50 resultados para Glycogen Staining

em Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)


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Kayotypes of four neotropical teiid lizard species (Tupinambinae) were herein studied after conventional as well as silver staining and CBG-banding: Crocodilurus amazonicus (2n = 34), Tupinambis teguixin (2n = 36), Tupinambis merianae and Tupinambis quadrilineatus (2n = 38). The karyological data for T. quadrilineatus as well as those obtained using differential staining for all species were unknown until now. The karyotypes of all species presented 12 macrochromosomes identical in morphology, but differed in the number of microchromosomes: 22 in C. amazonicus, 24 in T. teguixin and 26 in T. quadrilineatus and T. merianae. The Ag-NOR located at the secondary constriction at the distal end of pair 2 is shared by all species, contrasting with the variability observed for this character in species of the related Teiinae. CBG-banding revealed a species-specific pattern in T. quadrilineatus with conspicuous interstitial C-blocks at the proximal region of the long arm of pair 4 and the whole heterochromatic short arm of pair 6. The karyological data reported here corroborates the relationship hypothesis obtained for Tupinambis based on molecular characters. T. teguixin presents the putative ancestral karyotype for the genus with 2n = 36 whereas T. merianae and T. quadrilineatus exhibit 2n = 38, due to an additional pair of microchromosomes.

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The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

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IL-1 beta, TNF-alpha, cytokine-induced neutrophil chemoattractant-2 alpha/beta, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

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Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal Muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright (C) 2009 John Wiley & Sons, Ltd.

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Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

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Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

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Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

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P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

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Objective. This study evaluated histopathologically the response of pulp and periradicular tissues after pulp capping with an all-in-one self-etching adhesive system in dogs` teeth. Study design. Forty teeth of 4 dogs were assigned to 3 groups according to the pulp capping material: G1 (n = 20): self-etching adhesive system; G2 (n = 10): Ca(OH)(2); G3 (n = 10): zinc oxide-eugenol. The animals were killed 7 and 70 days after pulp capping. The pieces containing the pulp-capped teeth were removed and processed for histologic analysis. Results. At 7 days, no dentin bridge formation was observed; G1 and G3 exhibited inflammatory pulpal alterations, whereas G2 presented only mild inflammatory infiltrate in the pulp tissue adjacent to the capping material, the remainder being intact. At 70 days, no specimen in G1 or G3 presented dentin bridge formation. The remaining pulp tissue exhibited severe inflammatory alterations and areas of necrosis. In G2, all specimens showed dentin bridge formation and absence of inflammation and mineralized tissue resorption. No bacteria were identified using Brown and Brenn staining techniques in all 3 groups at any observation period. Conclusion. According to the conditions of this study, direct pulp capping with the self-etching adhesive system did not allow pulp tissue repair and failed histopathologically in 100% of the cases. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e34-e40)

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Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

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This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation. Reproduction (2011) 142 593-603

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This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

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Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or locomotor activity. Finally, we found exceptionally high levels of carbohydrates and lipids in the liver of S. rizibilis, a trait perhaps related to a long and demanding breeding period.

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In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

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Supernumerary marker chromosomes (sSMC) may or may not be associated with an abnormal phenotype, depending on the presence of euchromatin, on their chromosomal origin and whether they are inherited. Over 80% of sSMCs are derived from acrocentric chromosomes and half of them include the short arm of chromosome 15. Generally, they appear as bisatellited isodicentric marker chromosomes, most of them are symmetric. These chromosomes are normally originated de novo and are associated with mild to severe intellectual disability but not with physical abnormalities. We report on a patient with an SMC studied using classical and molecular cytogenetic procedures (G and C banding, NOR staining, painting and centromeric fluorescent in situ hybridization (FISH), BAC-FISH, and SKY). The MLPA technique and DNA polymorphic markers were used in order to identify its parental origin. The marker chromosome, monosatellited and monocentric, was found to be derived from a maternal chromosome 15 and was defined as 15pter-q21.2. This is the report of the largest de novo monosatellited 15q marker chromosome ever published presenting detailed cytogenetic and clinical data. It was associated with a phenotype including cardiac defect, absence of septum pellucidum, and dysplasia of the corpus callosum. (C) 2010 Wiley-Liss, Inc.