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Find video protocols related to scientific articles indexed in Pubmed.
Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-12-2014
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Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the "minor pocket," previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR(-/-) mice compared with WT mice. Furthermore, treatment of C5aR(-/-) mice with DF2593A did not produce any further antinociceptive effect compared with C5aR(-/-) mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.
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Alzheimer's Disease-Like Pathology Induced by Amyloid-? Oligomers in Nonhuman Primates.
J. Neurosci.
PUBLISHED: 10-10-2014
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Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-? (A?) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble A? oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, A? oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where A? oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with A? oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links A? oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.
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Effect of radiotherapy on osseointegration of dental implants immediately placed in postextraction sites of minipigs mandibles.
Implant Dent
PUBLISHED: 09-06-2014
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The aim of this study was to compare the osseointegration and the survival of dental implants (DIs) immediately placed in postextraction sites, in mandibles of minipigs that underwent radiotherapy (RT).
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Soil microbial properties after 5 years of consecutive amendment with composted tannery sludge.
Environ Monit Assess
PUBLISHED: 08-19-2014
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Composting has been recognised an alternative method to tannery sludge recycling and afterwards to be used in agriculture. As the tannery sludge contains salts and chromium, the application of composted tannery sludge (CTS) should be performed carefully to minimise negative effects on soil microbial properties. Therefore, this study evaluated the effects of 5-year repeated CTS amendment on soil microbial biomass (SMB) and enzyme activities in a tropical soil. CTS was applied during 5 years at 0, 2.5, 5, 10 and 20 Mg ha(-1), and at the fifth year, the microbial biomass C (MBC) and N (MBN), basal and substrate-induced respiration (SIR), metabolic quotient (qCO2) and dehydrogenase (DHA) and fluorescein diacetate (FDA) hydrolysis were determined in the soil samples. Soil MBC and MBN showed the highest values with the amendment of 5 Mg ha(-1) CTS. Soil respiration increased with the increase in CTS rates, while SIR showed the highest values with the amendment of 0, 2.5 and 5 Mg ha(-1) CTS. DHA activity showed the highest values with the amendment up to 2.5 Mg ha(-1), while FDA hydrolysis increased up to the rate of 5 Mg ha(-1) CTS. The results show that after 5 years of permanent amendment of CTS, soils amended with 2.5 Mg ha(-1) have SMB and enzymatic activities similar to those in unamended soil.
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Soil bacterial diversity in degraded and restored lands of Northeast Brazil.
Antonie Van Leeuwenhoek
PUBLISHED: 08-14-2014
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Land degradation deteriorates biological productivity and affects environmental, social, and economic sustainability, particularly so in the semi-arid region of Northeast Brazil. Although some studies exist reporting gross measures of soil microbial parameters and processes, limited information is available on how land degradation and restoration strategies influence the diversity and composition of soil microbial communities. In this study we compare the structure and diversity of bacterial communities in degraded and restored lands in Northeast Brazil and determine the soil biological and chemical properties influencing bacterial communities. We found that land degradation decreased the diversity of soil bacteria as indicated by both reduced operational taxonomic unit (OTU) richness and Shannon index. Soils under native vegetation and restoration had significantly higher bacterial richness and diversity than degraded soils. Redundancy analysis revealed that low soil bacterial diversity correlated with a high respiratory quotient, indicating stressed microbial communities. By contrast, soil bacterial communities in restored land positively correlated with high soil P levels. Importantly, however, we found significant differences in the soil bacterial community composition under native vegetation and in restored land, which may indicate differences in their functioning despite equal levels of bacterial diversity.
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Determination of Flavanones in Orange Juices Obtained from Different Sources by HPLC/DAD.
J Anal Methods Chem
PUBLISHED: 08-07-2014
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Flavanones (hesperidin, naringenin, naringin, and poncirin) in industrial, hand-squeezed orange juices and from fresh-in-squeeze machines orange juices were determined by HPLC/DAD analysis using a previously described liquid-liquid extraction method. Method validation including the accuracy was performed by using recovery tests. Samples (36) collected from different Brazilian locations and brands were analyzed. Concentrations were determined using an external standard curve. The limits of detection (LOD) and the limits of quantification (LOQ) calculated were 0.0037, 1.87, 0.0147, and 0.0066?mg 100?g(-1) and 0.0089, 7.84, 0.0302, and 0.0200?mg 100?g(-1) for naringin, hesperidin, poncirin, and naringenin, respectively. The results demonstrated that hesperidin was present at the highest concentration levels, especially in the industrial orange juices. Its average content and concentration range were 69.85 and 18.80-139.00?mg 100?g(-1). The other flavanones showed the lowest concentration levels. The average contents and concentration ranges found were 0.019, 0.01-0.30, and 0.12 and 0.1-0.17, 0.13, and 0.01-0.36?mg 100?g(-1), respectively. The results were also evaluated using the principal component analysis (PCA) multivariate analysis technique which showed that poncirin, naringenin, and naringin were the principal elements that contributed to the variability in the sample concentrations.
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Inflammation, defective insulin signaling, and mitochondrial dysfunction as common molecular denominators connecting type 2 diabetes to Alzheimer disease.
Diabetes
PUBLISHED: 06-15-2014
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A growing body of evidence supports an intriguing clinical/epidemiological connection between Alzheimer disease (AD) and type 2 diabetes (T2D). T2D patients have significantly increased risk of developing AD and vice versa. Recent studies have begun to reveal common pathogenic mechanisms shared by AD and metabolic disorders, notably obesity and T2D. In T2D and obesity, low-grade chronic inflammation is a key mechanism leading to peripheral insulin resistance, which progressively causes tissue deterioration and overall health decline. In the brain, proinflammatory signaling was recently found to mediate impaired neuronal insulin signaling, synapse deterioration, and memory loss. Here, we review evidence indicating that inflammation, insulin resistance, and mitochondrial dysfunction are common features in AD and T2D. We further propose the hypothesis that dementia and its underlying neuronal dysfunction are exacerbated or driven by peripheral inflammation. Identification of central and peripheral inflammation as potential mediators of brain dysfunction in AD may lead to the development of effective treatments for this devastating disease.
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Determination of copper in airborne particulate matter using slurry sampling and chemical vapor generation atomic absorption spectrometry.
Talanta
PUBLISHED: 04-03-2014
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The present paper describes the development of a method for the determination of copper in airborne particulate matter using slurry sampling and chemical vapor generation atomic absorption spectrometry (CVG AAS). Chemometric tools were employed to characterize the influence of several factors on the generation of volatile copper species. First, a two-level full factorial design was performed that included the following chemical variables: hydrochloric acid concentration, tetrahydroborate concentration, sulfanilamide concentration and tetrahydroborate volume, using absorbance as the response. Under the established experimental conditions, the hydrochloric acid concentration had the greatest influence on the generation of volatile copper species. Subsequently, a Box-Behnken design was performed to determine the optimum conditions for these parameters. A second chemometric study employing a two-level full factorial design was performed to evaluate the following physical factors: tetrahydroborate flow rate, flame composition, alcohol volume and sample volume. The results of this study demonstrated that the tetrahydroborate flow rate was critical for the process. The chemometric experiments determined the following experimental conditions for the method: hydrochloric acid concentration, 0.208 M; tetrahydroborate concentration, 4.59%; sulfanilamide concentration, 0.79%; tetrahydroborate volume, 2.50 mL; tetrahydroborate flow rate, 6.50 mL min(-1); alcohol volume, 200 µL; and sample volume, 7.0 mL. Thus, this method, using a slurry volume of 500 µL and a final dilution of 7 mL, allowed for the determination of copper with limits of detection and quantification of 0.30 and 0.99 µg L(-1), respectively. Precisions, expressed as RSD%, of 4.6 and 2.8% were obtained using copper solutions at concentrations of 5.0 and 50.0 µg L(-1), respectively. The accuracy was evaluated by the analysis of a certified reference material of urban particulate matter. The copper concentration obtained was 570±63 mg kg(-1), and the certified value was 610±70 mg kg(-1). This method was applied for the determination of copper in airborne particulate matter samples collected in two Brazilian regions of Bahia State, Brazil. The copper contents found varied from 14.46 to 164.31 ng m(-3).
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Nitroxyl inhibits overt pain-like behavior in mice: role of cGMP/PKG/ATP-sensitive potassium channel signaling pathway.
Pharmacol Rep
PUBLISHED: 03-28-2014
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Several lines of evidence have indicated that nitric oxide (NO) plays complex and diverse roles in modulation of pain/analgesia. However, the roles of charged and uncharged congeners of NO are less well understood. In the present study, the antinociceptive effect of the nitroxyl (HNO) donor, Angeli's salt (Na2N2O3; AS) was investigated in models of overt pain-like behavior. Moreover, whether the antinociceptive effect of nitroxyl was dependent on the activation of cGMP (cyclic guanosine monophosphate)/PKG (protein kinase G)/ATP-sensitive potassium channels was addressed.
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Accumulation of Intraneuronal Amyloid-? is Common in Normal Brain.
Curr Alzheimer Res
PUBLISHED: 02-22-2014
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Intraneuronal amyloid-? (iA?) accumulation has been demonstrated in Alzheimer disease (AD). Although extracellular amyloid plaques composed primarily of aggregated amyloid-? are one of the main pathological features of AD, functional characterization of iA? is still lacking. In this study, we identified the normal distribution of iA? through an analysis of hippocampal sections from a series of over 90 subjects with diverse antemortem clinical findings. In addition to AD cases, iA? in pyramidal neurons was readily and reproducibly demonstrated in the majority of control cases. Similar findings for controls were made across all ages, spanning from infants to the elderly. There was no correlation of iA? between gender, postmortem interval, or age. While the possible pathophysiological significance of iA? accumulation in AD remains to be elucidated, careful examination of iA? found in the normal brain may be informative for determining the biological role of iA? and how this function changes during disease. Current findings support a physiological role for iA? in neuronal function over the entire lifespan.
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Inflammation, defective insulin signaling, and neuronal dysfunction in Alzheimer's disease.
Alzheimers Dement
PUBLISHED: 02-18-2014
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A link between Alzheimer's disease (AD) and metabolic disorders has been established, with patients with type 2 diabetes at increased risk of developing AD and vice versa. The incidence of metabolic disorders, including insulin resistance and type 2 diabetes is increasing at alarming rates worldwide, primarily as a result of poor lifestyle habits. In parallel, as the world population ages, the prevalence of AD, the most common form of dementia in the elderly, also increases. In addition to their epidemiologic and clinical association, mounting recent evidence indicates shared mechanisms of pathogenesis between metabolic disorders and AD. We discuss the concept that peripheral and central nervous system inflammation link the pathogenesis of AD and metabolic diseases. We also explore the contribution of brain inflammation to defective insulin signaling and neuronal dysfunction. Last, we review recent evidence indicating that targeting neuroinflammation may provide novel therapeutic avenues for AD.
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How does brain insulin resistance develop in Alzheimer's disease?
Alzheimers Dement
PUBLISHED: 02-18-2014
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Compelling preclinical and clinical evidence supports a pathophysiological connection between Alzheimer's disease (AD) and diabetes. Altered metabolism, inflammation, and insulin resistance are key pathological features of both diseases. For many years, it was generally considered that the brain was insensitive to insulin, but it is now accepted that this hormone has central neuromodulatory functions, including roles in learning and memory, that are impaired in AD. However, until recently, the molecular mechanisms accounting for brain insulin resistance in AD have remained elusive. Here, we review recent evidence that sheds light on how brain insulin dysfunction is initiated at a molecular level and why abnormal insulin signaling culminates in synaptic failure and memory decline. We also discuss the cellular basis underlying the beneficial effects of stimulation of brain insulin signaling on cognition. Discoveries summarized here provide pathophysiological background for identification of novel molecular targets and for development of alternative therapeutic approaches in AD.
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Analytical strategies for determination of cadmium in Brazilian vinegar samples using ET AAS.
Food Chem
PUBLISHED: 02-08-2014
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This paper proposes two methods for determination of cadmium in vinegar employing electrothermal atomic absorption spectrometry. The optimization step was performed using two-level full factorial and Box-Behnken designs, being that a new multiple response function was established. Under experimental conditions of pyrolysis temperature of 640 °C and atomization temperature of 2000 °C, the direct method allows the analysis using the external calibration technique, with limit of quantification of 14 ng L(-1) and characteristic mass of 1.2 pg, having aluminium as chemical modifier. This method was applied in six samples of vinegar acquired from Salvador City, Brazil. The cadmium content varied from 20 to 890 ng L(-1). Other method was also proposed by digestion using nitric acid and hydrogen peroxide in reflux system employing cold finger, being cadmium determined by ETAAS. The results obtained with the complete digestion procedure were in agreement with those found by the direct method proposed herein.
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Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.
Br. J. Pharmacol.
PUBLISHED: 02-03-2014
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The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects.
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Anti-hypernociceptive effect of mangiferin in persistent and neuropathic pain models in rats.
Pharmacol. Biochem. Behav.
PUBLISHED: 01-31-2014
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The present study examines the possible effect of the glucosylxanthone mangiferin (MG) on pain-related behaviors in a tonic acute pain model (formalin test at 5%) and in a chronic constriction injury (CCI) model to clarify the underlying transient and long-term mechanisms. Acute administration of MG (10-100mg/kg, i.p.) reduced licking/biting exclusivity in the tonic phase of formalin test in a naloxone and yohimbine-sensitive manner. This effect was enhanced by a nonselective nitric oxide synthase (NOS) inhibitor (NG-monomethyl-L-arginine) and by a non-competitive N-methyl-D-aspartate (NMDA) antagonist (ketamine), but it was reversed by the NOS substrate (L-arginine). Pre-treatment with intrathecal yohimbine prevented the anti-hypernociceptive effect of systemic MG. Pre-treatment during 4 days before surgical and 3 days after CCI with MG (50mg/kg, i.p.) reduced mechanical hypernociception and decreased the signs of Wallerian degeneration (WD) of the sciatic nerve. MG improved the PC-12 cellular viability exposure to glutamate-mediated neuronal death, also involved in neuropathic pain. The findings of this study suggest that MG shows ability to decrease tonic pain in the formalin test. A transient activity of this xanthone on nociceptive pathways mediated by ?2 adrenergic receptors in cooperation with the opioid system could be involved, at least in part, in this effect. Its neuroprotective effect by preventing WD in mononeuropathic rats could be implicated in the mechano-antihypernociceptive long term mechanisms.
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Role of TNF-?/TNFR1 in intense acute swimming-induced delayed onset muscle soreness in mice.
Physiol. Behav.
PUBLISHED: 01-26-2014
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The injection of cytokines such as TNF-? induces muscle pain. Herein, it was addressed the role of endogenous TNF-?/TNFR1 signaling in intense acute swimming-induced muscle mechanical hyperalgesia in mice. Mice were exposed to water during 30 s (sham) or to a single session of 30-120 min of swimming. Intense acute swimming induced a dose-dependent (time of exercise-dependent) muscle mechanical hyperalgesia, which peaked after 24 h presenting characteristics of delayed onset muscle soreness (DOMS). The intense acute swimming (120 min)-induced muscle mechanical hyperalgesia was reduced in etanercept (soluble TNF receptor) treated and TNFR1 deficient ((-/-)) mice. TNF-? levels increased 2 and 4 h after intense acute swimming in soleus muscle (but not in gastrocnemius), and spinal cord, respectively. Exercise induced an increase of myeloperoxidase activity and decrease in reduced glutathione levels in an etanercept-sensitive and TNFR1-dependent manners in the soleus muscle, but not in the gastrocnemius muscle. Concluding, TNF-?/TNFR1 signaling mediates intense acute swimming-induced DOMS by an initial role in the soleus muscle followed by spinal cord, inducing muscle inflammatory hyperalgesia and oxidative stress. The knowledge of these mechanisms might contribute to improve the training of athletes, individuals with physical impairment and intense training such as military settings.
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Determination of the mineral composition of Caigua (Cyclanthera pedata) and evaluation using multivariate analysis.
Food Chem
PUBLISHED: 01-22-2014
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Caigua (in Brazil "maxixe do reino") is a fruit that is generally consumed either cooked or even raw as salad. This fruit has been used as a food and also in folk medicine. In this work, the mineral composition of Caigua was determined for the first time. Twenty-nine samples from five farms located in the southwestern region of Bahia, Brazil were acquired and analyzed using inductively coupled plasma optical emission spectrometry. The elements determined in this fruit included calcium, magnesium, sodium, potassium, phosphorus, manganese, iron, zinc, copper and vanadium. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to evaluate the obtained results. The average concentrations of the determined elements (expressed as mg 100 g(-1)) were as follows: 0.91 for sodium, 152 for potassium, 19.4 for phosphorus, 11.9 for calcium, 8.4 for magnesium, 0.074 for manganese, 0.21 for iron, 0.013 for copper, 0.13 for zinc and 0.015 for vanadium.
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Synergistic anti-inflammatory effect: simvastatin and pioglitazone reduce inflammatory markers of plasma and epicardial adipose tissue of coronary patients with metabolic syndrome.
Diabetol Metab Syndr
PUBLISHED: 01-16-2014
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The inappropriate secretion of adipocytokines plays a critical role in chronic inflammatory states associated with obesity-linked type 2 diabetes and atherosclerosis. The pleiotropic actions of simvastatin and pioglitazone on epicardial adipose tissue (EAT) are unknown. This study assessed the anti-inflammatory actions of simvastatin and pioglitazone on EAT in patients with coronary artery disease (CAD) and metabolic syndrome (MS).
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In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 01-15-2014
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Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.
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Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil.
Mol. Biol. Rep.
PUBLISHED: 01-07-2014
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Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HR-HPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p < 0.0106; OR = 5.31; 95 % IC 1.59-17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection.
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Targeting interleukin-1? reduces intense acute swimming-induced muscle mechanical hyperalgesia in mice.
J. Pharm. Pharmacol.
PUBLISHED: 01-01-2014
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The role of interleukin (IL)-1? in intense acute swimming-induced muscle mechanical hyperalgesia was investigated in mice.
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Lipopolysaccharide induces inflammatory hyperalgesia triggering a TLR4/MyD88-dependent cytokine cascade in the mice paw.
PLoS ONE
PUBLISHED: 01-01-2014
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Inflammatory pain can be triggered by different stimuli, such as trauma, radiation, antigen and infection. In a model of inflammatory pain caused by infection, injection in the mice paw of lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, produces mechanical hyperalgesia. We identify here the TLR4 linked signaling pathways that elicit this response. Firstly, LPS paw injection in wild type (WT) mice produced mechanical hyperalgesia that was not altered in TRIF-/- mice. On the other hand, this response was absent in TLR4 mutant and MyD88 null mice and reduced in TNFR1 null mice. Either an IL-1 receptor antagonist, anti-KC/CXCL1 antibody, indomethacin or guanethidine injection also lessened this response. Moreover, LPS-induced time dependent increases in TNF-?, KC/CXCL1 and IL-1? expression in the mice paw, which were absent in TLR4 mutant and MyD88 null mice. Furthermore, in TNFR1 deficient mice, the LPS-induced rises in KC/CXCL1 and IL-1? release were less than in their wild type counterpart. LPS also induced increase of myeloperoxidase activity in the paw skin, which was inhibited in TLR4 mutant and MyD88 null mice, and not altered in TRIF-/- mice. These results suggest that LPS-induced inflammatory pain in mice is solely dependent on the TLR4/MyD88 rather than the TLR4/TRIF signaling pathway. This pathway triggers pronociceptive cytokine TNF-? release that in turn mediates rises in KC/CXCL1 and IL-1? expression. Finally, these cytokines might be involved in stimulating production of directly-acting hyperalgesic mediators such as prostaglandins and sympathomimetic amine.
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The prion protein ligand, stress-inducible phosphoprotein 1, regulates amyloid-? oligomer toxicity.
J. Neurosci.
PUBLISHED: 10-18-2013
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In Alzheimers disease (AD), soluble amyloid-? oligomers (A?Os) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrP(C)). However, it is unknown whether other ligands of PrP(C) can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrP(C) in the vicinity of the A?O binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in A?O toxicity. We confirmed the specific binding of A?Os and STI1 to the PrP and showed that STI1 efficiently inhibited A?O binding to PrP in vitro (IC50 of ?70 nm) and also decreased A?O binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented A?O-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to A?O-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both A?O binding to PrP(C) and PrP(C)-dependent A?O toxicity were inhibited by TPR2A, the PrP(C)-interacting domain of STI1. Additionally, PrP(C)-STI1 engagement activated ?7 nicotinic acetylcholine receptors, which participated in neuroprotection against A?O-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrP(C) ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset A?O-induced toxicity.
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Electrochemical Derivatization-Capillary Electrophoresis-Contactless Conductivity Detection: A versatile strategy for simultaneous determination of cationic, anionic and neutral analytes.
Electrophoresis
PUBLISHED: 09-19-2013
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The simultaneous determination of cationic, anionic and neutral analytes in a real sample was demonstrated by coupling electrochemical (EC) derivatization with counter-electroosmotic flow capillary electrophoresis and capacitively coupled contactless conductivity detection (CE-C(4) D). An electrochemical flow cell was used to oxidize alcohols from an antiseptic mouthwash sample into carboxylic acids at a platinum electrode in acid medium. The carboxylates formed in the derivatization process and other sample ingredients, such as benzoate, saccharinate and sodium ions, were separated in counter-flow mode and detected in one run in Tris-HCl buffer, pH 8.6. Fewer than 5 min were needed to complete each analysis with the automated flow system comprising solenoid pumps for the management of solutions. Insights into the electrochemistry of benzoic acid, present in the sample matrix, were also gained by EC-CE-C(4) D; more specifically, by applying potentials higher than 1.4 V to the platinum electrode, some formiate and minute amounts of salicylate were detected.
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TNF-? Mediates PKR-Dependent Memory Impairment and Brain IRS-1 Inhibition Induced by Alzheimers ?-Amyloid Oligomers in Mice and Monkeys.
Cell Metab.
PUBLISHED: 09-17-2013
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Alzheimers disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2? (eIF2?-P), and AD brains exhibit elevated phospho-PKR and eIF2?-P levels. Whether and how PKR and eIF2?-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that ?-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor ? (TNF-?)-dependent manner, resulting in eIF2?-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2?-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2?-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2?-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.
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Development of a simple method for the determination of nitrite and nitrate in groundwater by high-resolution continuum source electrothermal molecular absorption spectrometry.
Anal. Chim. Acta
PUBLISHED: 08-31-2013
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In this work, it was developed a method for the determination of nitrite and nitrate in groundwater by high-resolution continuum source electrothermal molecular absorption spectrometry of NO produced by thermal decomposition of nitrate in a graphite furnace. The NO line at 215.360nm was used for all analytical measurements and the signal obtained by integrated absorbance of three pixels. A volume of 20?L of standard solution or groundwater sample was injected into graphite furnace and 5?L of a 1% (m/v) Ca solution was co-injected as chemical modifier. The pyrolisis and vaporization temperatures established were of 150 and 1300°C, respectively. Under these conditions, it was observed a difference of thermal stability among the two nitrogen species in the presence of hydrochloric acid co-injected. While that the nitrite signal was totally suppressed, nitrate signal remained nearly stable. This way, nitrogen can be quantified only as nitrate. The addition of hydrogen peroxide provided the oxidation of nitrite to nitrate, which allowed the total quantification of the species and nitrite obtained by difference. A volume of 5?L of 0.3% (v/v) hydrochloric acid was co-injected for the elimination of nitrite, whereas that hydrogen peroxide in the concentration of 0.75% (v/v) was added to samples or standards for the oxidation of nitrite to nitrate. Analytical curve was established using standard solution of nitrate. The method described has limits of detection and quantification of 0.10 and 0.33?gmL(-1) of nitrogen, respectively. The precision, estimated as relative standard deviation (RSD), was of 7.5 and 3.8% (n=10) for groundwater samples containing nitrate-N concentrations of 1.9 and 15.2?gmL(-1), respectively. The proposed method was applied to the analysis of 10 groundwater samples and the results were compared with those obtained by ion chromatography method. In all samples analyzed, the concentration of nitrite-N was always below of the limit of quantification of both the methods. The concentrations of nitrate-N varied from 0.58 to 15.5?gmL(-1). No significant difference it was observed between the results obtained by both methods for nitrate-N, at the 95% confidence level.
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To Shunt or not to Shunt ? An Experimental Study Comparing Temporary Vascular Shunts and Venous Ligation as Damage Control Techniques for Vascular Trauma.
Ann Vasc Surg
PUBLISHED: 07-29-2013
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To evaluate vascular flow through temporary vascular shunts inserted into peripheral arteries and veins and the repercussion on the arterial perfusion, of venous ligation and venous shunt insertion in an experimental model for damage control.
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Amyloid-? oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons.
Mol. Biol. Cell
PUBLISHED: 06-19-2013
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Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimers disease (AD). Soluble amyloid-? oligomers (A?Os), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. A?Os also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that A?Os reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3?, downstream targets of CaN, prevents BDNF transport defects induced by A?Os. We further show that A?Os induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for A?O-induced BDNF transport disruption.
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Fractalkine mediates inflammatory pain through activation of satellite glial cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-17-2013
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The activation of the satellite glial cells (SGCs) surrounding the dorsal root ganglion (DRG) neurons appears to play a role in pathological pain. We tested the hypothesis that fractalkine, which is constitutively expressed by primary nociceptive neurons, is the link between peripheral inflammation and the activation of SGCs and is thus responsible for the genesis of the inflammatory pain. The injection of carrageenin into the rat hind paw induced a decrease in the mechanical nociceptive threshold (hypernociception), which was associated with an increase in mRNA and GFAP protein expression in the DRG. Both events were inhibited by anti-fractalkine antibody administered directly into the DRG (L5) [intraganglionar (i.gl.)]. The administration of fractalkine into the DRG (L5) produced mechanical hypernociception in a dose-, time-, and CX3C receptor-1 (CX3CR1)-dependent manner. Fractalkines hypernociceptive effect appears to be indirect, as it was reduced by local treatment with anti-TNF-? antibody, IL-1-receptor antagonist, or indomethacin. Accordingly, the in vitro incubation of isolated and cultured SGC with fractalkine induced the production/release of TNF-?, IL-1?, and prostaglandin E2. Finally, treatment with i.gl. fluorocitrate blocked fractalkine (i.gl.)- and carrageenin (paw)-induced hypernociception. Overall, these results suggest that, during peripheral inflammation, fractalkine is released in the DRG and contributes to the genesis of inflammatory hypernociception. Fractalkines effect appears to be dependent on the activation of the SGCs, leading to the production of TNF?, IL-1?, and prostanoids, which are likely responsible for the maintenance of inflammatory pain. Thus, these results indicate that the inhibition of fractalkine/CX3CR1 signaling in SGCs may serve as a target to control inflammatory pain.
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Memantine rescues transient cognitive impairment caused by high-molecular-weight a? oligomers but not the persistent impairment induced by low-molecular-weight oligomers.
J. Neurosci.
PUBLISHED: 06-07-2013
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Brain accumulation of soluble amyloid-? oligomers (A?Os) has been implicated in synapse failure and cognitive impairment in Alzheimers disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) A? oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW A?Os (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW A?Os (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different A? oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.
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[Knowledge, attitudes and practices on tuberculosis in prisons and public health services].
Rev Bras Epidemiol
PUBLISHED: 05-18-2013
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To analyze the knowledge, attitudes and practices about TB in a prison and in public health services (PHS).
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5-lipoxygenase deficiency reduces acetaminophen-induced hepatotoxicity and lethality.
Biomed Res Int
PUBLISHED: 04-30-2013
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5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO(-/-)) mice and background wild type mice were challenged with APAP (0.3-6?g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3?g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO(-/-) mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1 ? , TNF- ? , IFN- ? , and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl- ? -D-glucosaminidase activity, Nrf2 and gp91(phox) mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO(-/-) mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.
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Deregulation of excitatory neurotransmission underlying synapse failure in Alzheimers disease.
J. Neurochem.
PUBLISHED: 04-26-2013
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Alzheimers disease (AD) is the most common form of dementia in the elderly. Memory loss in AD is increasingly attributed to soluble oligomers of the amyloid-? peptide (A?Os), toxins that accumulate in AD brains and target particular synapses. Glutamate receptors appear to be centrally involved in synaptic targeting by A?Os. Once bound to neurons, A?Os dysregulate the activity and reduce the surface expression of both N-methyl-D-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) types of glutamate receptors, impairing signaling pathways involved in synaptic plasticity. In the extracellular milieu, A?Os promote accumulation of the excitatory amino acids, glutamate and D-serine. This leads to overactivation of glutamate receptors, triggering abnormal calcium signals with noxious impacts on neurons. Here, we review key findings linking A?Os to deregulated glutamate neurotransmission and implicating this as a primary mechanism of synapse failure in AD. We also discuss strategies to counteract the impact of A?Os on excitatory neurotransmission. In particular, we review evidence showing that inducing neuronal hyperpolarization via activation of inhibitory GABA(A) receptors prevents A?O-induced excitotoxicity, suggesting that this could comprise a possible therapeutic approach in AD.
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2,4-Dinitrophenol induces neural differentiation of murine embryonic stem cells.
Stem Cell Res
PUBLISHED: 04-22-2013
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2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20 ?M DNP (or vehicle) for 4 days. Significant increases in the proportion of nestin- and ?-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.
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Heme oxygenase inhibition enhances neutrophil migration into the bronchoalveolar spaces and improves the outcome of murine pneumonia-induced sepsis.
Shock
PUBLISHED: 03-14-2013
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A reduction of the neutrophil migration into the site of infection during cecal ligation and puncture-induced sepsis increases host mortality. Inhibition of heme oxygenase (HO) prevents this neutrophil paralysis and improves host survival in the cecal ligation and puncture model. Taking into account that almost 50% of all sepsis cases are a consequence of pneumonia, we designed the present study to determine the role of HO in an experimental model of pneumonia-induced sepsis. The objective of this study was to evaluate whether the inhibition of HO improves the outcome and pathophysiologic changes of sepsis induced by an intratracheal instillation of Klebsiella pneumoniae. The pretreatment of mice subjected to pneumonia-induced sepsis with ZnDPBG (zinc deuteroporphyrin 2,4-bis glycol), a nonspecific HO inhibitor, increased the number of neutrophils in the bronchoalveolar spaces, reduced the bacterial load at the site of infection, and prevented the upregulation of CD11b and the downregulation of CXCR2 on blood neutrophils. Moreover, the pretreatment with ZnDPBG decreased alveolar collapse, attenuating the deleterious changes in pulmonary mechanics and gas exchanges and, as a consequence, improved the survival rate of mice from 0% to ?20%. These results show that heme oxygenase is involved in the pathophysiology of pneumonia-induced sepsis and suggest that HO inhibitors could be helpful for the management of this disease.
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The nitroxyl donor, Angelis salt, inhibits inflammatory hyperalgesia in rats.
Neuropharmacology
PUBLISHED: 02-22-2013
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Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO(-)) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angelis salt (AS; Na2N2O3). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 ?g/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-?, interleukin-1? and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K(+) channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO(+) donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K(+) channel signaling pathway in vivo.
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Skin vasodilation and analgesic effect of a topical nitric oxide-releasing hydrogel.
J Mater Sci Mater Med
PUBLISHED: 02-11-2013
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New approaches based on topical treatments are needed for treating pain and impaired dermal blood flow. We used a topical Pluronic F127 hydrogel containing S-nitrosoglutathione (GSNO) as a prodrug to generate free NO, an effector molecule that exerts both dermal vasodilation and antinociceptive effects. GSNO-containing hydrogels underwent gelation above 12 °C and released free NO at rates that were directly dependent on the GSNO concentration in the range of 50-150 mM. The topical application of this material led to dose-response dermal vasodilation in healthy volunteers and to a reduction of up to 50 % of the hypernociception intensity in Wistar rats that were subjected to inflammatory pain. Mechanistic investigations indicated that the antinociceptive effect of the topical F127/GSNO hydrogels is produced by the local activation of the cGMP/PKG/KATP channel-signaling pathway, which was stimulated by the free NO that diffused through the skin. These results expand the scope of the biomedical applications of this material and may represent a new approach for the topical treatment of inflammatory pain.
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Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-11-2013
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It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 ?L) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1? (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1?. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1? in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1(+) cells of the DRG, significantly increased after carrageenan or IL-1? administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKC? expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1? in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.
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Pressure-driven mesofluidic platform integrating automated on-chip renewable micro-solid-phase extraction for ultrasensitive determination of waterborne inorganic mercury.
Talanta
PUBLISHED: 01-25-2013
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A dedicated pressure-driven mesofluidic platform incorporating on-chip sample clean-up and analyte preconcentration is herein reported for expedient determination of trace level concentrations of waterborne inorganic mercury. Capitalizing upon the Lab-on-a-Valve (LOV) concept, the mesofluidic device integrates on-chip micro-solid phase extraction (?SPE) in automatic disposable mode followed by chemical vapor generation and gas-liquid separation prior to in-line atomic fluorescence spectrometric detection. In contrast to prevailing chelating sorbents for Hg(II), bare poly(divinylbenzene-N-vinylpyrrolidone) copolymer sorptive beads were resorted to efficient uptake of Hg(II) in hydrochloric acid milieu (pH=2.3) without the need for metal derivatization nor pH adjustment of prior acidified water samples for preservation to near-neutral conditions. Experimental variables influencing the sorptive uptake and retrieval of target species and the evolvement of elemental mercury within the miniaturized integrated reaction chamber/gas-liquid separator were investigated in detail. Using merely <10 mg of sorbent, the limits of detection and quantification at the 3s(blank) and 10s(blank) levels, respectively, for a sample volume of 3 mL were 12 and 42 ng L(-1) Hg(II) with a dynamic range extending up to 5.0 ?g L(-1). The proposed mesofluidic platform copes with the requirements of regulatory bodies (US-EPA, WHO, EU-Commission) for drinking water quality and surface waters that endorse maximum allowed concentrations of mercury spanning from 0.07 to 6.0 ?g L(-1). Demonstrated with the analysis of aqueous samples of varying matrix complexity, the LOV approach afforded reliable results with relative recoveries of 86-107% and intermediate precision down to 9% in the renewable ?SPE format.
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Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.
PLoS ONE
PUBLISHED: 01-01-2013
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Alzheimers disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-? peptide (A?) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble A? than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble A?40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble A?40 and blocking A?-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing ?7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of A?40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by ?7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble A?, IQ and analogues of IQ on ?3?4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble A?40 potently inhibit the function of ?3?4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of ?3?4 nAChRs by A?40 reversible. These findings indicate that A?40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.
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?-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid ? (A?)-associated cell death.
J. Biol. Chem.
PUBLISHED: 12-19-2011
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In physiological conditions, both ?-amyloid precursor protein (?APP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated ?-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-? (sAPP?) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid ? (A?)-mediated toxicity. We first show that both recombinant sAPP? and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated ?APP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated ?APP (APP(LDN)) that yields A? oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with A?-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPP? deficit taking place at the early asymptomatic phase of Alzheimer disease.
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Histological, biochemical and pharmacological characterization of the gastric muscular layer in Chagas disease.
Acta Cir Bras
PUBLISHED: 10-28-2011
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To assess in vitro the correlation between the number of neurons and the sensitivity to cholinergic drugs and acetylcholinesterase activity in chagasic patients.
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Peri-implant disease in subjects with and without preventive maintenance: a 5-year follow-up.
J. Clin. Periodontol.
PUBLISHED: 10-07-2011
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To determine the incidence of peri-implantitis in individuals with mucositis in a 5-year follow-up study.
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Homogeneity and stability studies during the preparation of a laboratory reference material of soy leaves for the determination of metals.
J AOAC Int
PUBLISHED: 09-28-2011
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The homogeneity and stability of metals were tested in a candidate laboratory reference material of soy leaves. Inductively coupled plasma-optical emission spectrometry was used to quantify calcium, magnesium, manganese, iron, zinc, copper, and vanadium. A 6 kg amount of the material, which was dried, ground, and classified as mesh 60, was distributed among 100 bottles. The between-bottle homogeneity test was established by analyzing two subsamples from nine bottles. For the within-bottle test, five determinations of each element of a single bottle were performed. The stability test was performed at temperatures of -10, +27, and +40 degrees C, and after storage times of 4, 12, 24, and 52 weeks. The obtained results indicated that the material was homogeneous and stable under the conditions studied.
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[Prevalence of human papillomavirus genotypes: comparison between three detection methods in patients of Pernambuco, Brazil].
Rev Bras Ginecol Obstet
PUBLISHED: 08-26-2011
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to compare three methods for the detection of HPV infection and to determine the prevalence of the genotypes found.
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Validation of a digestion system using a digester block/cold finger system for the determination of lead in vegetable foods by electrothermal atomic absorption spectrometry.
J AOAC Int
PUBLISHED: 07-30-2011
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This paper presents the validation of a system for sample digestion using a digester block/cold finger to determine the lead content in vegetables by electrothermal atomic absorption spectrometry (ETAAS). After mineralization, lead contents were determined by ETAAS using a calibration curve based on aqueous standards prepared in 2.60 M nitric acid solutions containing 5 microg ammonium phosphate as chemical modifier. A pyrolysis temperature of 900 degrees C and atomization temperature of 2000 degrees C were used. This method allowed the determination of lead with a characteristic mass of 35 pg; LOD and LOQ of 0.6 and 2 nglg, respectively, were found. The precision was investigated in terms of reproducibility and repeatability. Reproducibility was estimated by analysis of nine different portions of a certified reference material (CRM) of spinach leaves, and the repeatability was determined through the analysis of nine aliquots of the same solution. The reproducibility and repeatability were found to be 4.27 and 2.94% RSD, respectively. The accuracy was confirmed by analysis of whole meal flour, spinach leaves, and orchard leaves CRMs, all furnished by the National Institute of Standards and Technology. Lead contents were measured using the newly developed technique in 11 different potato samples. The lead contents ranged from 12.80 to 69.27 ng/g, with an average value of 28.59 ng/g. These values were in agreement with data reported in the literature.
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Endophytic Methylobacterium extorquens expresses a heterologous ?-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa.
World J. Microbiol. Biotechnol.
PUBLISHED: 07-26-2011
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Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.
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Selenite biotransformation during brewing. Evaluation by HPLC-ICP-MS.
Talanta
PUBLISHED: 06-20-2011
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Yeast (Saccharomyces cerevisiae) and lactic bacteria have shown their ability to accumulate and transform inorganic selenium into organo Se compounds. The objective of this work was to evaluate selenium biotransformation during brewing by using S. cerevisiae and Saccharomyces uvarum for Ale and Lager fermentation, respectively. Se-enriched beer was produced by the addition of sodium selenite (0, 0.2, 1.0, 2.0, 10.0, 20.0 ?g Se mL(-1), respectively) to the fermentation media composed of yeast, malt extract and water. The alcoholic fermentation process was not affected by the presence of selenium regardless of the type of Saccharomyces being used. The percentage of selenium incorporated into beer, added between 1.0 and 10 ?g mL(-1) was 55-60% of the selenium initially present. Se-compounds in post-fermentation (beer and yeast) products were investigated by using an analytical methodology based on HPLC-ICP-MS. For this purpose, several sample treatments, including ultrasonic-assisted enzymatic hydrolysis, in conjunction with different separation mechanisms like dialysis and anion exchange HPLC chromatography were applied for unambiguously identifying Se-species that produce during brewing. Selenomethionine was the main selenium compound identified in beer and yeast, being this species in the only case of the former not associated to peptides or proteins.
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Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.
PLoS ONE
PUBLISHED: 05-20-2011
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The amyloid precursor protein (APP) is well known for giving rise to the amyloid-? peptide and for its role in Alzheimers disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPP?(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPP?(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPP?(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)?8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPP? binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPP? modulates the biological action of semaphorins.
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The A? oligomer hypothesis for synapse failure and memory loss in Alzheimers disease.
Neurobiol Learn Mem
PUBLISHED: 04-04-2011
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Alzheimers disease (AD) is the 3rd most costly disease and the leading cause of dementia. It can linger for many years, but ultimately is fatal, the 6th leading cause of death. Alzheimers disease (AD) is fatal and affected individuals can sometimes linger many years. Current treatments are palliative and transient, not disease modifying. This article reviews progress in the search to identify the primary AD-causing toxins. We summarize the shift from an initial focus on amyloid plaques to the contemporary concept that AD memory failure is caused by small soluble oligomers of the A? peptide, toxins that target and disrupt particular synapses. Evidence is presented that links A? oligomers to pathogenesis in animal models and humans, with reference to seminal discoveries from cell biology and new ideas concerning pathogenic mechanisms, including relationships to diabetes and Fragile X. These findings have established the oligomer hypothesis as a new molecular basis for the cause, diagnosis, and treatment of AD.
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Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis.
PLoS ONE
PUBLISHED: 03-25-2011
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Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.
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Amyloid-beta oligomers increase the localization of prion protein at the cell surface.
J. Neurochem.
PUBLISHED: 03-23-2011
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In Alzheimers disease, the amyloid-? peptide (A?) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for A?. We show here that A? oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if A? oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with A? oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of A? oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. A? oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after A? oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of A? oligomers. Our experiments show for the first time that A? oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.
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Pain and analgesia: The dual effect of nitric oxide in the nociceptive system.
Nitric Oxide
PUBLISHED: 02-17-2011
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Nitric oxide (NO) is involved in many physiological processes and several lines of evidence have indicated that NO plays a complex and diverse role in the modulation of pain. Nitric oxide is an important neurotransmitter involved in the nociceptive process and, in the dorsal horn of the spinal cord, it contributes to the development of central sensitization. On the other hand, experimental data have also demonstrated that NO inhibits nociception in the peripheral and also in the central nervous system. In addition, it has been shown that nitric oxide mediates the analgesic effect of opioids and other analgesic substances. The information included in the present review aims to present and analyze data about the dual effect of NO on pain transmission and control, the molecular mechanisms involved in these effects and also the potential use of nitric oxide in pain therapy.
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Exogenous ornithine is an effective precursor and the ?-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves.
J. Plant Physiol.
PUBLISHED: 02-04-2011
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The role of the ?-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and ?(1)-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas ?(1)-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.
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Quercetin reduces neutrophil recruitment induced by CXCL8, LTB4, and fMLP: inhibition of actin polymerization.
J. Nat. Prod.
PUBLISHED: 01-28-2011
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Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Furthermore, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.
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Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: participation of TNF-?, IL-1? and CXCL1/KC.
Eur. J. Pharmacol.
PUBLISHED: 01-21-2011
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Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/HeJ mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-?, IL-1? and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-?, IL-1? and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis.
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Protein kinase C activity regulates D-serine availability in the brain.
J. Neurochem.
PUBLISHED: 01-21-2011
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D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.
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Granulocyte-colony stimulating factor (G-CSF) induces mechanical hyperalgesia via spinal activation of MAP kinases and PI3K in mice.
Pharmacol. Biochem. Behav.
PUBLISHED: 01-12-2011
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Granulocyte-colony stimulating factor (G-CSF) is a current pharmacological approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is most relevant side effect of G-CSF in healthy volunteers and cancer patients. Therefore, the mechanisms of G-CSF-induced hyperalgesia were investigated focusing on the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, and PI(3)K (phosphatidylinositol 3-kinase). G-CSF induced dose (30-300 ng/paw)-dependent mechanical hyperalgesia, which was inhibited by local post-treatment with morphine. This effect of morphine was reversed by naloxone (opioid receptor antagonist). Furthermore, G-CSF-induced hyperalgesia was inhibited in a dose-dependent manner by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmanin) inhibitors. The co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited G-CSF-induced hyperalgesia. Concluding, in addition to systemic opioids, peripheral opioids as well as spinal treatment with MAP kinases and PI(3)K inhibitors also reduce G-CSF-induced pain.
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Airway epithelium mediates the anti-inflammatory effects of exercise on asthma.
Respir Physiol Neurobiol
PUBLISHED: 01-04-2011
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Airway epithelium plays an important role in the asthma physiopathology. Aerobic exercise decreases Th2 response in murine models of allergic asthma, but its effects on the structure and activation of airway epithelium in asthma are unknown. BALB/c mice were divided into control, aerobic exercise, ovalbumin-sensitized and ovalbumin-sensitized plus aerobic exercise groups. Ovalbumin sensitization occurred on days 0, 14, 28, 42, and aerosol challenge from day 21 to day 50. Aerobic exercise started on day 22 and ended on day 50. Total cells and eosinophils were reduced in ovalbumin-sensitized group submitted to aerobic exercise. Aerobic exercise also reduced the oxidative and nitrosative stress and the epithelial expression of Th2 cytokines, chemokines, adhesion molecules, growth factors and NF-kB and P2X7 receptor. Additionally, aerobic exercise increased the epithelial expression of IL-10 in non-sensitized and sensitized animals. These findings contribute to the understanding of the beneficial effects of aerobic exercise for chronic allergic airway inflammation, suggesting an immune-regulatory role of exercise on airway epithelium.
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Synthesis of a new solid-phase extractor and its application to preconcentration and determination of lead in water samples.
J AOAC Int
PUBLISHED: 12-15-2010
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A procedure for preconcentration and determination of lead in water is described. The method is based on the sorption of Pb(II) in a minicolumn packed with a functionalized sorbent and subsequent elution with acidic solution. The determination of lead content in the eluate was carried out using flame atomic absorption spectrometry. The sorbent was prepared by immobilization of the ligand 4-(5-bromo-2-thiazolylazo)orcinol on polystyrene-divinylbenzene through an azo spacer. Diazotization and coupling reactions were used for synthesis of the sorbent. Some variables affecting the preconcentration were optimized using a full factorial design. Under optimized conditions, the method presented a detection limit of 0.5 microg/L and enrichment factor of 36 for a sample volume of 25 mL. The accuracy of the method was tested by the determination of lead in a standard reference material (National Institute of Standards and Technology 1643d Fresh Water). The proposed procedure was applied to the determination of lead in samples of natural and drinking waters.
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Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers.
J. Biol. Chem.
PUBLISHED: 11-29-2010
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Soluble oligomers of the amyloid-? peptide (A?Os) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. A?Os have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of A?Os. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by A?Os in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser(845), which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of A?Os, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by A?Os in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD.
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N-methyl-D-aspartate receptors are required for synaptic targeting of Alzheimers toxic amyloid-? peptide oligomers.
J. Neurochem.
PUBLISHED: 11-11-2010
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Soluble amyloid-? peptide (A?) oligomers, known to accumulate in Alzheimers disease brains, target excitatory post-synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimers disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N-methyl-d-aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock-down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by A? oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock-down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer-attacked and non-attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre-treatment of neurons with insulin down-regulates oligomer-binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by A? oligomers.
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Amyloid-? triggers the release of neuronal hexokinase 1 from mitochondria.
PLoS ONE
PUBLISHED: 10-01-2010
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Brain accumulation of the amyloid-? peptide (A?) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimers disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between A?-induced oxidative stress and HK activity. We found that A? triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. A? oligomers further impair energy metabolism by decreasing neuronal ATP levels. A?-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked A?-induced oxidative stress and neuronal death. Results suggest that A?-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.
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Amyloid-? decreases nitric oxide production in cultured retinal neurons: a possible mechanism for synaptic dysfunction in Alzheimers disease?
Neurochem. Res.
PUBLISHED: 09-29-2010
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The neurotoxicity of the amyloid-? peptide (A?) appears to be, at least in part, related to pathological activation of glutamate receptors by A? aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimers disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by A??? and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that L-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either A??? (20 ?M) or glutamate (1 mM). Surprisingly, at short incubation times both A? and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by A? and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by A? or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by A? and glutamate suggests a possible mechanism leading to synaptic failure in AD.
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Determination and evaluation of the mineral composition of Obi (Cola acuminate).
Biol Trace Elem Res
PUBLISHED: 08-19-2010
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The obi (Cola acuminate) is a native fruit from Africa, which has been mainly used in the production of soft drinks and also in rituals of African religions. In this paper, the mineral composition of obi collected in seven different cities from Bahia State, Brazil was determined and evaluated using multivariate analysis. The samples were digested using nitric acid and hydrogen peroxide and were analyzed using inductively coupled plasma optical emission spectrometry. The accuracy of the method was confirmed by analysis of a certified reference material of apple leaves, furnished by National Institute of Standard and Technology. The study involved 46 samples, being 18 of the red specie and 28 for the white specie. The results expressed as milligrams of element per 100 g(-1) of sample demonstrated that the concentration ranges varied of 21.28-548.77 for potassium, 15.73-129.85 for phosphorous, 27.95-286.92 for calcium, 7.67-134.45 for magnesium, 0.05-1.41 for manganese, 0.21-0.94 for iron, 0.11-0.39 for copper, 0.27-1.35 for zinc, and 0.025-0.517 for strontium. The principal component analysis and hierarchical cluster analysis evidenced that the mineral composition of the red specie is different of the white specie. The red obi has mineral content higher than white obi.
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[Knowledge and practice regarding tobacco use among pharmacy undergraduate students in Lima, Peru].
Rev Lat Am Enfermagem
PUBLISHED: 08-10-2010
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The aim of this study was to determine the knowledge and the practices regarding tobacco consumption in pharmacy students according to their demographic and academic characteristics. The sample consisted of 276 (55.2%) pharmacy students of a Peruvian Private University to whom the Global Youth Tobacco Survey (GYTS) was applied. In men, the prevalence of lifetime use of tobacco of 93.7%, of alcohol 70.8% and of illegal drugs 14.1% was identified. The prevalence of lifetime and current consumption of tobacco in the sample was very high. The majority of students began using drugs at 16 years old. There was a strong association between the consumption of tobacco and the fact that at least one of the parents smoked and the exposure to smoke inside the home.
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[Smoking among nurses of the national hospital in Lima, Peru].
Rev Lat Am Enfermagem
PUBLISHED: 08-10-2010
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Nurses have unique opportunities to help their patients to stop smoking. The aim of this study was to identify, among nurses of a hospital, the consumption of smoking products, their attitudes and information received on smoking. It was a descriptive study, carried out with 204 (48%) nurses of the Hospital Arzobispo Loayza, Lima, Peru. The Global Health Professional Survey (GHPS) was used. Among the sample the prevalence of smoking in life was 67.1% and 3% in the last month. The majority of nurses presented positive attitudes and knew the importance and responsibility of advising patients to quit. The majority had received general information regarding the topic, though half of them had not received specific information regarding treatment. In view of the importance of this theme, this study identified a need for further research of this nature as well as revision of the curricula regarding the approach toward the theme of smoking.
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Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons.
J. Neurosci.
PUBLISHED: 07-09-2010
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Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimers disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (AbetaOs), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 microm AbetaOs. NMDA receptor (NMDAR) antagonists, including d-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for AbetaO action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3beta (GSK-3beta) and transfection of neurons with a kinase-dead form of GSK-3beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that AbetaOs do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that AbetaOs negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3beta and establish a new connection between toxic Abeta oligomers and AD pathology.
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