NOX1 and PRDX6 synergistically support migration and invasiveness of hepatocellular carcinoma cells through enhanced NADPH oxidase activity.

The NADPH oxidase 1 (NOX1) complex formed by proteins NOX1, p22phox, NOXO1, NOXA1, and RAC1 plays an important role in the generation of superoxide and other reactive oxygen species (ROS) which are involved in normal and pathological cell functions due to their effects on diverse cell signaling pathways. Cell migration and invasiveness are at the origin of tumor metastasis during cancer progression which involves a process of cellular de-differentiation known as the epithelial-mesenchymal transition (EMT). During EMT cells lose their polarized epithelial phenotype and express mesenchymal marker proteins that enable cytoskeletal rearrangements promoting cell migration, expression and activation of matrix metalloproteinases (MMPs), tissue remodeling, and cell invasion during metastasis. In this work, we explored the importance of the peroxiredoxin 6 (PRDX6)-NOX1 enzyme interaction leading to NOXA1 protein stabilization and increased levels of superoxide produced by NOX in hepatocarcinoma cells. This increase was accompanied by higher levels of N-cadherin and MMP2, correlating with a greater capacity for cell migration and invasiveness of SNU475 hepatocarcinoma cells. The increase in superoxide and the associated downstream effects on cancer progression were suppressed when phospholipase A2 or peroxidase activities of PRDX6 were abolished by site-directed mutagenesis, reinforcing the importance of these catalytic activities in supporting NOX1-based superoxide generation. Overall, these results demonstrate a clear functional cooperation between NOX1 and PRDX6 catalytic activities which generate higher levels of ROS production, resulting in a more aggressive tumor phenotype.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3ß inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.

Clinical guide for the diagnosis and follow-up of myotonic dystrophy type 1, MD1 or Steinert's disease. / Guía clínica para el diagnóstico y seguimiento de la distrofia miotónica tipo 1, DM1 o enfermedad de Steinert.

BACKGROUND AND OBJECTIVES: Steinert's disease or myotonic dystrophy type 1 (MD1), (OMIM 160900), is the most prevalent myopathy in adults. It is a multisystemic disorder with dysfunction of virtually all organs and tissues and a great phenotypical variability, which implies that it has to be addressed by different specialities with experience in the disease. The knowledge of the disease and its management has changed dramatically in recent years. This guide tries to establish recommendations for the diagnosis, prognosis, follow-up and treatment of the complications of MD1. MATERIAL AND METHODS: Consensus guide developed through a multidisciplinary approach with a systematic literature review. Neurologists, pulmonologists, cardiologists, endocrinologists, neuropaediatricians and geneticists have participated in the guide. RECOMMENDATIONS: The genetic diagnosis should quantify the number of CTG repetitions. MD1 patients need cardiac and respiratory lifetime follow-up. Before any surgery under general anaesthesia, a respiratory evaluation must be done. Dysphagia must be screened periodically. Genetic counselling must be offered to patients and relatives. CONCLUSION: MD1 is a multisystemic disease that requires specialised multidisciplinary follow-up.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Regulation of Cell Survival, Apoptosis, and Epithelial-to-Mesenchymal Transition by Nitric Oxide-Dependent Post-Translational Modifications.

SIGNIFICANCE: Nitric oxide (NO) is a physiopathological messenger generating different reactive nitrogen species (RNS) according to hypoxic, acidic and redox conditions. Recent Advances: RNS and reactive oxygen species (ROS) promote relevant post-translational modifications, such as nitrosation, nitration, and oxidation, in critical components of cell proliferation and death, epithelial-to-mesenchymal transition, and metastasis. CRITICAL ISSUES: The pro- or antitumoral properties of NO are dependent on local concentration, redox state, cellular status, duration of exposure, and compartmentalization of NO generation. The increased expression of NO synthase has been associated with cancer progression. However, the experimental strategies leading to high intratumoral NO generation have been shown to exert antitumoral properties. The effect of NO and ROS on cell signaling is critically altered by factors modulating tumor progression such as oxygen content, metabolism, and inflammatory response. The review describes the alteration of key components involved in cell survival and death, metabolism, and metastasis induced by RNS- and ROS-related post-translational modifications. FUTURE DIRECTIONS: The identification of the molecular targets affected by nitrosation, nitration, and oxidation, as well as their interactions with other post-translational modifications, will improve the understanding on the complex signaling and cell fate decision in cancer. The therapeutic NO-based strategies have to address the complex crosstalk among NO and ROS with regard to critical components affecting tumor cell survival, metabolism, and metastasis in the progression of cancer, as well as close interaction with ionizing radiation and chemotherapy.

Effect of the route of nutrition and L-alanyl-L-glutamine supplementation in amino acids' concentration in trauma patients.

PURPOSE: Our purpose was to assess the amino acids' (AAs) profile in trauma patients and to assess the effect of the route of nutrition and the exogenous ALA-GLN dipeptide supplementation on plasma AAs' concentration. METHODS: This is a secondary analysis of a previous randomized controlled trial. On day 1 and day 6 after trauma, plasma concentration of 25 AAs was measured using reverse phase high-performance liquid chromatography. Results were analyzed in relation to the route of nutrition and supplementation of ALA-GLN dipeptide. Differences between plasma AAs' concentrations at day 1 and day 6 were evaluated using the Student's t test or Mann-Whitney-Wilcoxon test. One-way ANOVA and the Kruskal-Wallis test were used to compare groups. A two-sided p value less than 0.05 was considered statistically significant. RESULTS: Ninety-eight patients were analyzed. Mean plasma concentrations at day 1 were close to the lower normal level for most AAs. At day 6 we found an increase in the eight essential AAs' concentrations and in 9 out of 17 measured non-essential AAs. At day 6 we found no differences in plasma concentrations for the sum of all AAs (p = .72), glutamine (p = .31) and arginine (p = .23) distributed by the route of nutrition. Administration of ALA-GLN dipeptide increased the plasma concentration of alanine (p = .004), glutamine (p < .001) and citrulline (p = .006). CONCLUSIONS: We found an early depletion of plasma AAs' concentration which partially recovered at day 6, which was unaffected by the route of nutrition. ALA-GLN dipeptide supplementation produced a small increase in plasma levels of glutamine and citrulline.

High purity galacto-oligosaccharides enhance specific Bifidobacterium species and their metabolic activity in the mouse gut microbiome.

Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a ß(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1 ± 3.4 in stools and PD = 13.0 ± 0.6 in intestinal contents) than control males (PD = 7.8 ± 1.6 in stool samples and PD = 9.5 ± 1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1ß, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Redox regulation of metabolic and signaling pathways by thioredoxin and glutaredoxin in NOS-3 overexpressing hepatoblastoma cells.

Nitric oxide (NO) plays relevant roles in signal transduction in physiopathology and its effects are dependent on several environmental factors. NO has both pro-apoptotic and anti-apoptotic functions but the molecular mechanisms responsible for these opposite effects are not fully understood. The action of NO occurs mainly through redox changes in target proteins, particularly by S-nitrosylation of reactive cysteine residues. Thioredoxin (Trx) and glutaredoxin (Grx) systems are the main cellular controllers of the thiolic redox state of proteins exerting controversial effects on apoptosis with consequences for the resistance to or the development of cancer. The aim of this study was to ascertain whether Trx and/or Grx systems mediate the antiproliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO. Enzyme levels and cysteine redox state at several metabolic checkpoints were consistent with prominence of the pentose phosphate pathway to direct the metabolic flux toward NADPH for antioxidant defense and lowering of nucleotide biosynthesis and hence proliferation. Proteins involved in cell survival pathways, proteins of the redoxin systems and phosphorylation of MAPK were all significantly increased accompanied by a shift of the thiolic redox state of Akt1, Trx1 and Grx1 to more oxidized. Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and -8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells. These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new antitumoral therapies.

Redox Regulation Of Metabolic And Signaling Pathways By Thioredoxin And Glutaredoxin In Nitric Oxide Treated Hepatoblastoma Cells.

BACKGROUND: NO has an antiproliferative action on HepG2 cells and Thioredoxin (Trx) and Glutaredoxin (Grx) have denitrosilase and deglutathionylase activities. AIMS: To ascertain whether Trx and/or Grx systems intermediate the anti-proliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. METHODS: HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) were transfected with specific siRNA to silence Trx1 and Grx1. The expression and thiolic redox state of proteins were determined by Western blot and redox mobility shift assay. RESULTS: Overexpression of NOS3 increased the levels and activities of proteins of the redoxin systems, Trx1, Grx1, TrxR1 and TxnIP, and the levels of signaling proteins (Akt1, pAkt1-Ser473, MapK, pMapK, Stat3, Fas). The thiolic redox state of Trx1, Grx1 and Akt1 shifted to more oxidized. Increases were also observed in Pro-apoptotic Caspase-3 fragment levels; caspase 3, 8 and 9 activities; antiapoptotic (Bcl-2); mitochondrial energetic (Aco2) and heme (Urod) metabolism; Glycolysis (Pkm2); and pentose phosphate pathway (Tkt). However, two cytosolic proteins related to iron (Aco1) and one carbon (Mat2) metabolism decreased markedly. Moreover, the redox state of Urod and Aco1 shifted to more oxidized cysteines. Trx1 or Grx1 silencing augmented Tyr nitration and diminished cell proliferation in WT cells, but attenuated the antiproliferative effect on NO, the increase of Fas, Akt1 and pAkt1-Ser473 and the oxidative modification of Akt1 in NOS3 cells. CONCLUSIONS: Trx1 and Grx1 exert contradictory influences on HepG2 cells. They are required for proliferation but they also contribute to antiproliferative effect of NO, associated to Akt1 redox changes.

Hirayama disease: Is surgery an option?

INTRODUCTION: Hirayama disease is a rare cervical myelopathy, predominantly affecting young males, which presents with distal atrophy of the upper limbs as its first and main symptom. It must be differentiated from motor neuron diseases because its natural history is different and because HD tends to stabilise in less than 5 years. Diagnosis is based on clinical findings and dynamic flexion MRI showing segmental spinal muscular atrophy, detachment of the posterior dura mater and venous congestion in the epidural space. The tendency is to indicate conservative treatment and no indications for surgery have been established. PATIENTS: We present 4 cases meeting both clinical criteria and dynamic MRI imaging criteria for a diagnosis of Hirayama disease. Two have stabilised spontaneously over the course of many years, and MRI scans show that typical changes have disappeared. Another case also remains stable following a shorter observation time. The fourth case is a young man who developed severe myelopathy in just over a year, and therefore underwent surgery. While his follow-up time is still short, his condition remains stable. CONCLUSIONS: Our 4 cases suggest that the condition of most patients with Hirayama stabilises naturally; patients should be evaluated for surgery on an individual basis, and surgery should probably be limited to the most severe cases that have progressed quickly.

Stress hyperglycaemia in critically ill patients: potential role of incretin hormones; a preliminary study.

BACKGROUND: Stress hyperglycaemia is common in the intensive care unit (ICU) setting and has been related to a worst outcome. OBJECTIVE: The objective was to characterize the association of glucoregulatory hormones, mainly incretins, with the levels of glycaemia, and its relationship with outcome in ICU patients. METHODS: We prospectively studied 60 patients. Stress hyperglycaemia was diagnosed when glycaemia was < 115 mg/dL. At ICU admission we determined glycaemia, insulin, glucagon, cortisol, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) plasma levels. Groups were compared using Kruskal-Wallis test. The association between glycaemia levels and glucoregulatory hormones was evaluated using linear regression. RESULTS: Forty-five patients (75%) had hyperglycaemia. We observed no differences in glucoregulatory hormones levels between normo- and hyper- glycaemia groups. Glycaemia levels were not significantly correlated with insulin, glucagon, cortisol or GIP levels, but were correlated with GLP-1 (p = 0.04). GLP-1 was also correlated with cortisol (p = 0.01), but failed to show a significant correlation with insulin, glucagon or GIP levels. Lower levels of plasma GLP-1 were found in patients with stress hyperglycaemia requiring vasoactive support (p = 0.02). CONCLUSIONS: Glycaemia levels were correlated with GLP-1 levels in ICU patients. GLP-1 levels were also associated with cortisol. Patients with stress hyperglycaemia who required vasoactive support had lower incretin levels compared with those patients with stress hyperglycaemia who were hemodynamically stables. (ClinicalTrials.gov Identifier: NCT01087372).

Chimeric calicivirus-like particles elicit specific immune responses in pigs.

Virus-like particles (VLPs) have received considerable attention due to their potential application in veterinary vaccines and, in particular, VLPs from rabbit haemorrhagic disease virus (RHDV) have successfully shown to be good platforms for inducing immune responses against an inserted foreign epitope in mice. The aim of this study was to assess the immunogenicity of chimeric RHDV-VLPs as vaccine vectors in pigs. For this purpose, we have generated chimeric VLPs containing a well-known T epitope of 3A protein of foot-and-mouth disease virus (FMDV). Firstly, RHDV-VLPs were able to activate immature porcine bone marrow-derived dendritic cells (poBMDCs) in vitro. Secondly, pigs were inoculated twice in a two-week interval with chimeric RHDV-VLPs at different doses intranasally or intramuscularly. One intramuscularly treated group was also inoculated with adjuvant Montanide™ ISA 206 at the same time. Specific IgG and IgA antibodies against RHDV-VLPs were induced and such levels were higher in the adjuvanted group compared with other groups. Interestingly, anti-RHDV-VLP IgA responses were higher in groups inoculated intramuscularly than those that received the VLPs intranasally. Two weeks after the last immunisation, specific IFN-γ-secreting cells against 3A epitope and against RHDV-VLPs were detected in PBMCs by ELISPOT. The adjuvanted group exhibited the highest IFN-γ-secreting cell numbers and lymphoproliferative specific T cell responses against 3A epitope and RHDV-VLP. This is the first immunological report on the potential use of chimeric RHDV-VLPs as antigen carriers in pigs.

Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3).

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.

[Prognostic factors associated with postoperative complications in liver transplant]. / Factores pronósticos de complicaciones postoperatorias en el transplante hepático.

OBJECTIVES: the postoperative evolution of patients submitted to orthotopic liver transplant (OLT) is frequently associated with the appearance of different types of complications such as renal failure, graft rejection, infections, and neurological disorders. These complications are the most significant causes of early morbidity and mortality in patients undergoing OLT. The purpose of the present study was the identification of factors related to the different postoperative complications after OLT. EXPERIMENTAL DESIGN: a prospective study was carried out. PATIENTS: seventy-eight variables were analyzed in 32 consecutive patients undergoing OLT. The factors independently associated with the appearance of postoperative complications were identified using a stepwise logistic regression analysis. RESULTS: the multivariate analysis showed that malondialdehyde and creatinine pretransplant serum levels were associated with the development of renal dysfunction. The pretransplant levels of haemoglobin and the units of platelets administered during surgery were prognostic factors of infections. Acute graft rejection was predicted by ?-glutamyl transpeptidase and total bilirubin serum levels. The pretransplant sodium and glutaredoxin levels in serum were associated with neurological complications. CONCLUSIONS: we propose these markers for the identification of high-risk patients allowing an early surveillance and/or treatment to improve morbidity and survival in patients submitted to OLT.

Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV.

Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. approximately 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 microg of VP60r in the presence of adjuvant or 100 microg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use.

Glutaredoxins catalyze the reduction of glutathione by dihydrolipoamide with high efficiency.

Glutaredoxins (Grx) are small (approximately 12kDa) proteins which catalyze thiol disulfide oxidoreductions involving glutathione (GSH) and disulfides in proteins or small molecules. Here, we present data which demonstrate the ability of glutaredoxins to catalyze the reduction of oxidized glutathione (GSSG) by dihydrolipoamide (DHL), an important biological redox catalyst and synthetic antioxidant. We have designed a new assay method to quantify the rate of reduction of GSSG and other disulfides by reduced lipoamide and have tested a set of eight recombinant Grx from human, rat, yeast, and E. coli. Lipoamide dependent activity is highest with the large atypical E. coli Grx2 (k(cat)=3.235 min(-1)) and lowest for human mitochondrial Grx2a (k(cat)=96 min(-1)) covering a wider range than k(cat) for the standard reduction of hydroxyethyldisulfide (HED) by GSH (290-2.851 min(-1)). The lipoamide/HED activity ratio was highest for yeast Grx2 (1.25) and E. coli Grx2 and lowest for E. coli Grx1 (0.13). These results suggest a new role for Grxs as ancillary proteins that could shunt reducing equivalents from main catabolic pathways to recycling of GSSG via a lipoyl group, thus serving biochemical functions which involve GSH but without NAD(P)H consumption.