Polyglutamine-expanded ataxin-7 decreases nuclear translocation of NF-kappaB p65 and impairs NF-kappaB activity by inhibiting proteasome activity of cerebellar neurons

Our recent study indicated that polyglutamine-expanded ataxin-7-Q75 induced apoptotic death of cultured cerebellar neurons by downregulating Bcl-x(L) expression and activating mitochondrial apoptotic cascade. Mutant polyglutamine-expanded proteins are believed to impair the proteolytic function of ubiquitin-proteasome system by sequestering components of proteasomes. Proteasome degradation of IkappaBalpha permits nuclear translocation of NF-kappaB and is required for continuous NF-kappaB activity, which supports the survival of cultured cerebellar neurons by inducing Bcl-x(L) expression. Thus, we tested the hypothesis that mutant ataxin-7-Q75 causes proteasome dysfunction and impairs NF-kappaB activity, leading to reduced Bcl-x(L) expression, caspase activation and cerebellar neuronal death. EMSA assays indicate that DNA-binding activity of NF-kappaB was significantly decreased in cerebellar neurons expressing ataxin-7-Q75. Similar to mutant ataxin-7-Q75, NF-kappaB inhibitor APEQ induced cerebellar neuronal death by decreasing Bcl-x(L) expression and activating caspase-9. Mutant ataxin-7-Q75 inhibited the proteolytic activity of proteasomes in cerebellar neurons. Proteasome inhibitor MG132 also caused cerebellar neuronal death by decreasing Bcl-x(L) expression and activating caspase-9. Both ataxin-7-Q75 and MG132 caused the cytosolic accumulation of IkappaBalpha in cerebellar neurons. Mutant ataxin-7-Q75 or MG132 increased the cytosolic level of NF-kappaB p65 and decreased the nuclear NF-kappaB p65 level. Our study provides the evidence that polyglutamine-expanded ataxin-7-Q75 decreases nuclear translocation of NF-kappaB p65 and impairs NF-kappaB activity by inhibiting proteasome activity of cerebellar neurons.     

Taurine resumed neuronal differentiation in arsenite-treated N2a cells through reducing oxidative stress,endoplasmic reticulum stress,and mitochondrial dysfunction

The goal of the study is to investigate the preventive effect of taurine against arsenite-induced arrest of neuronal differentiation in N2a cells. Our results revealed that taurine reinstated the neurite outgrowth in arsenite-treated N2a cells. Meanwhile, arsenite-induced oxidative stress and mitochondrial dysfunction as well as degradation of mitochondria DNA (mtDNA) were also inhibited by co-treatment of taurine. Since oxidative stress and mitochondrial dysfunction is closely associated with endoplasmic reticulum (ER) stress, we further examined indicators of ER stress, 78 kDa glucose-regulated protein (GRP78), and C/EBP-homologous protein (CHOP) protein expression. The results demonstrated that taurine significantly reduced arsenite-induced ER stress in N2a cells. In the parallel experiment, arsenite-induced disruption of intracellular calcium homeostasis was also ameliorated by taurine. The proven bio-function of taurine preserved a preventive effect against deleteriously cross-talking between oxidative stress, mitochondria, and ER. Overall, the results of the study suggested that taurine reinstated neuronal differentiation by inhibiting oxidative stress, ER stress, and mitochondrial dysfunction in arsenite-treated N2a cells.     

Signaling pathways of LIGHT induced macrophage migration and vascular smooth muscle cell proliferation

The biological actions of LIGHT, a member of the tumor necrosis factor superfamily, are mediated by the interaction with lymphotoxin-beta receptor (LTbetaR) and/or herpes virus entry mediator (HVEM). Previous study demonstrated high-level expressions of LIGHT and HVEM receptors in atherosclerotic plaques. To investigate the role of LIGHT in the functioning of macrophages and vascular smooth muscle cells (VSMC) in relation to atherogenesis, we determined the effects of LIGHT on macrophage migration and VSMC proliferation. We found LIGHT through HVEM activation can induce both events. LIGHT-induced macrophage migration was associated with activation of signaling kinases, including MAPKs, PI3K/Akt, NF-kappaB, Src members, and FAK. Proliferation of VSMC was also shown relating to the activation of MAPKs, PI3K/Akt, and NF-kappaB, which consequently led to alter the expression of cell cycle regulatory molecules. Down-regulation of p21, p27, and p53, and inversely up-regulation of cyclin D and RB hyper-phosphorylation were demonstrated. In conclusion, LIGHT acts as a novel mediator for macrophage migration and VSMC proliferation, suggesting its involvement in the atherogenesis.     

Fibrin(ogen) peptide B beta 15-42 inhibits platelet aggregation and fibrinogen binding to activated platelets

The binding of fibrinogen to activated platelets leads to platelet aggregation. Fibrinogen has multiple binding sites to platelet membrane glycoprotein IIb-IIIa complex. At least two well-defined sequences in fibrinogen, Arg-Gly-Asp sequence of A alpha 95-97 and A alpha 572-574 and gamma 400-411, have been shown to interact with glycoprotein IIb-IIIa. A possible binding site on the amino-terminal end of fibrinogen to platelet glycoprotein IIb-IIIa has also been reported. In this paper the effect of synthetic peptides derived from the amino-terminal end of the B beta chain on platelet aggregation and fibrinogen binding has been examined. B beta 15-42 peptide inhibits platelet aggregation and 125I-fibrinogen binding to activated platelets in a dose-dependent manner. Since B beta 15-42 contains a previously identified fibrinogen binding site, B beta 15-18, exposed by thrombin cleavage of native fibrinogen, we also examined the effect of B beta 15-18, B beta 19-42, and B beta 1-14 (fibrinopeptide B) on platelet aggregation and fibrinogen binding. Synthetic fibrinopeptide B and B beta 15-18 had no effect on platelet aggregation and fibrinogen binding while B beta 19-42 retained the inhibitory effect. When fibrinogen is chromatographed on a column of agarose-bound B beta 15-42, a cation-dependent retention of fibrinogen on the peptide column was observed, and fibrinogen was eluted from the column by B beta 15-42 but not by B beta 1-14. Under the same conditions, platelet glycoprotein IIb-IIIa was not retained in the column. Thus, the observed inhibitory effect is due to its interaction with fibrinogen rather than to platelet glycoprotein IIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping the Fitness Landscape of Gene Expression Uncovers the Cause of Antagonism and Sign Epistasis between Adaptive Mutations

How do adapting populations navigate the tensions between the costs of gene expression and the benefits of gene products to optimize the levels of many genes at once? Here we combined independently-arising beneficial mutations that altered enzyme levels in the central metabolism of Methylobacterium extorquens to uncover the fitness landscape defined by gene expression levels. We found strong antagonism and sign epistasis between these beneficial mutations. Mutations with the largest individual benefit interacted the most antagonistically with other mutations, a trend we also uncovered through analyses of datasets from other model systems. However, these beneficial mutations interacted multiplicatively (i.e., no epistasis) at the level of enzyme expression. By generating a model that predicts fitness from enzyme levels we could explain the observed sign epistasis as a result of overshooting the optimum defined by a balance between enzyme catalysis benefits and fitness costs. Knowledge of the phenotypic landscape also illuminated that, although the fitness peak was phenotypically far from the ancestral state, it was not genetically distant. Single beneficial mutations jumped straight toward the global optimum rather than being constrained to change the expression phenotypes in the correlated fashion expected by the genetic architecture. Given that adaptation in nature often results from optimizing gene expression, these conclusions can be widely applicable to other organisms and selective conditions. Poor interactions between individually beneficial alleles affecting gene expression may thus compromise the benefit of sex during adaptation and promote genetic differentiation.     

Formalin-Inactivated EV71 Vaccine Candidate Induced Cross-Neutralizing Antibody against Subgenotypes B1, B4, B5 and C4A in Adult Volunteers

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16.

Methods

Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses.

Results

The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16.

Conclusion

EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials.

Trial Registration

ClinicalTrials.gov __NCT01268787     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.