92). Viral proteins encoded by poliovirus and equine arterivirus can trigger the formation of these autophagy-like vesicles (79, 80), and the expression of a single poliovirus protein, 2BC, is sufficient to induce lipidation of the host autophagy protein light chain 3 (LC3), encoded by the Atg8 gene (87). Taken together, these studies suggest that some viruses subvert the autophagy pathway to generate double-membraned vesicles that provide a surface for RNA replication (8, 37, 88). In addition, these vesicles may permit newly formed virions to escape from infected cells via a nonlytic route (36, 85).Although studies have demonstrated that the autophagic pathway may play an important role in virus infection in vitro, either to promote or to restrict viral replication, we are just beginning to appreciate and understand the function and effects of autophagy for virus infections in vivo. Autophagy acts in an antiviral fashion to limit tobacco mosaic virus replication and programmed cell death in plants (46), to prevent a pathogenic infection with vesicular stomatitis virus in flies (73), and to protect against fatal encephalitis in Sindbis virus- or herpes simplex virus type 1-infected mice (45, 59, 63). Nonetheless, to date there is a dearth of in vivo studies; animal models of virus infection are needed in order to better define the antiviral role of autophagy in vivo (41, 62). In addition, studies that address the role of viral subversion of autophagy in vivo are warranted. Does this process occur within infected animals, and is it required for viral replication in particular cell types or for viral pathogenesis? Recent studies have shown that autophagy not only promotes the replication of hepatitis B virus and enterovirus 71 in vitro but also may be induced by infection in vivo, potentially to benefit the virus rather than the host (28, 78).Type B coxsackieviruses (CVBs) are members of the Picornaviridae family and Enterovirus genus and, as such, are closely related to polioviruses. CVBs are important human pathogens that often induce severe acute and chronic diseases and cause morbidity and mortality (69, 91). CVBs are the most common cause of infectious myocarditis (38, 82) and frequently trigger pancreatitis and aseptic meningitis (7, 16, 29, 51). Tissue culture studies (93) have shown that CVB type 3 (CVB3) promotes LC3 conversion and autophagosome accumulation in virus-infected cells in vitro and that modulation of the autophagic pathway (using chemicals or small interfering RNA-mediated knockdown) to enhance or dampen autophagy results in an increase and a decrease, respectively, in viral protein expression and/or viral titers; however, the reported changes in viral titers were modest (2- to 4-fold). In the present study, we examine whether CVB3 activates the autophagic pathway in vivo, specifically in pancreatic acinar cells, which are a natural primary target for this virus. Using a mouse model of CVB3 infection, which faithfully recapitulates most aspects of CVB disease in humans, we demonstrate that this virus triggers LC3 conversion and also modulates other components of the autophagy machinery. In addition, using a recombinant CVB3 (rCVB3) that expresses Discosoma sp. red fluorescent protein (DsRed-CVB3), we identify virus-infected cells in situ and show that CVB3 infection increases autophagosome abundance in vivo. Lysosomal-associated membrane protein 1 (LAMP-1) immunostaining confirmed that amphisomes are generated in virus-infected cells but that autophagic flux was not substantially enhanced as the infection progressed; rather, there appears to be a substantial blockade in fusion with lysosomes. Finally, transmission electron microscopy (TEM) ultrastructural analysis of the infected pancreas confirmed that double-membraned autophagy-like vesicles as well as very large autophagic compartments (for which we have coined the term “megaphagosomes”) were generated in acinar cells following virus infection. Overall, these data provide compelling evidence that CVB3 induces autophagy in vivo and suggest that this picornavirus may subvert this process in a mammalian host.     

Betaine: A Promising Micronutrient in Diet Intervention for Ameliorating Maternal Blood Biochemical Alterations in Gestational Diabetes Mellitus

International Journal of Peptide Research and Therapeutics - Gestational diabetes mellitus (GDM) exact pathophysiology remain elusive to date, nevertheless most of the studies are compatible with...