Astrocyte swelling and protein tyrosine nitration in hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome during the course of acute or chronic liver disease. It is functional in nature, potentially reversible and precipitated by rather heterogeneous factors. Current evidence suggests that HE is the consequence of a low grade chronic glial edema with subsequent alterations of glioneuronal communication. Other lines of evidence support a role of NMDA receptor activation and oxidative/nitrosative stress in the pathogenesis of HE. Different HE-precipitating factors, such as ammonia, benzodiazepines, inflammatory cytokines and hyponatremia induce or aggravate astrocyte swelling and additionally increase oxidative/nitrosative stress and protein tyrosine nitration. Recent findings suggest a close interrelation between astrocyte swelling, NMDA receptor signaling, glutamate, oxidative stress and nitric oxide, which may result in mutual amplification of swelling and oxidative stress. Via such an autoamplificatory loop astrocytes may integrate HE-relevant signals triggered by HE-precipitating factors. This review focuses on the involvement of astrocyte swelling in protein tyrosine nitration and potential consequences in the setting of HE.     

A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.     

The Yin and Yang of redox regulation

Abstract

Mammalian cells produce reactive oxygen and nitrogen species (ROS/RNOS) in response to an oxidative environment. Powerful antioxidant mechanisms have been developed in order to avoid oxidative stress by contributing to the maintenance of redox homeostasis. Traditionally, accumulation of ROS/RNOS is considered deleterious for cells as it can lead to loss of cellular function, aging, and cell death. Consequently, ROS/RNOS imbalance has been implicated in the etiology and/or progression of numerous pathologies such as cardiovascular diseases, inflammation, and cancer. An interesting concept that has emerged more recently is that not only have cells developed efficient systems to cope with ROS/RNOS accumulation but they have also learned to profit of them under certain circumstances. This notion is supported by data showing that ROS/RNOS can act as signaling molecules affecting the function and activity of a multiplicity of protein kinases and phosphatases controlling cellular homeostasis. This review does not provide an exhaustive overview of molecular mechanisms linked to ROS/RNOS generation and processing but includes relevant examples highlighting the dichotomic nature of these small molecules and the multitude of effects elicited by their accumulation. This aspect of ROS/RNOS ought to be taken into account particularly in novel therapeutic setups that aim to achieve high efficiency and minimal or no side effects.     

Peripheral benzodiazepine receptors and mitochondrial function

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.     

A history of stress alters drought calcium signalling pathways in Arabidopsis

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (Bush, 1995; Knight et al., 1991). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress-responsive genes, including those encoding proteins of protective function (Knight et al., 1996; Knight et al., 1997). The kinetics and magnitude of the Ca2+ signal, or 'calcium signature', differ between different stimuli and are thought to contribute to the specificity of the end response (Dolmetsch et al., 1997; McAinsh and Hetherington, 1998). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of Arabidopsis thaliana. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress-induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre-treatment reduced the mannitol-induced [Ca2+]cyt response whereas osmotic stress pretreatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress-induced gene expression and, in the case of osmotic stress pre-treatment, the acquisition of stress-tolerance. This suggests that altered Ca2+ responses encode a 'memory' of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.     

环境胁迫下HOG-MAPK途径对真菌毒素形成的调控机制北大核心CSCD

真菌为了适应在生长侵染食品、饲料等农产品的过程中所面临的各种环境胁迫的考验,包括热胁迫、氧化胁迫、渗透压胁迫、紫外胁迫等,进化出一套高渗透性甘油促分裂原活化蛋白激酶(high osmolarity glycerol mitogen-activated protein kinase,HOG-MAPK)途径。该途径对真菌的生长发育、真菌毒素的产生和致病性都具有重要影响。HOG-MAPK途径共有两个分支,其中SLN1分支相比另一分支(SHO1分支)具有较为敏感的渗透压胁迫感应能力,能在高渗压和高盐浓度下进行渗透压胁迫反应。SHO1分支参与多种信号感应传导,比如氧化胁迫、热胁迫等。本文综述了真菌HOG-MAPK途径中关键基因sln1、sho1、ste11、ssk2、pbs2和hog1在应对渗透压胁迫、氧化胁迫等不同环境胁迫时所发挥的功能,说明HOG-MAPK途径可以响应多种环境信号,并参与调控黄曲霉、赭曲霉等致病真菌的生长和黄曲霉毒素(aflatoxin)、赭曲霉毒素(ochratoxin)等真菌毒素的产生。在不同环境胁迫下,HOG-MAPK途径对真菌毒素调控机制的研究可为食品和饲料等农产品真菌毒素的防控提供理论基础和指导方向。     

Impaired Purinergic Regulation of the Glial (Müller) Cell Volume in the Retina of Transgenic Rats Expressing Defective Polycystin-2

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD2_1/703 HA). Age-matched Sprague–Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD2_1/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y₁ or adenosine A₁ receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y₁ receptor antagonist was mediated by induction of oxidative–nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD2_1/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD2_1/703 rats, the gene expression levels of P2Y₁ and A₁ receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD2_1/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y₁ receptor activation serve to inhibit the induction of oxidative–nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

     

Peripheral benzodiazepine receptors in potatoes (Solanum tuberosum)

The peripheral benzodiazepine receptor (PBR), an internal protein of the mammalian mitochondrial membrane, is involved in several metabolic functions such as steroidogenesis, oxidative phosphorylation, and regulation of cell proliferation. Here we report the presence of PBRs in parenchymal and meristematic tissues of potato (Solanum tuberosum). PBRs are heterogeneously distributed in potato and are highly expressed in meristematic cells. In particular the receptor protein is mainly localised in the meristematic nuclear subcellular preparation. This 30-36 kDa protein, which corresponds to PBR, is increased, indeed, in meristematic compared to the parenchymal tissue. This suggests an involvement of this receptor in the regulation of cell plant growth. In addition, the demonstration that PBRs are also present in vegetables supports the hypothesis of a highly conserved receptor system during phylogenesis.     

Les endozépines, facteurs locaux de régulation de la stéroïdogenèse testiculaire

Testicular endocrine and exocrine functions are controlled by multiple signals including circulating gonadotropins and locally produced factors. Among these factors, endozepines (EZ), which are the endogenous ligands for benzodiazepine receptors, seem to exert an intracrine, autocrine and/or paracrine stimulatory effect on Leydig cell testosterone production. Benzodiazepine effects are mediated by two types of receptors, i.e. the central-type benzodiazepine receptor (CBR) associated with the GABAA-receptor complex, and the peripheral-type benzodiazepine receptor (PBR) principally located on the mitochondrial membrane and extremely abundant in steroidogenic cells. All EZ characterized to date are derived from an 86 amino acid polypeptide called diazepam binding inhibitor (DBI) that generates, via proteolytic cleavage, several biologically active peptides including the triakontatetraneuropeptide DBI17-50 (TTN) and the octadecaneuropeptide DBI33-50 (ODN). EZ are widely distributed in the brain and various peripheral organs, particularly in steroidogenic glands. A number of data suggest that, in rats, EZ could regulate testicular steroidogenesis. Firstly, DBI gene expression and the presence of DBI-like peptides have been shown in Sertoli cells, Leydig cells and in late-differentiated germ cells. Moreover, EZ are able to stimulate progesterone and testosterone production by rat Leydig cells and by MA-10 or R2C Leydig tumor cells. Finally, pharmacological studies have shown that EZ stimulate rat testicular steroidogenesis via activation of PBR. PBR appears to be an important component of a dynamic multistep process involving protein-protein interactions, to promote cholesterol translocation in the mitochondria, where it is converted into pregnenolone by cytochrome P450scc.     

Inhibition of poly(ADP-RIBOSE) polymerase (PARP) by nitric oxide and reactive nitrogen oxide species

The poly(ADP-ribose) polymerase (PARP) family of nuclear enzymes is involved in the detection and signaling of single strand breaks induced either directly by ionizing radiation or indirectly by the sequential action of various DNA repair proteins. Therefore, PARP plays an important role in maintaining genome stability. Because PARP proteins contain two zinc finger motifs, these enzymes can be targets for reactive nitrogen oxide intermediates (RNOS) generated as a result of nitric oxide (NO) biosynthesis in an aerobic environment. The effects of RNOS on the activity of purified PARP were examined using donor compounds. Both NO and nitroxyl (HNO) donors were found to be inhibitory in a similar time and concentration manner, indicating that PARP activity can be modified under both nitrosative and oxidative conditions. Moreover, these RNOS donors elicited comparable PARP inhibition in Sf21 insect cell extract and intact human MCF-7 cancer cells. The concentrations of donor required for 90% inhibition of PARP activity produce RNOS at a similar magnitude to those generated in the cellular microenvironment of activated leukocytes, suggesting that cellular scavenging of RNOS may not be protective against PARP modification and that inhibition of PARP may be significant under inflammatory conditions.     

Nuclear location-dependent role of peripheral benzodiazepine receptor (PBR) in hepatic tumoral cell lines proliferation

PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosphorylation and cell proliferation. The presence of PBR at the perinuclear/nuclear subcellular level has been demonstrated in aggressive breast cancer cell lines and human glioma cells where it seems to be involved in cell proliferation. In our study we investigated the presence of perinuclear/nuclear PBR in different hepatic tumor cell lines with regard to binding to [3H] PK 11195 and protein analysis. The results obtained by saturation binding experiments and scatchard analysis of perinuclear/nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the perinuclear/nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high perinuclear/nuclear PBR proliferated in response to PBR ligand, whereas that with low perinuclear/nuclear PBR did not. Our results reinforce the idea that the subcellular localisation of PBR defines its function and that this receptor could be a possible target for new strategies against cancer.     

Stress‐induced pyruvate accumulation contributes to cross protection in a fungus

It is commonly observed that microorganisms subjected to a mild stress develop tolerance not only to higher doses of the same stress but also to other stresses – a phenomenon called cross protection. The mechanisms for cross protection have not been fully revealed. Here, we report that heat shock induced cross protection against UV, oxidative and osmotic/salt stress conditions in the cosmopolitan fungus Metarhizium robertsii. Similarly, oxidative and osmotic/salt stresses also induced cross protection against multiple other stresses. We found that oxidative and osmotic/salt stresses produce an accumulation of pyruvate that scavenges stress‐induced reactive oxygen species and promotes fungal growth. Thus, stress‐induced pyruvate accumulation contributes to cross protection. RNA‐seq and qRT‐PCR analyses showed that UV, osmotic/salt and oxidative stress conditions decrease the expression level of pyruvate consumption genes in the trichloroacetic acid cycle and fermentation pathways leading to pyruvate accumulation. Our work presents a novel mechanism for cross protection in microorganisms.     

核黄素参与水稻非生物胁迫响应的分析

为探究核黄素在水稻非生物胁迫响应中的作用,以粳稻Kitaake和籼稻T98B为试验材料,考察了核黄素对2种材料的盐、高温、渗透、碱和氧化胁迫响应的影响,重点测定了盐和高温胁迫下水稻体内核黄素合成基因的表达和相关生理指标。结果表明,(1)施加外源核黄素有效提高了2种水稻材料的盐和高温胁迫耐受性,降低了渗透胁迫耐受性,而其氧化和碱胁迫耐受性不受影响。(2)逆境胁迫均不同程度地促进了核黄素在2种水稻材料中的积累,尤其在盐和高温胁迫下促进效果最明显。(3)盐和高温胁迫均诱导了核黄素合成酶基因的表达,促进了核黄素的生物合成,改善了水稻的胁迫耐受性。研究表明,非生物逆境胁迫能促进核黄素在水稻体内的合成和积累,外源核黄素也能明显提高水稻对盐和高温胁迫的耐受性,但却降低了其对渗透胁迫的耐受性。     

A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.     

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.     

Peripheral benzodiazepine receptor agonists exhibit potent antiapoptotic activities

The peripheral benzodiazepine receptor (PBR) has been implicated in several mitochondrial functions but the exact physiological role of this receptor is still under debate. Since the mitochondria have been attributed a central role in cell death, we have determined the effects of various PBR agonists and antagonists on the apoptosis of the human lymphoblastoid cell line U937. On this cell type, the PBR agonist Ro5-4864 was found to strongly protect the cells against apoptosis induced by TNFalpha. The antiapoptotic effect of PBR agonists was due to a selective interaction with the PBR as demonstrated by: (1) a close correlation between the antiapoptotic activity of various PBR agonists and their respective affinity for the PBR determined on the same cells, (2) a lack of effect of central benzodiazepine receptors agonists such as clonazepam on cell survival, (3) the lack of an antiapoptotic activity of Ro5-4864 on wild-type Jurkat cells (lacking the PBR receptor) and the reappearance of this effect on PBR-transfected Jurkat cells, and (4) the blockade of the antiapoptotic effect of PBR agonists by a selective PBR antagonist. The present results therefore indicate that PBR agonists are potent antiapoptotic compounds and show that this effect might represent a major function for this enigmatic receptor.     

Nitroxidation, nitration, and oxidation of a BODIPY fluorophore by RNOS and ROS.

BODIPY C11 581/591 (BODIPY11) represents a sensitive probe for quantification of relative antioxidant capacity. However, the mechanism of BODIPY11 fluorescence decay in the presence of reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) requires clarification. Azo-initiators provide a continuous source of peroxyl radicals that in simple, aerobic, homogeneous, buffered solution simulate lipid peroxyl radical formation. Inhibition of BODIPY11 fluorescence decay was assayed and quantified for several families of antioxidants, including phenols, NO donors, and thiols. Fluorescence decay of BODIPY11 in these systems demonstrated similar patterns of antioxidant activity to those observed in classical oxygen pressure measurements, and provided a readily applied quantification of antioxidant capacity and mechanistic information, which was analyzed by measurement of induction periods, initial rates, and net oxidation. LC/MS analysis confirmed that peroxyl radical-induced irreversible fluorescence decay of the BODIPY11 fluorophore is due to oxidative cleavage of the activated phenyldiene side chain. The behavior of BODIPY11 towards RNOS was more complex, even in these simple systems. Incubation of BODIPY11 with bolus peroxynitrite or a sydnonimine peroxynitrite source produced a variety of novel products, characterized by LC/MS, derived from oxidative cleavage, nitroxidation, and nitration reactions. The "NO scavenger" PTIO reinforced the antioxidant activity of NO, and inhibited BODIPY11 oxidation induced by the sydnonimine. These observations suggest that BODIPY11 is a well-behaved fluorescence probe for peroxidation and antioxidant studies, but that for study of RNOS even co-application of fluorescence decay with LC/MS measurements requires careful analysis and interpretation.