alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Existem diferenças nos parâmetros hematológicos e bioquímicos séricos entre fêmeas normais e portadoras do modelo experimental GRMD (Golden Retriever Muscular Dystrophy)?

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.

Should reproductively isolated populations of Lutzomyia longipalpis sensu lato receive taxonomically valid names?

A group of 18 research workers involved in different aspects of the biology of Lutzomyia longipalpis discussed whether or not it is important to give taxonomically valid names to populations that have been defined by biological, biochemical and molecular methods to be reproductively isolated. The type material of this medically important species has been lost and because of this it was recommended that a colony should be established from insects captured in the region of the type area and that their description should serve as the basis for future descriptions. It was pointed out that there is a lack of uniformity in the naming of closely related American sand flies and that some of the differences between populations of Lu. longipalpis are greater than those between accepted species. The majority of the participants agreed that the populations that have been defined in the literature as sibling species should be named.

Biópsia hepática por laparotomia paracostal em bovinos e búfalos

A técnica de biópsia hepática em ruminantes tem importante valor no diagnóstico clínico de doenças tóxicas e metabólicas, em especial nos desequilíbrios minerais. As técnicas mais comumente utilizadas restringem análises devido ao limitado volume de tecido obtido. No presente trabalho, avaliou-se o uso de uma técnica de biópsia hepática por laparotomia paracostal em bovinos e búfalos. Foram utilizados 10 bovinos e 10 búfalos hígidos. Os animais foram mantidos em estação, sedados com xilazina e infiltrados localmente com lidocaína e epinefrina. O acesso à cavidade abdominal foi realizado por meio de uma incisão dorso-ventral de 15cm no flanco direito, iniciada ventralmente (cerca de 4-5cm) ao processo transverso da 2a ou 3a vértebra lombar e situada caudalmente (cerca de 4cm) e paralelamente à 13a costela, obtendo-se visualização do fígado. Foi então realizado pinçamento do bordo caudal do órgão com pinça Doyen para remoção de fragmento hepático (2 a 4g). Procedeu-se o fechamento da cavidade abdominal como de rotina. Foram analisados os parâmetros bioquímicos e hematológicos antes do procedimento (tempo zero) e após 24 horas, 48 horas, 5 dias e 10 dias após a biópsia. Todas as variáveis bioquímicas estudadas retornaram aos valores basais 5 e 10 dias após o procedimento nos bovinos e búfalos, respectivamente. O tempo médio de cirurgia por animal foi de 25 minutos. A biópsia hepática por laparotomia paracostal demonstrou ser uma técnica eficaz e de baixo risco à saúde dos animais, permitindo a coleta de suficiente quantidade de tecido hepática para realização de múltiplas análises.

Effect of an energy-deficient diet on populations of ciliate protozoans in bovine rumen

Ten young rumen-cannulated crossbred steers were randomly divided into two groups: a control group (C; n=4), which was fed a balanced diet for daily weight gain of 900g; and a pronounced energy-deprived group (PED; n=6), receiving 30% less of the required energy for maintenance. After 140 days of these alimentary regimes, rumen fluid and urine samples were collected for biochemical and functional tests, before feeding and at 1, 3, 6, and 9 hours after feeding. The energy-deprivation diet caused a significant reduction in the number of Entodinium, Eodinium, Isotricha, Dasytricha, Eremoplastron, Eudiplodinium, Metadinium, Charonina, Ostracodinium, and Epidinium protozoa. There was no effect of the time of sampling in both groups on the total number of ciliates in rumen fluid. A higher number of protozoan forms in binary division were recorded in the control group, at the 6th and 9th hours after feeding (P<0.019). There was a high positive correlation between the total count of protozoans in rumen fluid and glucose fermentation, ammonia, and urinary allantoin excretion index; and a negative correlation between the total count of protozoa and metilene blue reduction, and a medium correlation between the total count of protozoa and total volatile fatty acids concentration. The determination of the protozoa populations does not imply in the use of complex and hard-to-execute techniques, although it is time consuming and needs practice. This exam particularly helps in clinical expected diagnosis.

Evaluation of the spouted bed dried leaf extract of Bauhinia forficata for the treatment of experimental diabetes in rats

Previously, we have demonstrated that treatment of experimental diabetes with a decoction of Bauhinia forficata leaves is beneficial. In this study, we prepared a two-fold concentrate of this extract and tested its effects on physiological, biochemical and toxicity markers in streptozotocin-diabetic rats. Dried and ground leaves were extracted with warm 70% hydroethanol and the filtrate concentrated by evaporation at 50 degrees C. This solution was mixed with colloidal silicon dioxide (Tixosil-333 (R)) and dried in a spouted bed (BfT). Rats were treated with water, insulin and Tixosil particles at low or high doses, alone or coated with dried BfT. Animals were periodically weighed and monitored for water and food intake; urinary volume, glucose, urea and protein; blood glucose, serum lipids, liver toxicity markers transaminase and phosphatase and masses of adipose tissue and skeletal muscle. Insulin treatment gave best rat growth and lowest values for all other markers. No other treatment affected any diabetic marker, but the enzyme activities were changed by diabetes and BfT. Thus, BfT toxicity could arise from secondary products of plant constituents or Tixosil interaction. Therefore, BfT prepared in the spouted bed as described, is unsuitable for treatment of diabetes, which implies that the method of preparation of any medicine is critical for its efficacy and toxicity.

Nutritional Status of Zinc in Children with Down Syndrome

Experimental and clinical studies have established that zinc metabolism is altered in individuals with Down syndrome (DS). The present study intends to evaluate the nutritional status of zinc in children with DS by determining their biochemical and dietary parameters. The investigation was carried out on a group of children with DS (n = 35) and compared with a control group (n = 33), both aging between 4 and 11 years. Weight-for-age, height-for-age, and weight-for-height indexes and diet were evaluated by using a 3-day dietary record. Zinc was evaluated in plasma, erythrocytes, and 24-h urine collection by using the method of atomic absorption spectroscopy. The frequency of short stature was higher in children with DS. Both groups presented high protein content, adequate concentrations of lipids and carbohydrates, and deficit in calories. Adequate zinc intake was observed in 40% of children with DS and in 67% of the control group. Zinc concentrations were significantly lower in plasma and urine and higher in erythrocytes of children with DS. The results allowed us to conclude that the altered zinc nutritional status of individuals with Down syndrome contributes to clinical disturbances that usually appear with aging in these patients.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-inserisitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG. (C) 2008 Elsevier Inc. All rights reserved.

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas` disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T cruzi strain Y solved at 2.2 angstrom resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation Of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design. (c) 2008 Elsevier Inc. All rights reserved.

Potentiation of dapsone induced methemoglobinemia by N-acetylcysteinc in rats

Dapsone (DDS) (4,4` diaminodiphenylsulfone), the drug of choice for the treatment of leprosy, frequently induces hemolytic anemia and methemoglobinemia. N-hydroxylation, one of the major pathways of biotransformation, has been constantly related to the methemeglobinemia after the use of the drug. In order to prevent the dapsone-induced hemotoxicity, N-acetylcysteine, a drug precursor of glutathione, was administered in combination with DDS to male Wistar rats, weighting 220-240 g. The animals were then anaesthetized and blood was collected from the aorta for determination of plasma DDS concentration by HPLC, determination of methemoglobinemia and glutathione by spectrophotometry, and for biochemical and hematological parameters. Our results showed that N-acetylcysteine enhanced dapsone-induced methemoglobinemia due to increased dapasone plasmatic concentration and consequent increased N-hydroxylamine formation. We concluded that drug interactions with dapsone require individually studies in order to avoid undesirable effects of dapsone.

Acute Toxicity of Schizolobium parahyba Aqueous Extract in Mice

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism. Copyright (C) 2009 John Wiley & Sons, Ltd.

Tobacco Smoke Induces Ventricular Remodeling Associated with an Increase in NADPH Oxidase Activity

Background: Recent studies have assessed the direct effects of smoking on cardiac remodeling and function. However, the mechanisms of these alterations remain unknown. The aim of this study was to investigate de role of cardiac NADPH oxidase and antioxidant enzyme system on ventricular remodeling induced by tobacco smoke. Methods: Male Wistar rats that weighed 200-230 g were divided into a control group (C) and an experimental group that was exposed to tobacco smoke for a period of two months (ETS). After the two-month exposure period, morphological, biochemical and functional analyses were performed. Results: The myocyte cross-sectional area and left ventricle end-diastolic dimension was increased 16.2% and 33.7%, respectively, in the ETS group. The interstitial collagen volume fraction was also higher in ETS group compared to the controls. In addition to these morphological changes, the ejection fraction and fractional shortening were decreased in the ETS group. Importantly, these alterations were related to augmented heart oxidative stress, which was characterized by an increase in NADPH oxidase activity, increased levels of lipid hydroperoxide and depletion of antioxidant enzymes (e.g., catalase, superoxide dismutase and glutathione peroxidase). In addition, cardiac levels of IFN-gamma, TNF-alpha and IL-10 were not different between the groups. Conclusion: Cardiac alterations that are induced by smoking are associated with increased NADPH oxidase activity, suggesting that this pathway plays a role in the ventricular remodeling induced by exposure to tobacco smoke. Copyright (C) 2011 S. Karger AG, Basel

Spinal Myoclonus after Subarachnoid Anesthesia with Bupivacaine

Abrao J, Bianco MP, Roma W, Krippa JAS, Hallak JE - Spinal Myoclonus after Subarachnoid Anesthesia with Bupivacaine. Background and objectives: It is presented in this case report a very rare complication after spinal anesthesia to provide subsidies to the management and therapeutic conduct. Case report: This is a 63-year old African-Brazilian patient, ASA I, scheduled for transurethral resection of the prostate (TURP). He underwent subarachnoid anesthesia with bupivacaine (15 mg) without adrenaline. Intercurrences were not observed during puncture, and the patient was positioned for surgery. Soon after positioning the patient, he complained of severe pain in the perineum region followed by involuntary tonic-clonic movements of the lower limbs. The patient was treated with a benzodiazepine to control the myoclonus without response. This episode was followed by significant agitation and the patient was intubated. He was maintained in controlled ventilation and transferred to the Intensive Care Unit. Despite all biochemical and imaging tests performed, an apparent cause was not detected. The medication was not changed and the same batch of anesthetic had been used in other patients that same day without intercurrences. Conclusions: After ruling out all possible causes, the diagnosis of spinal myoclonus after spinal anesthesia with bupivacaine was made by exclusion.

Evolutionary transition to freshwater by ancestral marine palaemonids: evidence from osmoregulation in a tide pool shrimp

The transition from marine/brackish waters to freshwater habitats constitutes a severe osmotic and ionic challenge, and successful invasion has demanded the selection of morphological, physiological, biochemical and behavioral adaptations. We evaluated short-term (1 to 12 h exposure) and long-term (5 d acclimation), anisosmotic extracellular (osmolality, [Na(+), Cl(-)]) and long-term isosmotic intracellular osmoregulatory capability in Palaemon northropi, a neotropical intertidal shrimp. F northropi survives well and osmo- and ionoregulates strongly during short- and long-term exposure to 5-45 parts per thousand salinity, consistent with its rocky tide pool habitat subject to cyclic salinity fluctuations, Muscle total free amino acid (FAA) concentrations decreased by 63% in shrimp acclimated to 5%. salinity, revealing a role in hypoosmotic cell volume regulation; this decrease is mainly a consequence of diminished glycine, arginine and proline. Total FAA contributed 31% to muscle intracellular osmolality at 20 parts per thousand, an isosmotic salinity, and decreased to 13% after acclimation to 5 parts per thousand. Gill and nerve tissue FAA concentrations remained unaltered. These tissue-specific responses reflect efficient anisosmotic and anisoionic extracellular regulatory mechanisms, and reveal the dependence of muscle tissue on intracellular osmotic effectors. FAA concentration is higher in P. northropi than in diadromous and hololimnetic palaemonids, confirming muscle FAA concentration as a good parameter to evaluate the degree of adaptation to dilute media. The osmoregulatory capability of P. northropi may reflect the potential physiological capacity of ancestral marine palaemonids to penetrate into dilute media, and reveals the importance of evaluating osmoregulatory processes in endeavors to comprehend the invasion of dilute media by ancestral marine crustaceans.

The macrophage-derived neutrophil chemotactic factor, MNCF: A lectin with TNF-alpha-like activities on neutrophils

The macro phage-derived neutrophil chemotactic factor (MNCF) is an alpha-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-alpha the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-alpha effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain. (C) 2008 Elsevier Inc. All rights reserved.