The ubiquitin-proteasome system is necessary for the replication of duck Tembusu virus.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.

Predictive and Descriptive CoMFA Models: The Effect of Variable Selection.

Aims & Scope: In this research, 8 variable selection approaches were used to investigate the effect of variable selection on the predictive power and stability of CoMFA models. MATERIALS & METHODS: Three data sets including 36 EPAC antagonists, 79 CD38 inhibitors and 57 ATAD2 bromodomain inhibitors were modelled by CoMFA. First of all, for all three data sets, CoMFA models with all CoMFA descriptors were created then by applying each variable selection method a new CoMFA model was developed so for each data set, 9 CoMFA models were built. Obtained results show noisy and uninformative variables affect CoMFA results. Based on created models, applying 5 variable selection approaches including FFD, SRD-FFD, IVE-PLS, SRD-UVEPLS and SPA-jackknife increases the predictive power and stability of CoMFA models significantly. RESULT & CONCLUSION: Among them, SPA-jackknife removes most of the variables while FFD retains most of them. FFD and IVE-PLS are time consuming process while SRD-FFD and SRD-UVE-PLS run need to few seconds. Also applying FFD, SRD-FFD, IVE-PLS, SRD-UVE-PLS protect CoMFA countor maps information for both fields.

Warhead biosynthesis and the origin of structural diversity in hydroxamate metalloproteinase inhibitors.

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are  potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining.

Cladosporol A triggers apoptosis sensitivity by ROS-mediated autophagic flux in human breast cancer cells.

BACKGROUND: Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression. RESULTS: Among NCI60 human cancer cell line panel Cladosporol A showed least IC50 value against human breast (MCF-7) cancer cells. The early events of apoptosis were characterized by phosphatidylserine exposure. It disrupts microtubule dynamics and also induces expression of pro-apoptotic protein p21. Moreover treatment of Cladosporol A significantly induced MMP loss, release of cytochrome c, Bcl-2 down regulation, Bax upregulation as well as increased monodansylcadaverine (MDC) staining and leads to LC3-I to LC3-II conversion. CONCLUSION: Our experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death. The proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.

Zonisamide attenuates lactacystin-induced parkinsonism in mice without affecting system xc.

Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc-, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc-. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6J mice receiving repeated intraperitoneal injections of either ZNS 30mgkg-1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post-surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc- was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6J mice, nor did it alter system xc- activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc- independent mechanisms.

The Glutaminase-1 Inhibitor 968 Enhances Dihydroartemisinin-Mediated Antitumor Efficacy in Hepatocellular Carcinoma Cells.

Reprogrammed metabolism and redox homeostasis are potential targets of cancer therapy. Our previous study demonstrated that the kidney form of glutaminase (GLS1) is highly expressed in hepatocellular carcinoma (HCC) cells and can be used as a target for effective anticancer therapy. Dihydroartemisinin (DHA) increases intracellular reactive oxygen species (ROS) levels leading to cytotoxicity in cancer cells. However, the heterogeneity of cancer cells often leads to differing responses to oxidative lesions. For instance, cancer cells with high ratio of GSH/GSSG, a critical ROS scavenger, are resistant to ROS-induced cytotoxicity. We postulate that a combinatorial strategy firstly disrupting redox homeostasis followed by DHA might yield a profound antitumor efficacy. In this study, when HCC cells were treated with a GLS1 inhibitor 968, the ROS elimination capacity was significantly reduced in HCC cells, which rendered HCC cells but not normal endothelial cells more sensitive to DHA-mediated cytotoxicity. We further confirmed that this synergistic antitumor efficacy was mediated by excessive ROS generation in HCC cells. NAC, a ROS inhibitor, partly rescued the combinatorial cytotoxic effect of 968 and DHA. Given that GLS1 is a potential antitumor target and DHA has been safely used in clinic, our findings provide new insight into liver cancer therapy targeting glutamine metabolism combined with the ROS generator DHA, which can be readily translated into cancer clinical trials.

Loss of C/EBPδ enhances IR-induced cell death by promoting oxidative stress and mitochondrial dysfunction.

Exposure of cells to ionizing radiation (IR) generates reactive oxygen species (ROS). This results in increased oxidative stress and DNA double strand breaks (DSBs) which are the two underlying mechanisms by which IR causes cell/tissue injury. Cells that are deficient or impaired in the cellular antioxidant response are susceptible to IR-induced apoptosis. The transcription factor CCAAT enhancer binding protein delta (Cebpd, C/EBPδ) has been implicated in the regulation of oxidative stress, DNA damage response, genomic stability and inflammation. We previously reported that Cebpd-deficient mice are sensitive to IR and display intestinal and hematopoietic injury, however the underlying mechanism is not known. In this study, we investigated whether an impaired ability to detoxify IR-induced ROS was the underlying cause of the increased radiosensitivity of Cebpd-deficient cells. We found that Cebpd-knockout (KO) mouse embryonic fibroblasts (MEFs) expressed elevated levels of ROS, both at basal levels and after exposure to gamma radiation which correlated with increased apoptosis, and decreased clonogenic survival. Pre-treatment of wild type (WT) and KO MEFs with polyethylene glycol-conjugated Cu-Zn superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) combination prior to irradiation showed a partial rescue of clonogenic survival, thus demonstrating a role for increased intracellular oxidants in promoting IR-induced cell death. Analysis of mitochondrial bioenergetics revealed that irradiated KO MEFs showed significant reductions in basal, adenosine triphosphate (ATP)-linked, maximal respiration and reserved respiratory capacity and decrease in intracellular ATP levels compared to WT MEFs indicating they display mitochondrial dysfunction. KO MEFs expressed significantly lower levels of the cellular antioxidant glutathione (GSH) and its precursor- cysteine as well as methionine. In addition to its antioxidant function, GSH plays an important role in detoxification of lipid peroxidation products such as 4-hydroxynonenal (4-HNE). The reduced GSH levels observed in KO MEFs correlated with elevated levels of 4-HNE protein adducts in irradiated KO MEFs compared to respective WT MEFs. We further showed that pre-treatment with the GSH precursor, N-acetyl L-cysteine (NAC) prior to irradiation showed a significant reduction of IR-induced cell death and increases in GSH levels, which contributed to the overall increase in clonogenic survival of KO MEFs. In contrast, pre-treatment with the GSH synthesis inhibitor- buthionine sulfoximine (BSO) further reduced the clonogenic survival of irradiated KO MEFs. This study demonstrates a novel role for C/EBPδ in protection from basal as well as IR-induced oxidative stress and mitochondrial dysfunction thus promoting post-radiation survival.

Soyasaponin Bb inhibits the recruitment of toll-like receptor 4 (TLR4) into lipid rafts and its signaling pathway by suppressing the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent generation of reactive oxygen species.

SCOPE: We and others recently showed that soyasaponin Bb (SSBb ) inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages. Since the recruitment of toll-like receptor 4 (TLR4) into lipid rafts is vital for LPS-initiated signaling, we investigated whether this process would be modulated by SSBb . METHODS AND RESULTS: By using sucrose gradient ultracentrifuge, we found that pretreatment of macrophages with SSBb inhibited LPS-induced recruitments of TLR4, myeloid differentiation primary response protein 88 (MyD88) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-ß (TRIF) into fractions enriched with lipid rafts marker flotillin-1. We also found SSBb decreased co-localization of TLR4 and lipid rafts by utilizing confocal immunofluorescence microscopy. Additionally, we observed that SSBb suppressed LPS-induced formation of TLR4/MyD88 and TLR4/TRIF complexes, production of pro-inflammatory molecules, and activation of nuclear factor kappa B (NF-κB). Furthermore, we found that these inhibitory effects of SSBb were associated with reduced reactive oxygen species (ROS) because pretreating cells with N-acetyl-L-cysteine and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited LPS-induced TLR4 recruitment into lipid rafts and NF-κB activation. SSBb also inhibited NADPH oxidase activation by blocking interaction between gp91(phox) and p47(phox) similarly as DPI. CONCLUSION: SSBb can inhibit TLR4 recruitment into lipid rafts and its signaling by suppressing the NADPH oxidase-dependent ROS generation.

Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce "protein denaturation". Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Anti-inflammatory and heme oxygenase-1 inducing activities of lanostane triterpenes isolated from mushroom Ganoderma lucidum in RAW264.7 cells.

Ganoderma lucidum is a popular medicinal mushroom used in traditional medicine for preventing or treating a variety of diseases. In the present study, we investigated the anti-inflammatory and heme oxygenase (HO)-1 inducing effects of 12 lanostane triterpenes from G. lucidum in RAW264.7 cells. Of these, seven triterpenes, butyl lucidenateE2, butyl lucidenateD2 (GT-2), butyl lucidenate P, butyl lucidenateQ, Ganoderiol F, methyl ganodenate J and butyl lucidenate N induced HO-1 expression and suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Inhibiting HO-1 activity abrogated the inhibitory effects of these triterpenes on the production of NO in LPS-stimulated RAW264.7 cells, suggesting the involvement of HO-1 in the anti-inflammatory effects of these triterpenes. We further studied the anti-inflammatory and HO-1 inducing effects of GT-2. Mitogen-activated protein kinase inhibitors or N-acetylcysteine, an antioxidant, did not suppress GT-2-mediated HO-1 induction; however, LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked GT-2-induced HO-1 mRNA and protein expression. GT-2 increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and knockdown of Nrf2 by small interfering RNA blocked GT-2-mediated HO-1 induction, suggesting that GT-2 induced HO-1 expression via the PI3K/AKT-Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, GT-2 inhibited the production of tumor necrosis factor-α and interleukin-6, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression. These findings suggest that HO-1 inducing activities of these lanostane triterpenes may be important in the understanding of a novel mechanism for the anti-inflammatory activity of G. lucidum.

The extent of neurodegeneration and neuroprotection in two chemical in vitro models related to Parkinson's disease is critically dependent on cell culture conditions.

The proteasome inhibition and mitochondrial dysfunction are involved in pathomechanism of Parkinson's disease. The main aim of this study was to assess how particular culture conditions of human dopaminergic neuroblastoma SH-SY5Y cells could affect the extent of neurodegeneration induced by proteasome inhibitor-lactacystin (LC) and mitochondrial toxin-rotenone (Rot). This study revealed that induction of neuronal differentiation of SH-SY5Y cells with retinoic acid (RA-SH-SY5Y) caused a higher resistance of these cells to LC-evoked cell death when compared to undifferentiated cells (UN-SH-SY5Y). In contrast, RA-SH-SY5Y cells were more vulnerable than the UN-SH-SY5Y to Rot-induced cell damage. Furthermore, we found that a prolonged incubation of the cells under low serum condition (PLSC) significantly increased the LC toxicity in both differentiated and undifferentiated cells. Next, the effects of combined treatment with LC and Rot on cell viability were studied in RA-SH-SY5Y cells under PLSC and normal low serum condition (NLSC). At a low concentration, Rot (0.001-1 µM) attenuated the LC-evoked cell death in RA-SH-SY5Y cells exposed to NLSC. In contrast, under PLSC low concentrations of Rot lacked neuroprotective action while its higher levels (10 µM) enhanced the LC toxicity. Further, we showed that low concentrations of celastrol (Cel; 0.001 µM), a putative neuroprotective agent with antioxidant and anti-inflammatory properties, were able to partially attenuate the Rot-evoked toxicity under both PLSC and NLSC. On the other hand, Cel (0.001 and 0.01 µM) attenuated the LC-induced cell damage only under PLSC. Interestingly, higher concentrations of Cel (>1 µM) reduced cell viability in both UN- and RA-SH-SY5Y but only in UN-SH-SY5Y cells the effect was enhanced under PLSC. The obtained data indicate that toxicity of LC and Rot in SH-SY5Y cell line depends on the stage of cell differentiation and is enhanced in cells cultured for a longer time in low serum medium. Moreover, the neuroprotective properties of Rot and Cel against the LC-induced cell damage can be observed only under particular low serum conditions.

AMPA glutamate receptors mediate the antidepressant-like effects of N-acetylcysteine in the mouse tail suspension test.

The aim of this study was to investigate the involvement of noradrenaline, serotonin, and subtypes of glutamate receptors in the antidepressant-like effects of N-acetylcysteine (NAC). The tail suspension test was used with male CF1 albino mice. D,L-α-methyl-ρ-tyrosine and ρ-chlorophenylalanine methyl ester hydrochloride were used as synthesis inhibitors of noradrenaline and serotonin, respectively. N-methyl-D-aspartate (NMDA) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione were used as an NMDA receptor agonist and an α-amino acid-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) receptor antagonist, respectively. NAC (10, 25, and 50 mg/kg intraperitoneally) significantly (P<0.05) decreased tail suspension test immobility time, whereas pretreatment with D,L-α-methyl-ρ-tyrosine, ρ-chlorophenylalanine methyl ester hydrochloride, and NMDA partially prevented (P<0.05) the effects of NAC (25 mg/kg), and pretreatment with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione completely abolished (P<0.01) this effect. The study corroborates the antidepressant-like effects of NAC in the TST, a model with a well-established predictive value. The results point to the key role of AMPA receptors in the mechanism of the antidepressant-like action of NAC. Like other AMPA potentiators, NAC indirectly modulates noradrenaline and serotonin pathways. It is suggested that the value of NAC as an antidepressant arises from combined and intertwined effects on a variety of pathways.

Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

p,p'-Dichlorodiphenoxydichloroethylene induced apoptosis of Sertoli cells through oxidative stress-mediated p38 MAPK and mitochondrial pathway.

p,p'-DDE, the major metabolite of dichlorodiphenoxytrichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. However, the mechanism underlying its male reproductive toxicity remains limited. Our previous studies have demonstrated that p,p'-DDE could induce mitochondria-mediated apoptosis of cultured rat Sertoli cells. In the present study, we investigated mitogen-activated protein kinase pathways as well as other mitochondria-related molecules including Bax family members and cytochrome c. Results showed that p,p'-DDE could induce oxidative stress-mediated p38 and JNK phosphorylation. In addition, elevated mRNA levels of cytochrome c and ratios of bax/bcl-w and bak/bcl-w were induced by p,p'-DDE treatment, which could be inhibited by RNA synthesis inhibitor (actinomycin D). p,p'-DDE-induced apoptosis was blocked by NAC (N-acetyl-L-cystein) preincubation and attenuated by pretreatment with p38 inhibitor (SB202190) or actinomycin D, but not with JNK inhibitor (SP600125). All of the findings suggested that oxidative stress-mediated p38 MAPK pathway and the balance between pro- and anti-apoptotic bax-gene family might play critical roles in p,p'-DDE-induced apoptosis.

Intrastriatal transplantation of GDNF-engineered BMSCs and its neuroprotection in lactacystin-induced Parkinsonian rat model.

The potential value of glial cell line-derived neurotrophic factor (GDNF) in treating Parkinson's disease (PD) remains controversial. In order to evaluate the therapeutic effect of GDNF-engineered bone marrow stromal cells (BMSCs) in parkinsonian rat model, GDNF-BMSCs and LacZ-BMSCs were transplanted into striatum and followed by Lactacystin lesioning at median forebrain bundles 1 week later. We observed that the intrastriatal transplantation of GDNF-BMSCs could significantly rescue the dopaminergic neurons from lactacystin-induced neurotoxicity with regard to behavioral recovery, tyrosine hydroxylase level in nigra and striatum, and striatal dopamine level. We interpret the outcomes that intrastriatal transplantation of GDNF-BMSCs might be beneficial in the treatment of PD.

Developmental etiology for neuroanatomical and cognitive deficits in mice overexpressing Galphas, a G-protein subunit genetically linked to schizophrenia.

Schizophrenia is a widespread psychiatric disorder, affecting 1% of people. Despite this high prevalence, schizophrenia is not well treated because of its enigmatic developmental origin. We explore here the developmental etiology of endophenotypes associated with schizophrenia using a regulated transgenic approach in mice. Recently, a polymorphism that increases mRNA levels of the G-protein subunit Galphas was genetically linked to schizophrenia. Here we show that regulated overexpression of Galphas mRNA in forebrain neurons of mice is sufficient to cause a number of schizophrenia-related phenotypes, as measured in adult mice, including sensorimotor gating deficits (prepulse inhibition of acoustic startle, PPI) that are reversed by haloperidol or the phosphodiesterase inhibitor rolipram, psychomotor agitation (hyperlocomotion), hippocampus-dependent learning and memory retrieval impairments (hidden water maze, contextual fear conditioning), and enlarged ventricles. Interestingly, overexpression of Galphas during development plays a significant role in some (PPI, spatial learning and memory and neuroanatomical deficits) but not all of these adulthood phenotypes. Pharmacological and biochemical studies suggest the Galphas-induced behavioral deficits correlate with compensatory decreases in hippocampal and cortical cyclic AMP (cAMP) levels. These decreases in cAMP may lead to reduced activation of the guanine exchange factor Epac (also known as RapGEF 3/4) as stimulation of Epac with the select agonist 8-pCPT-2'-O-Me-cAMP increases PPI and improves memory in C57BL/6J mice. Thus, we suggest that the developmental impact of a given biochemical insult, such as increased Galphas expression, is phenotype specific and that Epac may prove to be a novel therapeutic target for the treatment of both developmentally regulated and non-developmentally regulated symptoms associated with schizophrenia.

Inhibitory kappa B kinase-beta is a target for specific nuclear factor kappa B-mediated delayed cardioprotection.

OBJECTIVE: Myocardial ischemia/reperfusion injury remains a vexing problem. Translating experimental strategies that deliver protective agents before the ischemic insult limits clinical applicability. We targeted 2 proteins in the nuclear factor-kappaB pathway, inhibitory kappa B kinase-beta, and 26S cardiac proteasome to determine their cardioprotective effects when delivered during reperfusion. METHODS: C57BL/6 mice underwent left anterior descending artery occlusion for 30 minutes. An inhibitory kappa B kinase-beta inhibitor (Compound A), a proteasome inhibitor (PS-519), or vehicle was administered at left anterior descending artery release or 2 hours afterward. Infarct size was analyzed 24 hours later. Pressure-volume loops were performed at 72 hours. Serum and left ventricular tissue were collected 1 hour after injury to examine protein expression by enzyme-linked immunosorbent assay and Western blot. RESULTS: Inhibitory kappa B kinase-beta and proteasome inhibition significantly attenuated infarct size and preserved ejection fraction compared with the vehicle groups. When delivered even 2 hours after reperfusion, Compound A, but not PS-519, still decreased infarct size in mice. Finally, when delivered at reperfusion, successful inhibition of phosphorylated-p65 and decreased interleukin-6 and tumor necrosis factor-alpha levels occurred in mice given the inhibitory kappa B kinase-beta inhibitor, but not in mice with proteasome inhibition. CONCLUSION: Although inhibitory kappa B kinase-beta and proteasome inhibition at reperfusion attenuated infarct size after acute ischemia/reperfusion, only inhibitory kappa B kinase-beta inhibition provided cardioprotection through specific suppression of nuclear factor-kappaB signaling. This feature of highly targeted nuclear factor-kappaB inhibition might account for its delayed protective effects, providing a clinically relevant option for treating myocardial ischemia/reperfusion associated with unknown periods of ischemia and reperfusion as seen in cardiac surgery and acute coronary syndromes.

Comparison of neuroprotective and neurorestorative capabilities of rasagiline and selegiline against lactacystin-induced nigrostriatal dopaminergic degeneration.

Nigrostriatal neurodegeneration in Parkinson's disease (PD) has been postulated to be caused by various pathological conditions, such as mitochondrial defects, oxidative stress, and ubiquitin-proteasome system (UPS) dysfunction. Pharmacological strategies designed to interfere with these pathological pathways may effectively counteract the degeneration. Rasagiline and selegiline are selective and irreversible monoamine oxidase-B inhibitors that possess significant protective properties on dopamine neurons in various pre-clinical models of PD. In the present study, the neuroprotective and neurorestorative effects of rasagiline and selegiline were compared in an animal model of PD produced by inhibition of the UPS. C57BL/6 male mice were microinjected bilaterally with UPS inhibitor lactacystin (1.25 mug/side), into the medial forebrain bundle. Administration of rasagiline (0.2 mg/kg, i.p. once per day) or selegiline (1 mg/kg, i.p. once per day), started 7 days before or after (up to 28 days) after lactacystin microinjection. We found that both rasagiline and selegiline exerted a significant neuroprotective effect against lactacystin-induced neurodegeneration; but only rasagiline managed to restore the nigrostriatal degeneration. Furthermore, rasagiline showed a modest protection against lactacystin-induced inhibition of proteasomal activity. Our study indicates that compared with selegiline, rasagiline is more potent in protecting neurodegeneration induced by UPS impairment and may, therefore, exert disease-modifying effects in PD.

Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells.

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.

N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways.

During the oxidative stress generated by hydrogen peroxide (H2O2) in nerve growth factor (NGF)-differentiated PC12 cells, eIF4E binding protein (4E-BP1) and initiation factor 4E (eIF4E) phosphorylated levels decrease significantly, and an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreases eIF4F formation is observed. The treatment with N-acetyl-cysteine (NAC) completely abolishes the H2O2-induced decrease in eIF4E phosphorylated levels, whereas the decrease in 4E-BP1 phosphorylated levels and eIF4F activity inhibition are significantly but not fully reversed. Rapamycin, the mammalian target of rapamycin (FRAP/mTOR) inhibitor, prevents the effect of NAC on H2O2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H2O2. However, rapamycin has no effect on the NAC-induced recovery in phosphorylated eIF4E levels. Neither the MAP kinase inhibitors, PD98056 and SB203580, or the protein phosphatase 2A inhibitor, okadaic acid, mimic NAC effect on the H2O2-induced eIF4E dephosphorylation. Altogether our findings suggest that the effects caused by oxidative stress on eIF4s factors depends on two MAP kinase-independent signal transduction pathways, being at least one of them rapamycin-dependent.