The Ero1α-PDI Redox Cycle Regulates Retro-Translocation of Cholera Toxin

Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. Our previous analyses demonstrated that the ER oxidoreductase protein disulfide isomerase (PDI) acts as a redox-dependent chaperone to unfold CTA1, a reaction postulated to initiate toxin retro-translocation. In its reduced state, PDI binds and unfolds CTA1; subsequent oxidation of PDI by Ero1α enables toxin release. Whether this in vitro model describes events in cells that control CTA1 retro-translocation is unknown. Here we show that down-regulation of Ero1α decreases retro-translocation of CTA1 by increasing reduced PDI and blocking efficient toxin release. Overexpression of Ero1α also attenuates CTA1 retro-translocation, an effect due to increased PDI oxidation, which prevents PDI from engaging the toxin effectively. Interestingly, Ero1α down-regulation increases interaction between PDI and Derlin-1, an ER membrane protein that is a component of the retro-translocation complex. These findings demonstrate that an appropriate Ero1α-PDI ratio is critical for regulating the binding–release cycle of CTA1 by PDI during retro-translocation, and implicate PDI''s redox state in targeting it to the retro-translocon.     

The biological role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase‐activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth‐promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.     

Tamoxifen Enhances the Hsp90 Molecular Chaperone ATPase Activity

Background

Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.

Methodology/Principal Findings

Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.

Conclusions/Significance

Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.     

Baseline prediction of combination therapy outcome in hepatitis C virus 1b infected patients by discriminant analysis using viral and host factors

Background

Current treatment of chronic hepatitis C virus (HCV) infection has limited efficacy −especially among genotype 1 infected patients−, is costly, and involves severe side effects. Thus, predicting non-response is of major interest for both patient wellbeing and health care expense. At present, treatment cannot be individualized on the basis of any baseline predictor of response. We aimed to identify pre-treatment clinical and virological parameters associated with treatment failure, as well as to assess whether therapy outcome could be predicted at baseline.

Methodology

Forty-three HCV subtype 1b (HCV-1b) chronically infected patients treated with pegylated-interferon alpha plus ribavirin were retrospectively studied (21 responders and 22 non-responders). Host (gender, age, weight, transaminase levels, fibrosis stage, and source of infection) and viral-related factors (viral load, and genetic variability in the E1–E2 and Core regions) were assessed. Logistic regression and discriminant analyses were used to develop predictive models. A “leave-one-out” cross-validation method was used to assess the reliability of the discriminant models.

Principal Findings

Lower alanine transaminase levels (ALT, p = 0.009), a higher number of quasispecies variants in the E1–E2 region (number of haplotypes, nHap_E1–E2) (p = 0.003), and the absence of both amino acid arginine at position 70 and leucine at position 91 in the Core region (p = 0.039) were significantly associated with treatment failure. Therapy outcome was most accurately predicted by discriminant analysis (90.5% sensitivity and 95.5% specificity, 85.7% sensitivity and 81.8% specificity after cross-validation); the most significant variables included in the predictive model were the Core amino acid pattern, the nHap_E1–E2, and gamma-glutamyl transferase and ALT levels.

Conclusions and Significance

Discriminant analysis has been shown as a useful tool to predict treatment outcome using baseline HCV genetic variability and host characteristics. The discriminant models obtained in this study led to accurate predictions in our population of Spanish HCV-1b treatment naïve patients.     

Cost-effectiveness of intermittent preventive treatment of malaria in pregnancy in southern Mozambique

Background

Malaria in pregnancy is a public health problem for endemic countries. Economic evaluations of malaria preventive strategies in pregnancy are needed to guide health policies.

Methods and Findings

This analysis was carried out in the context of a trial of malaria intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP), where both intervention groups received an insecticide treated net through the antenatal clinic (ANC) in Mozambique. The cost-effectiveness of IPTp-SP on maternal clinical malaria and neonatal survival was estimated. Correlation and threshold analyses were undertaken to assess the main factors affecting the economic outcomes and the cut-off values beyond which the intervention is no longer cost-effective. In 2007 US$, the incremental cost-effectiveness ratio (ICER) for maternal malaria was 41.46 US$ (95% CI 20.5, 96.7) per disability-adjusted life-year (DALY) averted. The ICER per DALY averted due to the reduction in neonatal mortality was 1.08 US$ (95% CI 0.43, 3.48). The ICER including both the effect on the mother and on the newborn was 1.02 US$ (95% CI 0.42, 3.21) per DALY averted. Efficacy was the main factor affecting the economic evaluation of IPTp-SP. The intervention remained cost-effective with an increase in drug cost per dose up to 11 times in the case of maternal malaria and 183 times in the case of neonatal mortality.

Conclusions

IPTp-SP was highly cost-effective for both prevention of maternal malaria and reduction of neonatal mortality in Mozambique. These findings are likely to hold for other settings where IPTp-SP is implemented through ANC visits. The intervention remained cost-effective even with a significant increase in drug and other intervention costs. Improvements in the protective efficacy of the intervention would increase its cost-effectiveness. Provision of IPTp with a more effective, although more expensive drug than SP may still remain a cost-effective public health measure to prevent malaria in pregnancy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00209781","term_id":"NCT00209781"}}NCT00209781     

Anandamide Suppresses Proliferation and Cytokine Release from Primary Human T-Lymphocytes Mainly via CB2 Receptors

Background

Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB₁R and CB₂R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB₁ and CB₂ receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.

Methodology/Significance

In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-α and INF-γ from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB₂R, since it could be mimicked by the CB₂R selective agonist JWH-015, and could be blocked by the specific CB₂R antagonist SR144528. Instead the selective CB₁R agonist ACEA, or the selective CB₁R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17.

Conclusions/Significance

Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB₂R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.     

The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson''s disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.