The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.)

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway

The juice of fresh leaves of Catharanthus roseus Linn. reduces blood glucose in normal and alloxan diabetic rabbits

BACKGROUND: The leaf juice or water decoction of Catharanthus roseus L. (Apocyanaceae) is used as a folk medicine for the treatment of diabetes all over the world. In the present investigation, the leaf juice of C. roseus has been evaluated for its hypoglycemic activity in normal and alloxan-induced diabetic rabbits. METHODS: The blood glucose lowering activity of the leaf juice was studied in normal and alloxan-induced (100 mg/kg, i.v.) diabetic rabbits, after oral administration at doses of 0.5, 0.75 and 1.0 ml/kg body weight. Blood samples were collected from the marginal ear vein before and also at 4, 6, 8, 10, 12, 16, 18, 20 & 24 h after drug administration and blood glucose was analyzed by Nelson-Somogyi's method using a visible spectrophotometer. The data was compared statistically by using Student's t-test. RESULTS: The leaf juice of C. roseus produced dose-dependent reduction in blood glucose of both normal and diabetic rabbits and comparable with that of the standard drug, glibenclamide. The results indicate a prolonged action in reduction of blood glucose by C. roseus and the mode of action of the active compound(s) of C. roseus is probably mediated through enhance secretion of insulin from the β-cells of Langerhans or through extrapancreatic mechanism. CONCLUSIONS: The present study clearly indicated a significant antidiabetic activity with the leaf juice of Catharanthus roseus and supports the traditional usage of the fresh leaves by Ayurvedic physicians for the control of diabetes

Phenomenology of Light Sneutrino Dark Matter in cMSSM/mSUGRA with Inverse Seesaw

We study the possibility of a light Dark Matter (DM) within a constrained Minimal Supersymmetric Standard Model (cMSSM) framework augmented by a SM singlet-pair sector to account for the non-zero neutrino masses by inverse seesaw mechanism. Working within a 'hybrid' scenario with the MSSM sector fixed at high scale and the singlet neutrino sector at low scale, we find that, contrary to the case of the usual cMSSM where the neutralino DM cannot be very light, we can have a light sneutrino DM with mass below 100 GeV satisfying all the current experimental constraints from cosmology, collider as well as low-energy experiments. We also note that the supersymmetric inverse seesaw mechanism with sneutrino as the lightest supersymmetric partner can have enhanced same-sign dilepton final states with large missing transverse energy (mET) coming from the gluino- and squark-pair as well as the squark-gluino associated productions and their cascade decay through charginos. We present a collider study for the same-sign dilepton+jets+mET signal in this scenario and propose some distinctions with the usual cMSSM. We also comment on the implications of such a light DM scenario on the invisible decay width of an 125 GeV Higgs boson.Comment: 24 pages, 4 figures, 7 tables; matches published versio

Cytotoxic isolates of Helicobacter pylori from Peptic Ulcer Diseases decrease K(+)-dependent ATPase Activity in HeLa cells

BACKGROUND: Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases. However, not all H. pylori positive cases develop advanced disease. This discriminatory behavior has been attributed to the difference in virulence of the bacteria. Among all virulence factors, cytotoxin released by H. pylori is the most important factor. In this work, we studied variation in H. pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K(+)-dependent ATPase (one of its targets) enzyme activity in HeLa cells. METHODS: The patients were retrospectively grouped on the basis of endoscopic and histopathological observation as having gastritis or peptic ulcer. The HeLa cells were incubated with the broth culture filtrates (BCFs) of H. pylori isolates from patients of both groups and observed for the cytopathic effects: morphological changes and viability. In addition, the K(+)-dependent ATPase activity was measured in HeLa cells extracts. RESULTS: The cytotoxin production was observed in 3/7 (gastritis) and 4/4 (peptic ulcer) H. pylori isolates. The BCFs of cytotoxin producing H. pylori strains reduced the ATPase activity of HeLa cells to 40% of that measured with non-cytotoxin producing H. pylori strains (1.33 μmole Pi/mg protein and 3.36 μmole Pi/mg protein, respectively, p < 0.05). The decreased activity of ATPase enzyme or the release of cytotoxin also correlated with the increased pathogenicity indices of the patients. CONCLUSIONS: Our results suggest that the isolation of cytotoxic H. pylori is more common in severe form of acid peptic diseases (peptic ulcer) than in gastritis patients from India. Also the cytotoxin released by H. pylori impairs the ion-transporting ATPase and is a measure of cytotoxicity

The Contrasting Effect of Macromolecular Crowding on Amyloid Fibril Formation

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed

Evaluation of Analytical Methods to Study Aquifer Properties with Pumping Tests in Coastal Aquifers with Numerical Modelling (Motril-Salobreña Aquifer)

Two pumping tests were performed in the unconfined Motril-Salobreña detrital aquifer in a 250 m-deep well 300 m from the coastline containing both freshwater and saltwater. It is an artesian well as it is in the discharge zone of this coastal aquifer. The two observation wells where the drawdowns are measured record the influence of tidal fluctuations, and the well lithological columns reveal high vertical heterogeneity in the aquifer. The Theis and Cooper-Jacob approaches give average transmissivity (T) and storage coefficient (S) values of 1460 m2 /d and 0.027, respectively. Other analytical solutions, modified to be more accurate in the boundary conditions found in coastal aquifers, provide similar T values to those found with the Theis and Cooper-Jacob methods, but give very different S values or could not estimate them. Numerical modelling in a synthetic model was applied to analyse the sensitivity of the Theis and Cooper-Jacob approaches to the usual boundary conditions in coastal aquifers. The T and S values calculated from the numerical modelling drawdowns indicate that the regional flow, variable pumping flows, and tidal effect produce an error of under 10 % compared to results obtained with classic methods. Fluids of different density (freshwater and saltwater) cause an error of 20 % in estimating T and of over 100 % in calculating S. The factor most affecting T and S results in the pumping test interpretation is vertical heterogeneity in sediments, which can produce errors of over 100 % in both parameters.This research has been financed by Project CGL2012-32892 (Ministerio de Economía y Competitividad of Spain) and by the Research Group Sedimentary Geology and Groundwater (RNM-369) of the Junta de Andalucía

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals

Superoxide Dismutase 1 and tgSOD1G93A Mouse Spinal Cord Seed Fibrils, Suggesting a Propagative Cell Death Mechanism in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects motor neurons and leads to a progressive and ultimately fatal loss of function, resulting in death typically within 3 to 5 years of diagnosis. The disease starts with a focal centre of weakness, such as one limb, and appears to spread to other parts of the body. Mutations in superoxide dismutase 1 (SOD1) are known to cause disease and it is generally accepted they lead to pathology not by loss of enzymatic activity but by gain of some unknown toxic function(s). Although different mutations lead to varying tendencies of SOD1 to aggregate, we suggest abnormal proteins share a common misfolding pathway that leads to the formation of amyloid fibrils.Methodology/Principal Findings: Here we demonstrate that misfolding of superoxide dismutase 1 leads to the formation of amyloid fibrils associated with seeding activity, which can accelerate the formation of new fibrils in an autocatalytic cascade. The time limiting event is nucleation to form a stable protein "seed" before a rapid linear polymerisation results in amyloid fibrils analogous to other protein misfolding disorders. This phenomenon was not confined to fibrils of recombinant protein as here we show, for the first time, that spinal cord homogenates obtained from a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (the TgSOD1(G93A) mouse) also contain amyloid seeds that accelerate the formation of new fibrils in both wildtype and mutant SOD1 protein in vitro.Conclusions/Significance: These findings provide new insights into ALS disease mechanism and in particular a mechanism that could account for the spread of pathology throughout the nervous system. This model of disease spread, which has analogies to other protein misfolding disorders such as prion disease, also suggests it may be possible to design assays for therapeutics that can inhibit fibril propagation and hence, possibly, disease progression

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins

Discordant Gene Expression Signatures and Related Phenotypic Differences in Lamin A- and A/C-Related Hutchinson-Gilford Progeria Syndrome (HGPS)

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder displaying features reminiscent of premature senescence caused by germline mutations in the LMNA gene encoding lamin A and C, essential components of the nuclear lamina. By studying a family with homozygous LMNA mutation (K542N), we showed that HGPS can also be caused by mutations affecting both isoforms, lamin A and C. Here, we aimed to elucidate the molecular mechanisms underlying the pathogenesis in both, lamin A- (sporadic) and lamin A and C-related (hereditary) HGPS. For this, we performed detailed molecular studies on primary fibroblasts of hetero- and homozygous LMNA K542N mutation carriers, accompanied with clinical examinations related to the molecular findings. By assessing global gene expression we found substantial overlap in altered transcription profiles (13.7%; 90/657) in sporadic and hereditary HGPS, with 83.3% (75/90) concordant and 16.7% (15/90) discordant transcriptional changes. Among the concordant ones we observed down-regulation of TWIST2, whose inactivation in mice and humans leads to loss of subcutaneous fat and dermal appendages, and loss of expression in dermal fibroblasts and periadnexial cells from a LMNAK542N/K542N patient further confirming its pivotal role in skin development. Among the discordant transcriptional profiles we identified two key mediators of vascular calcification and bone metabolism, ENPP1 and OPG, which offer a molecular explanation for the major phenotypic differences in vascular and bone disease in sporadic and hereditary HGPS. Finally, this study correlates reduced TWIST2 and OPG expression with increased osteocalcin levels, thereby linking altered bone remodeling to energy homeostasis in hereditary HGPS