Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1 beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray diffraction analysis of suramin, a highly charged polysulfonated napthylurea, complexed with a myotoxic PLA(2) from Bothrops asper venom

Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA(2) from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 degreesC. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a=49.05, b=63.84 and c=85.67 Angstrom, Diffraction data was collected to 1.78 Angstrom. (C) 2004 Elsevier B.V. All rights reserved.

A Simple Method Based on ICP-MS for Estimation of Background Levels of Arsenic, Cadmium, Copper, Manganese, Nickel, Lead, and Selenium in Blood of the Brazilian Population

Throughout the world, biomonitoring has become the standard for assessing exposure of individuals to toxic elements as well as for responding to serious environmental public health problems. However, extensive biomonitoring surveys require rapid and simple analytical methods. Thus, a simple and high-throughput method is proposed for the determination of arsenic (As), cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni), lead (Pb), and selenium (Se) in blood samples by using inductively coupled plasma-mass spectrometry (ICP-MS). Prior to analysis, 200 l of blood samples was mixed with 500 l of 10% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 10 min, and subsequently diluted to 10 ml with a solution containing 0.05% w/v ethylenediamine tetraacetic acid (EDTA) + 0.005% v/v Triton X-100. After that, samples were directly analyzed by ICP-MS (ELAN DRC II). Rhodium was selected as an internal standard with matrix-matching calibration. Method detection limits were 0.08, 0.04, 0.5, 0.09, 0.12, 0.04, and 0.1 g//L for As, Cd, Cu, Mn, Ni, Pb, and Se, respectively. Validation data are provided based on the analysis of blood samples from the trace elements inter-\comparison program operated by the Institut National de Sante Publique du Quebec, Canada. Additional validation was provided by the analysis of human blood samples by the proposed method and by using electrothermal atomic absorption spectrometry (ETAAS). The method was subsequently applied for the estimation of background metal blood values in the Brazilian population. In general, the mean concentrations of As, Cd, Cu, Mn, Ni, Pb, and Se in blood were 1.1, 0.4, 890, 9.6, 2.1, 65.4, and 89.3 g/L, respectively, and are in agreement with other global populations. Influences of age, gender, smoking habits, alcohol consumption, and geographical variation on the values were also considered. Smoking habits influenced the levels of Cd in blood. The levels of Cu, Mn, and Pb were significantly correlated with gender, whereas Cu and Pb were significantly correlated with age. There were also interesting differences in Mn and Se levels in the population living in the north of Brazil compared to the south.

Functional and structural analysis of two fibrinogen-activating enzymes isolated from the venoms of Crotalus durissus terrificus and Crotalus durissus collilineatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of chromatin and nuclear area alterations in triatomines during starvation

In the present study changes in nuclear area and chromatin distribution in the Malpighian tubules of a blood-feeding insect, the vector of Chagas' disease were analysed. Male and female adult insects were dissected, after a starving period of up to 28 days. The Malpighian tubules were fixed and stained by orcein lacto-acetic. The nuclei were analyzed by photomicrography, karyometry and a statistical analysis was performed. It was observed that with the increase of the starvation period there were alterations in the nuclear phenotype, that is, changes in the nuclear area and chromatin extenuation. In males, starting from the second week of starvation, chromatin extenuation was accentuated, and the beginning of nuclear coalition occurred more frequently in the third and fourth starvation weeks. In females, the accentuated chromatin extenuation was observed after the third starvation week, and irregularities in nuclear borders were more frequent. The descriptive statistical analysis of the male and temale nuclear areas revealed that the nuclear areas continued to increase during starvation reaching highest values in the third week. Another outstanding aspect is that in the third starvation week karyometry presented larger heterogeneity with higher standard deviation values for both genders. Therefore, nuclear alterations related to the distribution and extenuation chromatin and increase in nuclear area were observed. These aspects suggest the presence of nuclear metabolic mechanisms that lead to synthesis of specific proteins necessary for the protection and maintenance of the cell during physiologic stress.

Structural, cytochemical, immunocytochemical and ultrastructural characterization of blood granulocytes of the roadside hawk Buteo magnirostris (Gmelin, 1788) (Avian, Falconiform)

In the peripheral blood of the roadside hawk, Buteo magnirostris, the following types of granulocytic leucocytes were identified: heterophil, eosinophil and basophil. The heterophils presented acidophilic and spindle shaped granules, the eosinophils possess spherical eosinophilic granules and the basophils showed spherical and basophilic granules. The heterophils and eosinophils presented positive cytochemical reaction to glycogen and basic polyaminoacid, while the eosinophils presented sudanophilic granules, which were also positive for myeloperoxidase. The heterophils, alone, presented positivity for acid phosphatase in some granules and immunoreactivity to TGF-β1 was observed only in the cytoplasm of the eosinophils. Electron microscopy demonstrated the heterophil granules as predominantly spindle shaped, being strongly electron-dense, while the eosinophils had numerous uniformly electron-dense spherical granules and the basophils presented three different types of granules identified according to their electron-density and the aspect of their matrix.

Serum iron analysis of adults receiving three different iron compounds

Iron deficiency is one of the most prevalent nutritional problems in the world. Iron compounds are used for the prevention and treatment of this deficiency. A double-blind randomized crossover study with 12 volunteers was carried out to compare the effect of three iron compounds, ferrous glycine chelate (GLY), ferric EDTA (EDTA), and ferrous sulfate (FS) on serum iron, and to examine serial serum iron levels. The products were offered as tablets, and blood samples were drawn hourly for 4 hours. FS produced higher serum iron levels than EDTA or GLY (P <0.05). Data for GLY and EDTA were similar. The areas under the curve (AUC) for serum iron for the different compounds gave AUCFS > AUCGLY = AUCEDTA (P < 0.05). Iron from FS had a significantly greater bioavailability than for the other two compounds. © 2004 Elsevier Inc. All rights reserved.

The relationship between onset of blood lactate accumulation, critical velocity, and maximal lactate steady state in soccer players

The objective of this study was to analyze the validity of the velocity corresponding to the onset of blood lactate accumulation (OBLA) and critical velocity (CV) to determine the maximal lactate steady state (MLSS) in soccer players. Twelve male soccer players (21.5 ± 1.0 years) performed an incremental treadmill test for the determination of OBLA. The velocity corresponding to OBLA (3.5 mM of blood lactate) was determined through linear interpolation. The subjects returned to the laboratory on 7 occasions for the determination of MLSS and CV. The MLSS was determined from 5 treadmill runs of up to 30-minute duration and defined as the highest velocity at which blood lactate did not increase by more than 1 mM between minutes 10 and 30 of the constant velocity runs. The CV was determined by 2 maximal running efforts of 1,500 and 3,000 m performed on a 400-m running track. The CV was calculated as the slope of the linear regression of distance run versus time. Analysis of variance revealed no significant differences between OBLA (13.6 ± 1.4 km·h-1) and MLSS (13.1 ± 1.2 km·h-1) and between OBLA and CV (14.4 ± 1.1 km·h-1). The CV was significantly higher than the MLSS. There was a significant correlation between MLSS and OBLA (r = 0.80), MLSS and CV (r = 0.90), and OBLA and CV (r = 0.80). We can conclude that the OBLA can be utilized in soccer players to estimate the MLSS. In this group of athletes, however, CV does not represent a sustainable steady-state exercise intensity. © 2005 National Strength & Conditioning Association.

Analysis of DNA damage induced by aflatoxin B1 in Dunkin-Hartley guinea pigs

Aflatoxin B1 (AFB1) is among the most potent naturally occurring carcinogens and classified as a group I carcinogen. Since the ingestion of aflatoxin-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the effect of 2, 20, and 200 ppb of AFB1 on DNA damage in peripheral blood lymphocytes and liver cells in Dunkin-Hartley guinea pigs. The animals were divided into four groups according to the given diet. After the treatment the lymphocytes and liver cells were isolated and DNA damage determined by Comet assay. The levels of DNA damage in lymphocytes were higher animals treated with 200 ppb of AFB1-enriched diet (P = 0.02). In the liver cells there were a relationship between the levels of DNA damage and the consumption of AFB1 in all studied groups. These results suggest that Comet assay performed on lymphocytes is a valuable genotoxic marker for high levels of exposure to AFB1 in guinea pig. Additionally our results indicate that the exposure to this toxin increases significantly and increases the level of DNA damage in liver cells, which is a key step on liver cancer development. We also suggest that the Comet assay is an useful tool for monitoring the genotoxicity of AFB1 in liver. © 2007 Springer Science+Business Media B.V.

Responses of blood lactate concentration in aerobic and anaerobic training protocols at different swimming exercise intensities in rats

A swimming periodized experimental training model in rats in which different training protocols (TP) were classified in aerobic (A) and anaerobic (AN) intensity levels. The purpose of the present study was to verify if the classification of the TP used in the periodized training experimental model presented the blood lactate concentration [La] response adequate to the aerobic and anaerobic intensities levels. Twenty three male Wistar rats were divided into three groups. Two groups of swimming training (continuous, CT, n = 7, and periodized training, PET, n = 7) rats were evaluated during 5 weeks in eight different TP (TP-1 to TP-8) through the analysis of the [La] response. The third group was the sedentary control (SC, n = 9). The TP were classified in five intensity levels, three aerobic (A-1, A-2, A-3) and two anaerobic (AN-1, AN-2). Analysis of variance (ANOVA one-way, P<0.05) indicated significant differences in the [La] among the TP and among the five intensity levels. All TP of the A-2 and A-3 intensity levels differed from the A-1 and AN-1. The A-1 and AN-1 also differed among them. These findings demonstrate that the TP were classified properly at different levels of aerobic and anaerobic intensities, as based on the [La] response in a way similar to that of high performance swimming with humans. The results offer new perspectives for the study of exercise training in swimming rats at different levels intensity for performance or for health.

Effect of instrumentation using curettes, piezoelectric ultrasonic scaler and er,cr:Ysgg laser on the morphology and adhesion of blood components on root surfaces - A SEM study

This study used scanning electron microscopy (SEM) to evaluate the morphology and adhesion of blood components on root surfaces instrumented by curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser. One hundred samples from 25 teeth were divided into 5 groups: 1) Curettes; 2) Piezoelectric ultrasonic scaler; 3) Curettes plus piezoelectric ultrasonic scaler; 4) Er,Cr:YSGG laser; 5) Curettes plus Er,Cr:YSGG laser. Ten samples from each group were used for analysis of root morphology and the other 10 were used for analysis of adhesion of blood components on root surface. The results were analyzed statistically by the Kruskall-Wallis and Mann-Whitney tests with a significance level of 5%. The group treated with curettes showed smoother surfaces when compared to the groups were instrumented with piezoelectric ultrasonic scaler and the Er,Cr:YSGG laser. The surfaces instrumented with piezoelectric ultrasonic scaler and Er,Cr:YSGG laser, alone or in combination with hand scaling and root planing, did not differ significantly (p>0.05) among themselves. No statistically significant differences (p>0.05) among groups were found as to the adhesion of blood components on root surface. Ultrasonic instrumentation and Er,Cr:YSGG irradiation produced rougher root surfaces than the use of curettes, but there were no differences among treatments with respect to the adhesion of blood components.

Histochemical and ultrastructural analysis of hepatic glycogen and collagen fibers in alloxan-induced diabetic rats submitted to long-term physical training

Alterations in liver functions are common among diabetic patients, and many symptoms in the liver have been reported, including changes in glycogen stores and in the amount of collagen fibers. The practice of physical training and its morphological effects in this organ, however, are scarcely studied. In order to observe the morphological effects of alloxan-induced diabetes and the alterations arising from the practice of long-term chronic physical training in the liver, samples were collected and processed, and then analyzed by means of the histochemical techniques Periodic Acid-Schiff and Picrosirius-Hematoxylin, and ultrastructural cytochemical test of Afzelius. Through evaluation of the tissue, it was observed a drastic reduction in hepatic glycogen stores of sedentary diabetics, recovered in trained diabetic rats. Furthermore, it was detected a decrease in the content of perisinusoidal collagen fibers in the diabetic liver, also recovered due to the development of a training protocol. On ultrastructural level, cytochemical analysis confirmed the loss of glycogen and the recovery obtained by training. In conclusion, the practice of a long-term chronic physical training protocol may be considered an important assistant in the treatment of diabetes, mitigating the occurrence of possible damages to liver tissue. © 2011 Elsevier Ltd.

Leucometric analysis of 1-day-old chicks inoculated with Salmonella Typhimurium or Lactobacilli

Salmonella infection is responsible for major economic losses in poultry industry. Consequently, the development of new methods for fighting such disease is desirable, such as the use of acid-lactic bacteria. However, reference values of chicks in such conditions are dissimilar to those of other species. Leucometry reference values for chicks have not been reported. The aim of this article was to evaluate and determine the leucometric values of chicks inoculated with Salmonella Typhimurium or treated with Lactobacilli probiotics. In this study, 144 1-day-old birds were divided in three groups of 48 animals each (non-treated group, Salmonella Typhimurium (ST)-inoculated group, and Lactobacilli inoculated group). A total of four blood collections were made with the first one performed 3 h after inoculation with ST or treatment with Lactobacilli. Subsequent samples were obtained every 48 h for 7 days. Leucometric evaluation was performed immediately after each collection. All birds presented an initial decrease pattern in general leukocyte values, and the chicks inoculated with ST revealed lymphomonoheteropaenic leukopaenia, eosinophilia and basophilia in conjunction with convalescence after 96 h of inoculation. The animals inoculated with Lactobacilli revealed leucocytosis with monocytosis, lymphocytosis and marked eosinopaenia. We conclude that there is no efficient bone marrow response in 1-day-old chicks challenged with Salmonella Typhimurium; additionally, an immunostimulatory effect in 1-day-old chicks treated with Lactobacilli-modulated probiotics could be stated. © 2011 Springer-Verlag London Limited.