Müller cells and diabetic retinopathy.

Müller cells provide nutrition for neural cells. We studied the structure and ultrastructure of Müller cells in the retina of thirty 3-month old Wistar rats, divided equally into 3 groups: normal rats, alloxan diabetic rats and treated alloxan diabetic rats, 1 and 12 months after induction of diabetes. We observed that the Müller cell nuclei under light microscope examination had hexagonal shape and higher density than the other nuclei. Differences between groups could be observed only by electron microscopy. In the diabetic rats, Müller cells presented dispersion of nuclear chromatin and electrondense nuclear granulations, with the presence of increased glycogen, dense bodies and lysosomes in the cytoplasm. The alterations were more frequent in the perivascular region and at 12 months. The treated diabetic rats exhibited some alterations we observed in diabetic rats, but these alterations were less intense. We conclude that, despite the treatment, the diabetic retinopathy continues to evolve.

Histochemical study of muscle fiber types in Synbranchus marmoratus Bloch, 1795

The myotomal muscle of Synbranchus marmoratus was investigated using histochemical and immunohistochemical reactions. This musculature is composed of a superficial red compartment, uniformly distributed around the trunk circumferentially and also in the lateral line. The red compartment fibers are small in diameter and have an oxidative metabolism, a high rate of glycogen and a negative reaction to alkaline and acid myofibrillar ATPase (mATPase). The white muscle forms the bulk of the muscle mass. Its fibers are large in diameter and have a glycolytic metabolism, a negative reaction to glycogen, a strong reaction to alkaline mATPase and a negative reaction to acid mATPase. Between these two compartments there is an intermediate layer of fibers presenting a mosaic metabolism pattern with a high rate of glycogen. These fibers stained moderately for alkaline and acid m-ATPase. Several clusters of red muscles were observed inside the white muscle. Each cluster is composed of three fiber types, with a predominance of red and intermediate fibers. Reactivity to anti-MHC BA-D5 was positive only in the intermediate fibers. Reactivity to anti-MHC SC-71 was negative in all fiber types.

Ultrastructural and morphometric aspects of ageing in the retinal capillaries of rats

Age-related morphological, ultrastructural and morphometric changes in the capillaries of the superficial and deep plexuses of the rat retina were studied in animals aged from 3 to 15 months. Our results suggest that age-related morphological alterations start occurring in the retina of rats at about 12 months of age. Increased glycogen deposits, pinocytotic vesicles, residual bodies and cell debris were observed in both the endothelial and pericytic cells of 12- and 15-month-old animals. In addition, heterogeneous osmiophilic accumulations, electron-transparent spaces were observed in the basement membrane as well as projections of the basement membrane towards the neighboring cells. Morphometric examination of the two vascular plexuses studied did not show differences in the area of the endothelial or pericytic cells, basement membrane or vascular lumen between rats of different ages.

Effects of Chronic Physical Activity and of Ultrasound Treatment on Bone Consolidation

The objective of this study was to investigate the effects of ultrasound treatment and physical exercise on the velocity of bone consolidation and resistance to deformation. We performed osteotomy in the upper third of the right tibia of rats. Physical training consisted of swimming 1 h per day with a load of 5% b.w. for 30 days. Therapy with medium-intensity ultrasound was applied daily on the damaged area. Wistar rats were divided into the following groups: osteotomized sedentary animals with no ultrasound treatment (1.OSnUS), osteotomized trained animals with no ultrasound treatment (2.OTnUS), osteotomized sedentary animals with ultrasound treatment (3.OSwUS). and osteotomized trained animals with ultrasound treatment (4.OTwUS). The animals were sacrificed for the following analyses: muscle glycogen, serum alkaline phosphatase at the 5th, 10th, 20th, and 30th days, test of maximum resistance to flexion, rupture flexion and mean tibial rigidity at the 30th day. Muscle glycogen was increased at the 20th day: alkaline phosphatase was elevated at the 5th and 20th days in groups 3.OSwUS and 4.OTwUS. and decreased at the 10th day. Groups1.OSnUS and 2.OTnUS did not show significant variations. In the mechanical resistance tests, we noted that ultrasound therapy and the association of physical activity used in the present study showed significant differences in bone resistance and bone rigidity after 30 days of treatment. These facts suggest that ultrasound or physical activity, or their combination may accelerate the process of bone tissue repair.

Regression of the lateral oviducts during the larval-adult transformation of the reproductive system of Melipona quadrifasciata and Frieseomelitta varia

Toward the end of the larval phase (pre-pupa), the reproductive systems of Melipona quadrifasciata and Frieseomelitta varia workers are anatomically similar. Scanning electron microscopy showed that during this developmental phase the right and left ovaries are fused and form a heart-shaped structure located above the midgut. Each ovary is connected to the genital chamber by a long and slender lateral oviduct. During pupal development, the lateral oviducts of workers from both species become extremely reduced due to a drastic process of cell death, as shown by transmission electron microscopy. During the lateral oviduct shortening, their simple columnar epithelial cells show some signs of apoptosis in addition to necrosis. Cell death was characterized by cytoplasmic vesiculation, peculiar accumulation of glycogen, and dilation of cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum. The nuclei, at first irregularly contoured, became swollen, with chromatin flocculation and various areas of condensed chromatin next to the nuclear envelope. At the end of the pupal phase, deep recesses marked the nuclei. At emergence, worker and queen reproductive systems showed marked differences, although reduction in the lateral oviducts was an event occurring in both castes. However, in queens the ovarioles increased in length and the spermatheca was larger than that of workers. At the external anatomical level, the reproductive system of workers and queens could be distinguished in the white- and pink-eyed pupal phase. The metamorphic function of the death of lateral oviduct cells, with consequent oviduct shortening, is discussed in terms of the anatomical reorganization of the reproductive system and of the ventrolateral positioning of adult worker bee ovaries. (C) 2000 Wiley-Liss, Inc.

Endocytic activity in the thrombocytes of the turtle Phrynopys hilarii (freshwater South American species)

The phagocytic process in cells depends on lysosomal enzymes, high-energy metabolism and cellular recognition. In this paper, we investigated the presence of energy and recognition factors in thrombocytes of turtle Phrynopys hilarii (a freshwater South American species). Turtle thrombocytes (P. hilarii) present glycogen - possibly β particles - dispersed in their cytoplasm and glycoproteins in the cell surface, as well as a large number of enzymes involved in the endocytic process (Pellizzon, 1996). The activity of these enzymes depends on high-energy metabolism and on cellular recognition provided by specific glycoconjugates (Alberts et al., 1994). This metabolic characterization is demonstrated by the large amount of glycogen particles observed in the cytoplasm by Thiéry's method. Glycogen labeling was also observed when concanavalin A-peroxidase was used as a marker for thrombocytes and for endocyted charcoal particles. Our results show that these cells have phagocytic ability, suggesting that their function in blood circulation is not limited to aggregation but may also involve a great potential for phagocytosis.

Toxic mechanism of cadmium exposure on cardiac tissue

The presence of toxic substances in the workplace environment requires systematic evaluation of exposure and health status in exposed subjects. Cadmium is a highly toxic element found in water. Although free mediated cellular damage and reactive oxygen species (ROS), had been theorized as contributing to the cadmium mechanism of toxicity, and recent investigations have established that free radicals may be important contributors to cardiac dysfunction, there is little information on the effect of cadmium exposure on markers of oxidative stress in cardiac tissue. Cadmium exposure (Cd2+ - 100 mg/1-from CdCl2) in drinking water, during 15 days, significantly increased lipoperoxide and decreased the activities of superoxide dismutase and glutathione peroxidase. No alterations were observed in catalase activity in heart of rats with cadmium exposure. We also observed decreased glycogen and glucose concentration and increased total lipid content in cardiac tissue of rats with cadmium exposure. The decreased activities of alanine transaminase and aspartate transaminase reflected decreased metabolic protein degradation, and increased lactate dehydrogenase activity was related with increases in capacity of glycolysis. Since the metabolic pathways were altered by cadmium exposure, we can conclude that Cd2+ exposure induced ROS and initiate some series of events that occur in the heart and resulted in metabolic pathways alterations.

Hepatic gluconeogenesis in rats trained to eat a single meal daily. Role of eating periodicity and the amount of food ingested in the last meal

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The adverse effect of a high energy dense diet on cardiac tissue

Purpose: To determine whether a high energy dense diet intake increases oxidative stress and alters antioxidant enzymes in cardiac tissue. Design: A randomized, controlled study. Ninety-day-old female rats were randomly divided into two groups: one fed with a low energy dense diet (LE; 3.0 kcal g-1) and one with a high energy dense diet (HE; 4.5 kcal g-1). Materials and Methods: After 8 weeks of treatment, the animals were fasted overnight and sacrificed by decapitation. The serum was used for glucose, triacylglycerol, cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol determinations. The glycogen, lipoperoxide, lipid hydroperoxide, superoxide dismutase, glutathione peroxidase, lactate dehydrogenase, citrate synthase, total and non-protein sulphhydryl groups were determined in cardiac tissue. Results: HE decreased the myocardial glycogen content and increased the lactate dehydrogenase/citrate synthase ratio, indicating an increased glycolytic pathway and a shift from myocardial aerobic metabolism. HE-treated female rats showed increased lipoperoxide and hydroperoxide levels in cardiac tissue. Although no alterations were observed in the total sulphhydryl group and superoxide dismutase activities, glutathione peroxidase and the non-protein sulphhydryl group were significantly decreased in HE-treated animals. Conclusions: Although no alterations were observed in energy intake, HE induced an increased intake of fat and carbohydrate and an increased rate of weight gain. HE intake induced alterations in markers of oxidative stress in cardiac tissue. Hydrogen peroxide is an important toxic intermediate in the development of cardiac oxidative stress by HE. The specific nutrient content, such as fat and carbohydrate, rather than caloric intake, appears to be the main process inducing oxidative stress in HE-treated female rats.

The monosodium glutamate (MSG) obese rat as a model for the study of exercise in obesity

Obesity is an increasing problem in several countries, leading to health problems. Physical exercise, in turn, can be used effectively by itself or in combination with dietary restriction to trigger weight loss. The present study was designed to evaluate the effects of aerobic exercise training on lipid profile of obese male Wistar rats in order to verify if this model may be of value for the study of exercise in obesity. Obesity was induced by MSG administration (4mg/g, each other day, from birth to 14 days old) After 14 from drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (exercise trained). Exercise training consisted in 1h/day, 5 days/week, with an overload of 5% bw, for 10 weeks. Rats of the same age and strain, receiving saline at birth, were used as control (C), and subdivided into two groups: C-S and C-T. At the end of the experimental period, MSG-T and C-T rats showed similar blood lactate and muscle glycogen responses to exercise training and acute exercise. MSG-S rats showed significantly higher carcass fat, serum triacylglycerol, serum insulin and liver total fat than C-S rats. On the other hand, MSG-T rats had lower carcass fat, serum triacylglycerol and liver total fat than MSG-S rats. There were no statistical differences in food intake and serum free fatty acids among the groups studied. These data indicate that this model may be of value for the study of exercise effects on tissue and circulating lipid profile in obesity.

Dietary restriction: Metabolic shifting for cardiac health

Purpose: To determine the effect of dietary restriction on metabolic pathways and the relationship of the metabolic shifting on antioxidant enzymes in cardiac tissue. Design: Randomized, controlled study. Male rats at 60 days old were randomly divided into four groups. Materials and Methods: The rats of control groups C30 and C60 were given free access to the diet over 30 and 60 days. The rats of the DR30 group were fed 60% of the chow consumed by the control groups over 30 days. The animals of the DR60 group ate 60% of the amount consumed by the C60 group over 60 days. Serum was used for total protein, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Protein, glycogen, total lipids, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), LDH, AST and ALT were determined in cardiac tissue. Results: Dietary restriction induced diminished serum and cardiac LDH activities. AST activities were lower in the serum and cardiac muscle of the DR60 animals. Dietary restriction induced elevated total lipid concentrations in cardiac muscle. No significant differences were observed in total protein and glycogen content among the groups. Antioxidant enzyme determinations demonstrated increased cardiac GSH-Px activities in the DR60 animals and increased SOD activities in the cardiac tissue of both feed-restricted groups. Conclusions: Dietary restriction was protective against oxidative stress in the heart by improving cardiac endogenous antioxidant defences and shifting the metabolic pathway for energy production.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aro A-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg-1. The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory. © 2002 Elsevier Science (USA). All rights reserved.

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.

Dietary restriction and fibre supplementation: Oxidative stress and metabolic shifting for cardiac health

Dietary modification ought to be the first line of strategy in prevention of the development of cardiac disease. The purpose of this study was to investigate whether dietary restriction, dietary-fibre-enriched diet, and their interactions might affect antioxidant capacity and oxidative stress in cardiac tissue. Male Wistar rats (180-200 g; n = 10) were divided into four groups: control ad libitum diet (C), 50% restricted diet (DR), fed with fibre-enriched diet (F), and 50% restricted fibre-enriched diet (DR-F). After 35 days of the treatments, F, DR, and DR-F rats showed low cholesterol, LDL-cholesterol, and triacylglycerol, and high HDL-cholesterol in serum. The DR, DR-F, and F groups had decreased myocardial lipoperoxide and lipid hydroperoxide. The DR-F and F treatments increased superoxide dismutase and glutatione peroxidase (GSH-Px). The DR treatment increased GSH-Px and catalase activities. Dietary fibre beneficial effects were related to metabolic alterations. The F and DR-F groups showed high cardiac glycogen and low lactate dehydrogenase/citrate synthase ratios, indicating diminished anaerobic and elevated aerobic myocardial metabolism in these animals. There was no synergistic effect between dietary restriction and dietary fibre addition, since no differences were observed in markers of oxidative stress in the F and DR-F groups. Dietary fibre supplementation, rather than energy intake and dietary restriction, appears to be the main process retarding oxidative stress in cardiac tissue.

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.