Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na+ channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.

Effects of simvastatin on bone regeneration in the mandibles of ovariectomized rats and on blood cholesterol levels.

The purpose of this study was to evaluate the effects of simvastatin on guided bone regeneration in the mandibles of ovariectomized rats, and to observe their blood cholesterol levels. Seventy female rats were divided into two groups: control and treated, both groups containing normal and ovariectomized rats. A month after ovariectomy a bone defect was created in the mandible, and was covered by a polytetrafluoroethylene membrane. The treated groups received simvastatin orally for 15 or 30 days. The rats were sacrificed 15, 30 or 60 days after surgery, at which time a blood sample was extracted for blood cholesterol level analysis and the mandible was extracted for densitometric, histological and morphometric analysis. All specimens underwent analysis of variance. The ovariectomized animals had higher cholesterol levels than the treated normal animals, and no significant difference was found between the different treatment periods and the sacrifice times. The densitometric, histological and morphometric analysis showed that the treated ovariectomized animals developed more new bone than the control ovariectomized rats, but no significant difference was observed between the treatment periods. It can be concluded that the deficiency of estrogen increased the level of blood cholesterol and that the simvastatin aided new bone formation in the ovariectomized animals.

Histoenzymology and morphometry of the masticatory muscles of tufted capuchin monkey (Cebus apella Linnaeus, 1758)

Samples of the anterior and posterior regions of the masseter and temporal muscles and of the anterior belly of the digastric muscle of 4 adult male tufted capuchin monkeys (Cebus apella) were removed and stained with HE and submitted to the m-ATPase reaction (with alkaline and acid preincubation) and to the NADH-TR and SDH reactions. The results of the histoenzymologic reactions were similar, except for acid reversal which did not occur in fibers of the fast glycolytic (FG) type in the mandibular locomotor muscles. FG fibers had a larger area and were more frequent in all regions studied. No significant differences in frequency or area of each fiber type were detected, considering the anterior and posterior regions of the masseter and temporal muscles. The frequency of fibers of the fast oxidative glycolytic (FOG) and slow oxidative (SO) types and of FOG area differed significantly between the anterior belly of the digastric muscle and the mandibular locomotor muscle. The predominance of fast twitch (FG and FOG) fibers and the multipenniform and bipenniform internal architecture of the masseter and temporal muscles, respectively, are characteristics that permit the powerful bite typical of tufted capuchin monkeys.

Evaluation of a protein deficient diet in rats through blood oxidative stress biomarkers

Protein malnutrition leads to functional impairment in several organs, which is not fully restored with nutritional recovery. Little is known about the role of oxidative stress in the genesis of these alterations. This study was designed to assess the sensitivity of blood oxidative stress biomarkers to a dietary protein restriction. Male Wistar rats were divided into two groups, according to the diet fed from weaning (21 days) to 60 day old: normal protein (17% protein) and low protein (6% protein). Serum protein, albumin, free fatty acid and liver glycogen and lipids were evaluated to assess the nutritional status. Blood glutathione reductase (GR) and catalase (CAT) activities, plasma total sulfhydryl groups concentration (TSG) as well as plasma thiobarbituric acid reactive substances (TBARs) and reactive carbonyl derivatives (RCD) were measured as biomarkers of the antioxidant system and oxidative damage, respectively. The glucose metabolism in soleus muscle was also evaluated as an index of stress severity imposed to muscular mass by protein malnutrition. No difference was observed in muscle glucose metabolism or plasma RCD concentration between both groups. However, our results showed that the low protein group had higher plasma TBARs (62%) concentration and lower TSG (44%) concentration than control group, indicating increased reactive oxygen species production in low protein group. The enhancement of erythrocyte GR (29%) and CAT (28%) activities in this group also suggest an adaptation to the stress generated by the protein deficiency. Taken together, the results presented here show that the biomarkers used were able to reflect the oxidative stress level induced by this specific protein deficient diet.

Morphometric and quantitative evaluation of the NADH-diaphorase positive myenteric neurons of the jejunum of streptozotocin-diabetic rats supplemented with acetyl-L-carnitine

In this study we investigated the effect of the acetyl-L-carnitine (ALC) supplementation on the myenteric neurons of the jejunum of rats made diabetic at the age of 105 days by streptozotocin (35 mg/kg body weight). Four groups were used: non-diabetic (C), non-diabetic supplemented with ALC (CC), diabetic (D), diabetic supplemented with ALC (DC). After 15 weeks of diabetes induction the blood was collected by cardiac puncture to evaluate glycaemia and glycated haemoglobin. Next the animals were killed and the jejunum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The neuronal counts were made in 80 microscopic fields, in tissue samples of five animals of each group. The profiles of the cell bodies of 1000 neurons per group were analysed. Diabetes induced a significant increase in the area of the cell body and decrease in the number of NADH-diaphorase positive myoenteric neurons. ALC suplementation to the diabetic group promoted smaller hypertrophic effects and less neuronal loss than in the myoenteric neurons of the diabetic rats, and in addition diminished the body weight decrease and reduced the fasting glycaemia. © 2005 Blackwell Verlag.

Eficácia e segurança da atorvastatina no tratamento de pacientes com hipercolesterolemia primária

Introduction: Hypercholesterolemia is an important risk factor for cardiovascular disease, the first cause of death and third reason for hospital admissions in Brazil. The reduction of serum cholesterol levels reduces morbidity and mortality from cardiovascular disease. The present study evaluated the efficacy and safety of atorvastatin in the treatment of Brazilian patients with primary hypercholesterolemia (types IIA and IIB dyslipidemias). Patients and methods: After a 4-week wash-out period, 152 patients were treated with atorvastatin at the initial dose of 10 mg/day. According to treatment efficacy within the first 8 weeks this dose could be increased to 20 mg/day. Treatment lasted for a total of 16 weeks, and its efficacy was evaluated by the reduction of serum levels of LDL-cholesterol, total cholesterol, HDL-cholesterol, and triglycerides, as well as by the propotion of patients that achieved the target levels recommended by the National Cholesterol Education Program - Adult Treatment Panel II (NCEP ATP II) Results: The analysis of efficacy was conducted in 145 patients. Atorvastatin led to significant reductions in the levels of LDL-cholesterol after 8 and 16 weeks of treatment (P<0.001 for both comparisons). The relative reduction of such levels was 38% (P<0.001 after 8 and 16 weeks). Atorvastatin also led to significant reductions of total cholesterol and triglycerides. At the end of the study, 81% of patients achieved the target LDL-cholesterol levels recommended by NCEP ATP II. Treatment was well tolerated, and was interrupted due to creatine phosphokinase elevation in only one patient. Conclusion: Atorvastatina is efficacious and safe in the treatment of patients with primary hypercholesteromia. © Copyright Moreira Jr. Editora. Todos os direitos reservados.

Transmission analysis of candidate genes for nonsyndromic oral clefts in Brazilian parent-child triads with recurrence

Cleft lip and/or palate (CL/P) is a major congenital defect with complex etiology, including multiple genetic and environmental factors. Approximately two thirds of the cases are not accompanied by other anomalies and are called nonsyndromic (NS). In the present study, we performed transmission distortion analysis of the MSX1-CA, TGFB3-CA and MTHFR-C677T polymorphisms in 60 parent-child triads, in which the NS-CL/ P affected child had at least one affected parent. No association with genes MSX1 or TGFB3 was found, but the results were suggestive of an association of the MTHFR-C677T polymorphism with NS-CL/P. © 2006 Sociedade Brasileira de Genética.

Morphological and histochemical analysis of the human vestibular fold

A morphological and histochemical study of the human vestibular fold was carried out using routine histological techniques. Seven μm-thick histological sections stained with hematoxylin-eosin (HE) and Calleja showed the presence of elastic collagen fibers and seromucous glands in the vestibular fold. Muscle fibers forming the ventricular muscle were also identified. Ultrastructural analyses of the epithelial layer by scanning electron microscopy (SEM) revealed ciliated cells and gland ducts opening on the epithelial surface. Histochemical analyses were performed on ventricular muscles submitted to nicotinamide-adenine-dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), and myofibrillar adenosine triphosphatase (mATPase) reactions. Based on these reactions, it was observed that the muscle is formed by three types of muscle fibers: slow-twitch oxidative (SO), fast-twitch oxydative glycolytic (FOG) and fast-twitch glycolytic (FG) fibers distributed in a mosaic pattern. The fiber frequency was 22.7%, 69.9% and 7.4%, respectively. The higher frequency of SO and FOG fibers characterized the muscle as having aerobic metabolism and resistance to fatigue. The ventricular muscle was considered fast. The study of the neuromuscular junctions performed after nonspecific esterase reaction showed that they are of the en-plaque type and have multiple occurrences in the ventricular muscle.

The influence of ovariectomy, simvastatin and sodium alendronate on alveolar bone in rats

Bisphosphonates are currently used in the treatment of many diseases involving increased bone resorption such as osteoporosis. Statins have been widely used for the treatment of hypercholesterolemia and recent studies have shown that these drugs are also capable of stimulating bone formation. The purpose of this study was to evaluate thel influence of an estrogen deficient state and the effects of simvastatin and sodium alendronate therapies on alveolar bone in female rats. Fifty-four rats were either ovariectomized (OVX) or sham operated. A month later, the animals began to receive a daily dose of simvastatin (SIN - 25 mg/kg), sodium alendronate (ALN - 2 mg/kg) or water (control) orally. Thirty-five days after the beginning of the treatment, the rats were sacrificed and their left hemimandibles were removed and radiographed using digital X-ray equipment. The alveolar radiographic density under the first molar was determined with gray-level scaling and the values were submitted to analysis of variance (α = 5%). Ovariectomized rats gained more weight (mean ± standard deviation: 20.06 ± 6.68%) than did the sham operated animals (12.13 ± 5.63%). Alveolar radiographic density values, expressed as gray levels, were lowest in the OVX-water group (183.49 ± 6.47), and differed significantly from those observed for the groups receiving alendronate (sham-ALN: 193.85 ± 3.81; OVX-ALN: 196.06 ± 5.11) and from those of the sham-water group (193.66 ± 4.36). Other comparisons between groups did not show significant differences. It was concluded that the ovariectomy reduced alveolar bone density and that alendronate was efficient for the treatment of this condition.

Treatment of atherosclerotic renovascular disease

Treatment of atherosclerotic renovascular disease is controversial and revascularization is not a beneficial approach to all patients. Conditions as progressive deterioration of renal function, refractory hypertension or accelerated cardiovascular disease, especially recurrent pulmonary edema, could profit from renal angioplasty with stent placement. Surgical revascularization is a good option for patients who will need concomitant surgical corrections of abdominal aortic lesions. Treatment of all other patients must be individualized. Medical therapy is indicated for all patients with atherosclerotic renovascular disease. Observational studies pointed out to the beneficial effect of controlling blood pressure (<130/80 mm Hg), glucose and lipids profile, lifestyle modifications, specific use of platelet antiaggregant therapy, Angiotensin Conversion Enzyme Inhibitors (ACEI) and statins. All others cardiovascular risk factors must be controlled. The evaluation and management of other systemic atherosclerotic vascular lesions is important, especially coronary, carotid and abdominal aortic. This paper presents a review of evidences to rationale the atherosclerotic renovascular disease treatment. © 2008 Bentham Science Publishers Ltd.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Polimorfismo nos genes metilenotetrahidrofolato redutase e cistationina-beta-sintase e a sua relação com eventos vaso-oclusivos na doença falciforme

Sickle cell disease is an inflammatory condition with a pathophysiology that involves vaso-occlusive episodes. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS) genes are risk factors for vascular disease. Due to the importance of identifying risk factors for vaso-occlusive events in sickle cell patients, we investigated the frequencies of the C677T and 844ins68 mutations of the MTHFR and CBS genes, respectively. Three hundred patients with Hb SS, HB SC and HbS/Beta thalassemia, from Brasília, Goiânia, Rio de Janeiro, São Jose do Rio Preto and São Paulo were evaluated. Samples of 5 mL of venous blood were collected in EDTA after informed consent was received from patients. Classical diagnostic methods were used to confirm the hemoglobin phenotypes. The hemoglobin genotypes and polymorphisms studied were evaluated by Restriction Fragment Length Polymorphism and Allele Specific amplification. The results showed that 93 patients (31.00%) were heterozygous and 13 (4.33%) homozygous for the C677T mutation and 90 were heterozygotes (30.00%) and 8 homozygous (2.66%) for the 844ins68 mutation, both with significant differences for genotype frequency between the localities. The allelic frequencies are in Hardy-Weinberg equilibrium for both polymorphisms. The frequency of mutations was significant and the presence of related vaso-occlusive events was more common in patients with Hb SS (p = 0007). The 844ins68 mutation was approximately three times more frequent in patients with vaso-occlusive complications (p = 0011). The C677T mutation did not prove to be associated with risk of vaso-occlusive events (p = 0.193). A C677T-844ins68 interaction occurred in 12.08% of the patients, doubling the risk of vaso-occlusive manifestations. The frequencies of the polymorphisms are consistent with those expected in the Brazilian population. The presence of the 844ins68 mutation of the CBS gene proved to be a potential risk factor for vaso-occlusive events in sickle cell patients.

Participation of genes involved in the process of anaerobic respiration of infection in chickens by salmonella typhimurium

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells are priority for the invasion of the pathogen. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. Vitamin B12 (cobalamin) is synthesized by Salmonella Typhimurium only under anaerobic growth conditions used as a cofactor in four known reactions. The deletion of cobS and cbiA genes prevent any form of cobalamin production. In the present study we evaluate the infection of birds by mutants of STM, with the anaerobic respiratory system committed by mutations in the genes: narG, napA, cobS, cbiA, frdA, dmsA, and torC. Virulence was assessed by oral inoculation of groups of one-day-old broilers with 0.1 mL of culture contained 10 8 colony forming units (CFU)/mL or diluted at 10 -3 and 10 -2 of strains mutants of Salmonella Typhimurium. Clinical signs and mortality were recorded over a period of 21 days. In general, the symptoms of chickens infected with the mutant strains were similar to those presenting by control birds. Except for STMNalr cbiA, all showed reduced capacity to cause mortality in comparison with the original strain. The mortality of group of chickens infected with STMNal r △narG, STMNal r △frdA, STMNal r △dmsA and STMNal r △cobS△cbiA showed significant decrease in mortality compared to control group (p<0.05).

Antioxidant enzyme activity and hydrogen peroxide content during the drying of Arabica coffee beans

The development of the germination process and drought stress during the drying of coffee can generate reactive oxygen species, which can be neutralized by way of antioxidant mechanisms. No studies related to antioxidant enzymes during the drying of coffee were found in the literature, and considering their importance, the enzymatic activities of superoxide dismutase (SOD), guaiacol peroxidase (GPOX) and glutathione reductase (GR), and also the hydrogen peroxide content were evaluated during the drying of two types of coffee bean, one processed as natural coffee and the other as pulped natural coffee. The results showed a reduction in the SOD, GPOX and GR enzymatic activities of the natural coffee as compared to the pulped natural coffee during the drying period. Moreover, the hydrogen peroxide content of the natural coffee was greater than that of the pulped natural coffee. These results suggest the development of oxidative stress during the coffee drying process, controlled more efficiently in pulped natural coffee by the early action of GPOX during the drying process. Nevertheless, differential responses by SOD isoenzymes and possibly the role of other peroxidases also appear to be involved in the responses observed. © 2013 Springer-Verlag Berlin Heidelberg.

Biochemical biomarkers in Scinax fuscovarius tadpoles exposed to a commercial formulation of the pesticide fipronil

One of the main pesticides used in the cultivation of sugarcane in São Paulo State, Brazil, is Regent®800WG, the main active compound of which is fipronil. Fipronil is a potent insecticide that eliminates pests, including insects resistant to pyrethroids, organophosphates (OP) and carbamates (CA). There is little known on the toxic effects of fipronil on non-target organisms, such as tadpoles of frogs. It is possible that this compound carries a high toxicity for these organisms, since the pesticide can be incorporated into aquatic environments during the rainy season, a time which coincides with the time of amphibian reproduction and the occurrence of tadpoles in the aquatic environment in this region. Thus, the pesticide could be contributing to the decline of amphibians in the northwest region of São Paulo state due to its wide use. This study aimed to test the influence of Regent®800WG on some biochemical systems of tadpoles (such as antioxidant defense systems) at different stages of development. The results of analysis from in vivo exposures demonstrated that only a few parameters in the groups exposed to fipronil responded to exposure to Regent®800WG, results which indicate that the pesticide instigates biochemical responses in tadpoles. Although catalase and glucose-6-phosphate dehydrogenase (G6PDH) were unchanged during the experiments, glutathione-S-transferase (GST) was inhibited in tadpoles, and the activity of glutathione reductase (GR) varied according to the exposure period and pesticide concentration. This data demonstrated the influence of the fipronil formulation on the metabolism of tadpoles, and showed that it can increase their susceptibility to environmental contaminants. © 2013 Elsevier Ltd. All rights reserved.