The common-866G > A variant in the promoter of UCP2 is associated with decreased risk of coronary artery disease in type 2 diabetic men

OBJECTIVE-Uncoupling protein 2 (UCP2) is a physiological downregulator of reactive oxygen species generation and plays an antiatherogenic role in the vascular wall. A common variant in the UCP2 promoter (-866G>A) modulates mRNA expression, with increased expression associated with the A allele. We investigated association of this variant with coronary artery disease (CAD) in two cohorts of type 2 diabetic subjects. RESEARCH DESIGN AND METHODS-We studied 3,122 subjects from the 6-year prospective Non-Insulin-Dependent Diabetes, Hypertension, Microalbuminuria, Cardiovascular Events, and Ramipril (DIABHYCAR) Study (14.9% of CAD incidence at follow-up). An independent, hospital-based cohort of 335 men, 52% of whom had CAD, was also studied. RESULTS-We observed an inverse association of the A allele with incident cases of CAD in a dominant model (hazard risk 0.88 [95% CI 0.80-0.96]; P = 0.006). Similar results were observed for baseline cases of CAD. Stratification by sex confirmed an allelic association with CAD in men, whereas no association was observed in women. All CAD phenotypes considered-myocardial infarction, angina pectoris, coronary artery bypass graft (CABG), and sudden death-contributed significantly to the association. Results were replicated in a cross-sectional study of an independent cohort (odds ratio 0.47 [95% CI 0.25-0.89]; P = 0.02 for a recessive model). CONCLUSIONS-The A allele of the -866G>A variant of UCP2 was associated with reduced risk of CAD in men with type 2 diabetes in a 6-year prospective study. Decreased risk of myocardial infarction, angina pectoris, CABG, and sudden death contributed individually and significantly to the reduction of CAD risk. This association was independent of other common CAD risk factors.

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 A mu g rhBMP-2 + monoolein; II: 3 A mu g rhBMP-2 + monoolein; III: 7 A mu g rhBMP-2 + monoolein; IV: 1 A mu g rhBMP-2 + poloxamer; V: 3 A mu g rhBMP-2 + poloxamer; VI: 7 A mu g rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 A mu g) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 angstrom resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date. (C) 2008 Elsevier Inc. All rights reserved.

The impact of obstructive sleep apnea on metabolic and inflammatory markers in consecutive patients with metabolic syndrome

Background: Obstructive Sleep Apnea (OSA) is tightly linked to some components of Metabolic Syndrome (MetS). However, most of the evidence evaluated individual components of the MetS or patients with a diagnosis of OSA that were referred for sleep studies due to sleep complaints. Therefore, it is not clear whether OSA exacerbates the metabolic abnormalities in a representative sample of patients with MetS. Methodology/Principal Findings: We studied 152 consecutive patients (age 48 +/- 9 years, body mass index 32.3 +/- 3.4 Kg/m(2)) newly diagnosed with MetS (Adult Treatment Panel III). All participants underwent standard polysomnography irrespective of sleep complaints, and laboratory measurements (glucose, lipid profile, uric acid and C-reactive protein). The prevalence of OSA (apnea-hypopnea index >= 15 events per hour of sleep) was 60.5%. Patients with OSA exhibited significantly higher levels of blood pressure, glucose, triglycerides, cholesterol, LDL, cholesterol/HDL ratio, triglycerides/HDL ratio, uric acid and C-reactive protein than patients without OSA. OSA was independently associated with 2 MetS criteria: triglycerides: OR: 3.26 (1.47-7.21) and glucose: OR: 2.31 (1.12-4.80). OSA was also independently associated with increased cholesterol/HDL ratio: OR: 2.38 (1.08-5.24), uric acid: OR: 4.19 (1.70-10.35) and C-reactive protein: OR: 6.10 (2.64-14.11). Indices of sleep apnea severity, apnea-hypopnea index and minimum oxygen saturation, were independently associated with increased levels of triglycerides, glucose as well as cholesterol/HDL ratio, uric acid and C-reactive protein. Excessive daytime sleepiness had no effect on the metabolic and inflammatory parameters. Conclusions/Significance: Unrecognized OSA is common in consecutive patients with MetS. OSA may contribute to metabolic dysregulation and systemic inflammation in patients with MetS, regardless of symptoms of daytime sleepiness.

Application of Low-Level Laser Irradiation (LLLI) and rhBMP-2 in Critical Bone Defect of Ovariectomized Rats: Histomorphometric Evaluation

Objectives: The aim of this study was to evaluate the osteogenic potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser irradiation (LLLI), isolated or combined in critical bone defects (5mm) in parietal bone using ovariectomized female rats as an experimental animal model. Materials and Methods: Forty-nine female Wistar rats, bilaterally ovariectomized (OVX), were divided into seven treatment groups of seven animals each: (I) laser in a single application, (II) 7 mu g of pure rhBMP-2, (III) laser and 7 mu g of pure rhBMP-2, (IV) 7 mu g of rhBMP-2/monoolein gel, (V) laser and 7 mu g of rhBMP-2/monoolein gel, (VI) laser and pure monoolein gel, and (VII) critical bone defect controls. The low-level laser source used was a gallium aluminum arsenide semiconductor diode laser device (lambda = 780 nm, D = 120 J/cm(2)). Results: Groups II and III presented higher levels of newly formed bone than all other groups with levels of 40.57% and 40.39%, respectively (p < 0.05). The levels of newly formed bone of groups I, IV, V, and VI were similar with levels of 29.67%, 25.75%, 27.75%, and 30.64%, respectively (p > 0.05). The area of new bone formation in group VII was 20.96%, which is significantly lower than groups I, II, III, and VI. Conclusions: It was concluded that pure rhBMP-2 and a single dose of laser application stimulated new bone formation, but the new bone formation area was significantly increased when only rhBMP-2 was used. Additionally, the laser application in combination with other treatments did not influence the bone formation area.

Expression of genes related to muscle plasticity after strength and power training regimens

The purpose of our study was to compare the effects of 8-week progressive strength and power training regimens on strength gains and muscle plasticity [muscle fiber hypertrophy and phenotype shift, mammalian target of rapamycin (mTOR), regulatory-associated protein of mTOR (RAPTOR), rapamycin-insensitive companion of m-TOR (RICTOR), calcineurin and calcipressin gene expression]. Twenty-nine physically active subjects were divided into three groups: strength training (ST), power training (PT) and control (C). Squat 1 RM and muscle biopsies were obtained before and after the training period. Strength increased similarly for both ST and PT groups (P < 0.001). Fiber types I, IIa and IIb presented hypertrophy main time effect (P < 0.05). Only type IIb percentage decreased from pre- to post-test (main time effect, P < 0.05). mTOR and RICTOR mRNA expression increased similarly from pre- to post-test (P < 0.01). RAPTOR increased after training for both groups (P < 0.0001), but to a greater extent in the ST (P < 0.001) than in the PT group. 4EBP-1 decreased after training when the ST and PT groups were pooled (P < 0.05). Calcineurin levels did not change after training, while calcipressin increased similarly from pre- to post-test (P < 0.01). In conclusion, our data indicate that these training regimens produce similar performance improvements; however, there was a trend toward greater hypertrophy-related gene expression and muscle fiber hypertrophy in the ST group.

The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim: Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. there is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1, ABCG2 and ABCC2) and uptake (SLCO1B1, SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated. Methods: Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results: Differences in mRnA basal levels of the transporters were as follows: ABCC2>ABCG2>ABCB1>SLCO1B1>>>SLC22A1>SLC O2B1 for HepG2 cells, and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABCG2 and SLCO2B1 mRnA levels were significantly up-regulated at 1, 10 and 20 mu mol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRnA levels after 12 or 24 h treatment. Conclusion: These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

Evaluation of rhBMP-2 and Natural Latex as Potential Osteogenic Proteins in Critical Size Defects by Histomorphometric Methods

This in vivo study evaluated the osteogenic potential of two proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and a protein extracted from natural latex (Hevea brasiliensis, P-1), and compared their effects on bone defects when combined with a carrier or a collagen gelatin. Eighty-four (84) Wistar rats were divided into two groups, with and without the use of collagen gelatin, and each of these were divided into six treatment groups of seven animals each. The treatment groups were: (1) 5 mu g of pure rhBMP-2; (2) 5 mu g of rhBMP-2/monoolein gel; (3) pure monoolein gel; (4) 5 mu g of pure P-1; (5) 5 mu g of P-1/monoolein gel; (6) critical bone defect control. The animals were anesthetized and a 6 mm diameter critical bone defect was made in the left posterior region of the parietal bone. Animals were submitted to intracardiac perfusion after 4 weeks and the calvaria tissue was removed for histomorphometric analysis. In this experimental study, it was concluded that rhBMP-2 allowed greater new bone formation than P-1 protein and this process was more effective when the bone defect was covered with collagen gelatin (P < 0.05). Anat Rec, 293:794-801, 2010. (C) 2010 Wiley-Liss, Inc.

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased Mortality Due to Sepsis in Mice

Rationale: The reduction of neutrophil migration to the bacterial focus is associated with poor outcome in sepsis. Objectives: The objective of this study was to identify soluble substances in the blood of septic mice that inhibit neutrophil migration. Methods: A pool of serum obtained from mice 2 hours after the induction of severe sepsis by cecal ligation and puncture inhibited the neutrophil migration. The proteins with inhibitory activity on neutrophil migration were isolated by Blue-Sepharose chromatography, high-performance liquid chromatography, and electrophoresis, and identified by mass spectrometry. Measurements and Main Results: Hemopexin was identified as the serum component responsible for the inhibition of neutrophil migration. In sepsis, the pretreatment of wild-type mice with hemopexin inhibited neutrophil migration to the focus of infection and decreased the survival rate from 87.5 to 50.0%. Hemopexin-null mice subjected to severe sepsis presented normal neutrophil migration, low bacteremia, and an improvement of 40% in survival rate. Moreover, hemopexin inhibited the neutrophil chemotaxis response evoked by C5a or macrophage inflammatory protein-2 and induced a reduction of CXCR2 and L-selectin as well as the up-regulation of CD11b expression in neutrophil membranes. The inhibitory effect of hemopexin on neutrophil chemotaxis was prevented by serine protease inhibitors or ATP. In addition, serum levels of ATP were decreased 2 hours after severe sepsis. Conclusions: These data demonstrate for the first time the inhibitory role of hemopexin in neutrophil migration during sepsis and suggest that the therapeutic inhibition of hemopexin or its protease activity could improve neutrophil migration to the focus of infection and survival in sepsis.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1 beta, MIP-2, and LTB(4)

Objective. The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design. MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1 beta (IL-1 beta) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1 beta and MIP-2 exudates was measured by ELISA. Results. MTA induced dose-and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1 beta antibodies. In the exudates, IL-1 beta and MIP-2 were detected. Conclusions. This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1 beta, MIP-2, and LTB(4).

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Background Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures Purpose The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-D, L-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental Implants Materials and methods Nine adult female sheep were submitted to bilateral sinus-floor elevation In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest Three different implants (Branemark (R), 31 (R) and Straumann (R)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated Results BD and BIC were significantly higher at 12 weeks in the test group if the Implants were placed at the time of the sinus lift (p < 0 05) No difference was observed between the different implant systems or positions Conclusions The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone Different implant systems and positions of the implants had no effect on BIC or BD (C) 2010 European Association for Cranio-Maxillo-Facial Surgery

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.