Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue. (C) 2012 Elsevier Inc. All rights reserved.

Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease

Alzheimer's disease (AD) is the most common cause of dementia in the human population, characterized by a spectrum of neuropathological abnormalities that results in memory impairment and loss of other cognitive processes as well as the presence of non-cognitive symptoms. Transcriptomic analyses provide an important approach to elucidating the pathogenesis of complex diseases like AD, helping to figure out both pre-clinical markers to identify susceptible patients and the early pathogenic mechanisms to serve as therapeutic targets. This study provides the gene expression profile of postmortem brain tissue from subjects with clinic-pathological AD (Braak IV, V, or V and CERAD B or C; and CDR >= 1), preclinical AD (Braak IV, V, or VI and CERAD B or C; and CDR = 0), and healthy older individuals (Braak <= II and CERAD 0 or A; and CDR = 0) in order to establish genes related to both AD neuropathology and clinical emergence of dementia. Based on differential gene expression, hierarchical clustering and network analysis, genes involved in energy metabolism, oxidative stress, DNA damage/repair, senescence, and transcriptional regulation were implicated with the neuropathology of AD; a transcriptional profile related to clinical manifestation of AD could not be detected with reliability using differential gene expression analysis, although genes involved in synaptic plasticity, and cell cycle seems to have a role revealed by gene classifier. In conclusion, the present data suggest gene expression profile changes secondary to the development of AD-related pathology and some genes that appear to be related to the clinical manifestation of dementia in subjects with significant AD pathology, making necessary further investigations to better understand these transcriptional findings on the pathogenesis and clinical emergence of AD.

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether. LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. (C) 2012 Elsevier Inc. All rights reserved.

Assessing the benefits of rosiglitazone in women with polycystic ovary syndrome through its effects on insulin-like growth factor 1, insulin-like growth factor-binding protein-3 and insulin resistance: a pilot study

Federal University of Sao Paulo

Molecular Surveillance of the Newcastle Disease Virus in Domestic and Wild Birds on the North Eastern Coast and Amazon Biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Autoimmune disease and multiple autoantibodies in 42 patients with RASopathies

The association of RASopathies [Noonan syndrome (NS) and Noonan-related syndromes] and autoimmune disorders has been reported sporadically. However, a concomitant evaluation of autoimmune diseases and an assessment of multiple autoantibodies in a large population of patients with molecularly confirmed RASopathy have not been performed. The clinical and laboratory features were analyzed in 42 RASopathy patients, the majority of whom had NS and five individuals had Noonan-related disorders. The following autoantibodies were measured: Anti-nuclear antibodies, anti-double stranded DNA, anti-SS-A/Ro, anti-SS-B/La, anti-Sm, anti-RNP, anti-Scl-70, anti-Jo-1, anti-ribosomal P, IgG and IgM anticardiolipin (aCL), thyroid, anti-smooth muscle, anti-endomysial (AE), anti-liver cytosolic protein type 1 (LC1), anti-parietal cell (APC), anti-mitochondrial (AM) antibodies, anti-liver-kidney microsome type 1 antibodies (LKM-1), and lupus anticoagulant. Six patients (14%) fulfilled the clinical criteria for autoimmune diseases [systemic lupus erythematous, polyendocrinopathy (autoimmune thyroiditis and celiac disease), primary antiphospholipid syndrome (PAPS), autoimmune hepatitis, vitiligo, and autoimmune thyroiditis]. Autoimmune antibodies were observed in 52% of the patients. Remarkably, three (7%) of the patients had specific gastrointestinal and liver autoantibodies without clinical findings. Autoimmune diseases and autoantibodies were frequently present in patients with RASopathies. Until a final conclusion of the real incidence of autoimmunity in Rasopathy is drawn, the physicians should be alerted to the possibility of this association and the need for a fast diagnosis, proper referral to a specialist and ultimately, adequate treatment. (c) 2012 Wiley Periodicals, Inc.

Serum C-reactive protein levels predict neurological outcome after aneurysmal subarachnoid hemorrhage

Objectives: Our aim was to evaluate the relationship between serum C-reactive protein (CRP) levels and the neurological prognosis and development of vasospasm in patients with aneurysmal subarachnoid hemorrhage (aSAH). Methods: Eighty-two adult patients with aSAH diagnoses were prospectively evaluated. Glasgow Coma Scale (GCS) score, Hunt and Hess grade, Fisher grade, cranial CT scans, digital subtraction angiography studies and daily neurological examinations were recorded. Serial serum CRP measurements were obtained daily between admission and the tenth day. Glasgow Outcome Scale (GOS) and the modified Rankin Scale (mRS) were used to assess the prognosis. Results: Serum CRP levels were related to severity of aSAH. Patients with lower GCS scores and higher Hunt and Hess and Fisher grades presented statistically significant higher serum CRP levels. Patients with higher serum CRP levels had a less favorable prognosis. Conclusions: Increased serum CRP levels were strongly associated with worse clinical prognosis in this study.

Expression of calcium binding protein S100 A7 (psoriasin) in laryngeal carcinoma

Many studies have reported increased expression of S100 A7 (psoriasin) in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. Objective: This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. Material and Methods: Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. Results: The group with highly differentiated tumors had the highest treatment failure scores. Moderately differentiated tumors had higher treatment failure scores than poorly differentiated tumors. Higher scores were predominantly seen on stages I and II in moderately differentiated tumors, whereas score distribution was more homogeneous in advanced stage disease (III and IV). Regarding failure in treatment, the group scoring zero (3/4 complications: 75%) differed significantly from the remaining groups (13/59: 22%). Conclusions: S100 A7 marker was expressed in 93.7% of laryngeal cancer cases, with higher positive correlation rates in more differentiated tumors and significantly lower rates of treatment failure. Scores had no impact on survival rates.

Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

Purpose: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. Methods: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). Results: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. Conclusions: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 mu M tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

Protein Oxidative Stress and Dyslipidemia in Dialysis Patients

Our aim was to investigate and determine the associations between oxidative stress (OS), dyslipidemia and inflammation in patients treated with continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) using observational cross-sectional study. Twenty patients in CAPD and 48 in HD for at least 8 weeks and aged =18 years were included in the study. Individuals with malignant or acute inflammatory disease were excluded. A control group of 17 healthy individuals was also recruited. The biochemical parameter evaluations were analyzed using colorimetric kits for albumin, serum glucose, total cholesterol (TC) and lipid fractions. To determine the inflammatory status, CRP, IL-6 and TNF-a were analyzed by automated chemiluminescence kits. Plasma advanced oxidation protein products (AOPP) were determined by spectrophotometry. Mean AOPP levels were significantly higher for the HD group compared to the control, and there was no difference in AOPP concentrations between the control and CAPD groups. Dialysis patients had levels of inflammatory parameters higher than controls, and showed a high prevalence of patients with dyslipidemia, especially in CAPD. In the HD group, AOPP was positively correlated with triglycerides (TG) and inversely associated with HDL. Also the HD group was observed to have negative associations between TNF-a and HDL, LDL and TC. In the CAPD group, CRP was inversely correlated with HDL. Hemodialysis patients had increased protein OS and associations of inflammation and dyslipidemia were also observed in these dialysis groups. A more detailed characterization of the relations between oxidative stress and other more traditional risk factors has therapeutic importance, since cardiovascular diseases are the leading cause of death among dialysis patients.

Protein quality control disruption by PKC beta II in heart failure: rescue by the selective PKC beta II inhibitor, beta IIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform beta II (PKC beta II) in disrupting PQC. We show that active PKC beta II directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKC beta II, using a selective PKC beta II peptide inhibitor (beta IIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKC beta II increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, beta IIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKC beta II activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKC beta II as a novel inhibitor of proteasomal function. PQC disruption by increased PKC beta II activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKC beta II inhibition may benefit patients with heart failure. (218 words)

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. Methodology/Principal Findings: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. Conclusions/Significance: Herein it is shown, for the first time, that paraflagellar rod proteins and alpha-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Lipid transfers to HDL are predictors of precocious clinical coronary heart disease

Background: High-density-lipoprotein (HDL) has several antiatherogenic properties and, although the concentration of HDL-cholesterol negatively correlates with incidence of coronary artery disease (CAD), this is not sufficient to evaluate the overall HDL protective role. The aim was to investigate whether precocious CAD patients show abnormalities in lipid transfers to HDL, a fundamental step in HDL metabolism and function. Methods: Thirty normocholesterolemic CAD patients aged <50 y and 30 controls paired for sex, age and B.M.I. were studied. Fasting blood samples were collected for the in vitro lipid transfer assay and plasma lipid determination. A donor nanoemulsion labeled with radioactive free-cholesterol. cholesteryl esters, phospholipids and triglycerides was incubated with whole plasma and after chemical precipitation of non-HDL fractions, supernatant was counted for radioactivity in HDL. Results: LDL and HDL-cholesterol and triglycerides were equal in both groups. Transfers of free-cholesterol (3.8 +/- 1.2%vs 7.0 +/- 33%,p<0.0001) and triglycerides (3.7 +/- 1.7%vs 4.9 +/- 1.9%, p = 0.0125) were diminished in CAD patients whereas cholesteryl ester transfer increased (6.5 +/- 1.9%vs 4.8 +/- 1.8%, p = 0.0008); phospholipid transfer was equal (17.8 +/- 3.5% vs19.5 +/- 3.9%). Conclusion: Alterations in the transfer of lipids to HDL may constitute a new marker for precocious CAD and relation of this metabolic alteration with HDL antiatherogenic function should be investigated in future studies. (C) 2011 Published by Elsevier B.V.