Biomedical subjects

C R Carvalho

Publications and source records attributed to C R Carvalho.

At least 37 records · Page 2Linked to original sources

PubMed1999

Growth hormone stimulates the tyrosine kinase activity of JAK2 and induces tyrosine phosphorylation of insulin receptor substrates and Shc in rat tissues.

GH stimulates the tyrosine phosphorylation of various cellular polypeptides, including the GH receptor itself, in an early part of the intracellular response. Some of these phosphorylations are catalyzed by a GH receptor-associated kinase identified as JAK2, a member of the Janus family of tyrosine kinases. In cultured cells, GH stimulates the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc. This study investigated whether GH could cause the tyrosine phosphorylation of IRSs and Shc proteins in fasted rat tissues in vivo. GH was administered to fasted Wistar rats via a portal vein, and extracts of different tissues were immunoprecipitated with specific antibodies. GH increased the tyrosine phosphorylation of IRS-1, IRS-2, JAK2, and Shc proteins in the liver, heart, kidney, muscle, and adipose tissue of rats. The roles of these substrates as signaling molecules for GH were further demonstrated by the finding that GH stimulated the association of IRS-1/2 with phosphatidylinositol 3-kinase, Grb2, and phosphotyrosine phosphatase and of Shc with Grb2. The correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation in response to GH together with the results of the in vitro tyrosine kinase assay are consistent with the hypothesis that JAK2 may mediate GH-induced phosphorylation of IRS-1.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome.

BACKGROUND: In patients with the acute respiratory distress syndrome, massive alveolar collapse and cyclic lung reopening and overdistention during mechanical ventilation may perpetuate alveolar injury. We determined whether a ventilatory strategy designed to minimize such lung injuries could reduce not only pulmonary complications but also mortality at 28 days in patients with the acute respiratory distress syndrome. METHODS: We randomly assigned 53 patients with early acute respiratory distress syndrome (including 28 described previously), all of whom were receiving identical hemodynamic and general support, to conventional or protective mechanical ventilation. Conventional ventilation was based on the strategy of maintaining the lowest positive end-expiratory pressure (PEEP) for acceptable oxygenation, with a tidal volume of 12 ml per kilogram of body weight and normal arterial carbon dioxide levels (35 to 38 mm Hg). Protective ventilation involved end-expiratory pressures above the lower inflection point on the static pressure-volume curve, a tidal volume of less than 6 ml per kilogram, driving pressures of less than 20 cm of water above the PEEP value, permissive hypercapnia, and preferential use of pressure-limited ventilatory modes. RESULTS: After 28 days, 11 of 29 patients (38 percent) in the protective-ventilation group had died, as compared with 17 of 24 (71 percent) in the conventional-ventilation group (P<0.001). The rates of weaning from mechanical ventilation were 66 percent in the protective-ventilation group and 29 percent in the conventional-ventilation group (P=0.005): the rates of clinical barotrauma were 7 percent and 42 percent, respectively (P=0.02), despite the use of higher PEEP and mean airway pressures in the protective-ventilation group. The difference in survival to hospital discharge was not significant; 13 of 29 patients (45 percent) in the protective-ventilation group died in the hospital, as compared with 17 of 24 in the conventional-ventilation group (71 percent, P=0.37). CONCLUSIONS: As compared with conventional ventilation, the protective strategy was associated with improved survival at 28 days, a higher rate of weaning from mechanical ventilation, and a lower rate of barotrauma in patients with the acute respiratory distress syndrome. Protective ventilation was not associated with a higher rate of survival to hospital discharge.

Adult↗

PubMed1998

Regulation of insulin-stimulated tyrosine phosphorylation of Shc and IRS-1 in the muscle of rats: effect of growth hormone and epinephrine.

Insulin receptor substrate-1 (IRS-1) and Shc protein have the same binding site at the insulin receptor and compete in their association with the phosphorylated receptor. The present study demonstrates that a decrease in the level of muscle insulin receptor phosphorylation induced by chronic growth hormone (GH) treatment or acute epinephrine infusion is accompanied by a reduction in the level of IRS-1 phosphorylation and in the association with phosphatidylinositol 3-kinase. In contrast, no change is observed in insulin-stimulated Shc tyrosine phosphorylation, or in the association of this substrate with Grb2. These data suggest that a reduction in insulin receptor phosphorylation may affect post-receptor processes differentially by preserving the phosphorylation of some substrates and pathways, but not of others.

Amino Acid Sequence↗

PubMed1998

Increased tyrosine phosphorylation of band 3 in hemoglobinopathies.

In order to investigate the tyrosine phosphorylation of band 3, we performed immunoblotting of intact red cells using anti-phosphotyrosine antibody of 21 patients with sickle cell disorders (11 SS, 5 Sbeta, 5 SC), 7 patients with beta thalassemias (5 beta thal intermedia, 2 deltabeta thal), 10 normal controls, and 1 patient with hereditary spherocytosis. They had not received transfusion for the last 4 months and all were clinically stable. Our results showed an increased tyrosine phosphorylation of two proteins, in the 100 and 80 kD regions, in sickle cell and beta-thalassemic red cells when compared to the normal controls and to the patient with hereditary spherocytosis. Immunoprecipitation of the lysed red cells with anti-band 3 antibody and immunoblotting with anti-phosphotyrosine antibody confirmed that the 100 kD tyrosine phosphorylated protein was band 3. In the sickle cell disease group, the band 3 tyrosine phosphorylation varied from 2- to 10-fold increase compared to control (x +/- SD; SS = 7.8- +/- 2.7-fold; SC = 3.8- +/- 1.3-fold; Sbeta = 5.2- +/- 2.0-fold). It was also higher in the beta-thalassemic group (beta-thal = 4.3- +/- 3.7-fold). There was no significant difference in tyrosine phosphorylation among the various groups tested, except when we compared the phosphorylation in intact red cells of patients with sickle cell anemia and hemoglobinopathy SC (U = 6, P < 0.02). The tyrosine phosphorylation of band 3 was increased in hemoglobinopathies even in the absence of high reticulocyte count. At least two mechanisms might be involved in the increased tyrosine phosphorylation of band 3 in these hemoglobin disorders, probably related to the endogenous reactive oxygen intermediates generated by the abnormal erythrocyte: an inhibition of protein tyrosine phosphatase activity or an activation of the protein tyrosine kinase p72syk.

Adolescent↗

PubMed1998

Insulin signalling in heart involves insulin receptor substrates-1 and -2, activation of phosphatidylinositol 3-kinase and the JAK 2-growth related pathway.

OBJECTIVE: Hyperinsulinemia is a common feature of obesity and hypertension and may be associated with abnormal metabolism and growth of heart muscle and vascular wall. Most of the known actions of insulin were characterised in muscle, adipose tissue and liver. In this study we investigate the initial steps of insulin signalling in rat heart. METHODS: After insulin infusion in the cava vein of male Wistar rats, the insulin receptor, insulin receptor substrates-1 and -2, phosphatidylinositol 3-kinase activity and Janus kinase (JAK) 2 engagement were studied by immunoprecipitation and immunoblot of heart extracts. RESULTS: An insulin load induces rapid autophosphorylation of the insulin receptor which is followed by the phosphorylation of insulin receptor substrates-1 and -2. The phosphorylation of these early intracellular substrates leads to the association of the p85 subunit of phosphatidylinositol 3-kinase and subsequent activation of its catalytic p110 subunit. Besides activation of the lipid metabolising enzyme phosphatidylinositol 3-kinase, the phosphorylation of insulin receptor substrates-1 and -2 engages the intracellular kinase JAK 2 and induces JAK 2-STAT 1 complex formation. CONCLUSIONS: We demonstrate that the early steps of insulin signalling in heart include the phosphorylation-activation of the insulin receptor, engagement of insulin receptor substrates-1 and -2 with the consequent activation of phosphatidylinositol 3-kinase and the involvement of the recently discovered growth related pathway--JAK 2-STAT 1.

Animals↗

PubMed1998

Angiotensin-converting enzyme inhibitor increases insulin-induced pp185 phosphorylation in liver and muscle of obese rats.

The clinical use of angiotensin-converting enzyme (ACE) inhibitors has been associated with increased insulin sensitivity. However, the molecular mechanism is unknown. The authors examined the early steps in insulin action, i.e., the phosphorylation status of the insulin receptor and of the pp185 in liver and muscle of obese rats treated acutely with captopril, using immunoblotting with antiphosphotyrosine antibodies. Following treatment with captopril there was an improvement in insulin-induced insulin receptor and pp185 phosphorylation in the liver and muscle of obese rats. This finding contribute to an explanation of the mechanism by which ACE inhibitors appear to improve insulin sensitivity.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Hyperoxia and lung disease.

Experimental studies and few human reports demonstrate that hyperoxia increases the level of reactive oxygen-derived free radicals and that these substances can produce oxidative cellular injury. However, available data suggest that the human lung is more resistant to hyperoxic oxidative damage than previously expected and demonstrate that absorption atelectasis is the most frequently pulmonary effect of inhalation of a high inspired oxygen fraction. Practically, the therapeutic use of high inspired oxygen fractions is limited to patients with acute lung injury and severe hypoxemia. Recent studies demonstrated that ventilator-induced lung injury is a more important cause of pulmonary damage in these patients than hyperoxic toxicity. New protective ventilatory strategies are associated with increased survival.

Absorption↗

PubMed1998

Pinealectomy causes glucose intolerance and decreases adipose cell responsiveness to insulin in rats.

Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.

Adipose Tissue↗

PubMed1998

Thyroxine 5'-deiodination mediates norepinephrine-induced lipogenesis in dispersed brown adipocytes.

In euthyroid rats, maximal sympathetic nervous system stimulation (e.g. during cold exposure) results in a 3- to 4-fold increase in brown adipose tissue lipogenesis, a response that is blunted in hypothyroid rats. To further investigate this phenomenon, the role of local type II 5'-deiodinase (5'-DII) was studied in freshly isolated brown adipocytes. In a typical experiment, 1.5 x 10(6) cells were incubated for up to 48 h in a water-saturated 5% CO2-95% O2 atmosphere. After incubation with medium alone or with different concentrations of T4, T3, and/or norepinephrine (NE), lipogenesis was studied by measuring 1) the rate of fatty acid synthesis as reflected by 3H2O incorporation into lipids and 2) the activity of key rate-limiting enzymes, i.e. acetyl coenzyme A carboxylase and malic enzyme, and the results are reported in terms of DNA content per tube. Lipogenesis decreased progressively over time (approximately 40%) when no additions were made to the incubation medium. T4 or T3 partially prevented that inhibition at physiological concentrations (65 x 10[-9] and 0.77 x 10[-9] M, respectively), whereas a receptor-saturating concentration of T3, (154 x 10[-9] M) doubled the lipogenesis rate. The addition of 10(-6) M NE inhibited lipogenesis acutely (approximately 50% by 12 h) and was followed by a progressive stimulation that reached approximately 2-fold by 48 h, but only in the presence of T4. Furthermore, NE did not attenuate T3 (154 x 10[-9] M)-induced lipogenesis. Both the inhibition and the stimulation of lipogenesis caused by NE showed a strong dose-response relationship within the range of 10(-11)-10(-5) M. The role of local 5'-DII was further tested by incubating brown adipocytes with 10(-6) M NE and T4 (65 x 10[-9] M) in the presence of 100 microM iopanoic acid, a potent inhibitor of 5'-DII. Although iopanoic acid did not affect the T3 stimulation of lipogenesis, it did block the approximately 2-fold stimulation of lipogenesis triggered by NE in the presence of T4, confirming the mediation of 5'-DII in this process. In conclusion, lipogenesis in brown adipose tissue is under complex hormonal control, with key roles played by NE, thyroid hormones, and local 5'-DII. As in other tissues, NE-generated signals acutely (12 h) inhibited lipogenesis. However, the presence of the 5'-DII generated enough T3 to stimulate lipogenesis and gradually reverse the short-lived NE-induced inhibition, leading to the 2- to 3-fold response observed at later time points.

Adipocytes↗

PubMed1998

Insulin induces tyrosine phosphorylation of Shc and stimulates Shc/GRB2 association in insulin-sensitive tissues of the intact rat.

Shc is a novel type of tyrosine-phosphorylated protein activated in response to a wide variety of polypeptide ligands. In this study, we used immunoprecipitation and immunoblotting to examine the effect of insulin on Shc tyrosine phosphorylation and Shc/GRB2 association in insulin-sensitive tissues of the intact rat. Following an infusion of insulin, Shc was tyrosine-phosphorylated in the liver, skeletal muscle, and adipose tissue in a time- and dose-dependent fashion, which peaked 5 min after exposure to the hormone and, except in the case of adipose tissue, returned to basal values after 15 min. There was coimmunoprecipitation of Shc and the insulin receptor after stimulation with insulin. Receptor tyrosine kinase activity toward Shc was also observed. Following an infusion of insulin, Shc was found to associate with GRB2. These results demonstrate that after stimulation of rat tissues with insulin, Shc binds to the insulin receptor, is tyrosine-phosphorylated, and subsequently associated with GRB2.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The insulin receptor substrate 1 associates with phosphotyrosine phosphatase SHPTP2 in liver and muscle of rats.

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.

Animals↗

PubMed1998

Insulin receptor has tyrosine kinase activity toward Shc in rat liver.

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Effect of chronic growth hormone treatment on insulin signal transduction in rat tissues.

Growth hormone (GH) is known to produce insulin resistance, but the exact molecular mechanism remains unclear. We have chronically treated rats with GH and observed that the levels of insulin receptor in the liver or muscle were similar in both the GH-treated and non-treated rats. Insulin-stimulated receptor autophosphorylation was unaltered in the liver, but was reduced in the muscle of rats treated with GH. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol (PI) 3-kinase protein levels decreased in the liver but not muscle of GH-treated rats. There was no change in hepatic and muscle IRS-2 concentrations. A common finding in liver and muscle was the decrease in IRS-1 and IRS-2 tyrosine phosphorylation associated with a reduction in the interaction between these substrates and PI 3-kinase. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in rats exposed to an excess of GH.

Animals↗

PubMed1997

Defects in insulin signal transduction in liver and muscle of pregnant rats.

Pregnancy is known to induce resistance, but the exact molecular mechanism involved is unknown. In the present study, we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1 (IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of pregnant rats (day 20 of gestation) by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and anti-phosphotyrosine antibodies. There were no changes in the insulin receptor concentration in the liver and muscle of pregnant rats. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody, was reduced by 30 +/- 6% (p < 0.02) in muscle and 36 +/- 5% (p < 0.01) in liver at day 20 of gestation. IRS-1 protein levels decreased by 45 +/- 6% (p < 0.002) in liver and by 56 +/- 9% (p < 0.002) in muscle of pregnant rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle and liver of pregnant rats decreased by 70 +/- 9% (p < 0.01) and 75 +/- 8% (p < 0.01), respectively. The insulin-stimulated IRS-1 association with PI 3-kinase decreased by 81 +/- 6% in muscle (p < 0.01) and 79 +/- 11% (p < 0.01) in the liver during pregnancy. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in pregnancy.

Animals↗

PubMed1997

Immaturity, ageing and oral tolerance.

Founding studies of cellular immunology emphasized that tolerance to allografts could only be achieved early in the embryonic or neonatal period, suggesting that the establishment of self-tolerance, a main event in the organization of the immune system, would necessarily take place in immature hosts. Contradicting these ideas, oral tolerance is a common, daily phenomenon, easily achieved by a physiological route in adult immunocompetent animals. Furthermore, there is solid evidence that, after the neonatal period, the susceptibility to oral tolerance induction also wanes and that it may be restored by adoptive transfer of cells from young hosts. These findings are briefly reviewed here to emphasize that immunological activity is a continuous and ongoing epigenesis extending throughout the entire life of the organism, far beyond the early phases of ontogenesis.

Aging↗

PubMed1997

Indirect effects of oral tolerance cannot be ascribed to bystander suppression.

The addition of tolerated antigens to immunizing doses of unrelated antigens blocks antibody responses to these unrelated antigens. This inhibition, which the authors have called the indirect effects of tolerated antigens, occurs even when the mixture of proteins is injected as soon as 24 h after the oral tolerance induction. The indirect effects also do not require the simultaneous injection of the two proteins: they are still present 72 h after an injection of Ova in Ova tolerant mice, but do not occur if the unrelated protein is injected 24 h before the tolerated protein. In addition, indirect effects do not block secondary responses to unrelated proteins if the primary immunization is made in the absence of the tolerated protein. These results cannot be explained by innocent bystander suppression, which is believed to result from the action of suppressive cytokines released by specific tolerant lymphocytes upon unrelated lymphocytes that would otherwise respond to the second, non-tolerated antigen. Indirect effects may be better understood in terms of network models.

Administration, Oral↗

PubMed1997

Pulmonary schistosomiasis.

Schistosomiasis is a very important infectious disease, and pulmonary involvement is not very rare. There may be two forms of pulmonary involvement, acute and chronic. The acute form usually occurs about 6 weeks after the infection (Katayama syndrome) and seems to be due to an allergic manifestation to the presence of Schistosoma spp. worm or eggs. The chronic form is more commonly seen in endemic areas and may cause pulmonary hypertension and cor pulmonale, pulmonary granulomatous schistosomiasis, and pulmonary arteriovenous fistulas. Recurrence of pulmonary infiltrations may appear after treatment. The globalization of the world with international travel makes it necessary for clinicians around the world to be aware of some "old" diseases from endemic areas of the globe.

Acute Disease↗

PubMed1997

Temporal hemodynamic effects of permissive hypercapnia associated with ideal PEEP in ARDS.

The associated use of permissive hypercapnia (PHY) and high PEEP levels (PEEP(IDEAL)) has been recently indicated as part of a lung-protective-approach (LPA) in acute respiratory distress syndrome (ARDS). However, the net hemodynamic effect produced by this association is not known. We analyzed the temporal hemodynamic effects of this combined strategy in 48 patients (mean age 34 +/- 13 yr) with ARDS, focusing on its immediate (after 1 h), early (first 36 h), and late (2nd-7th d) consequences. Twenty-five patients were submitted to LPA--with the combined use of permissive hypercapnia (PHY), VT < 6 ml/kg, distending pressures above PEEP < 20 cm H2O, and PEEP 2 cm H2O above the lower inflection point on the static inspiratory P-V curve (P(FLEX))- and 23 control patients were submitted to conventional mechanical ventilation. LPA was initiated at once, resulting in an immediate increase in heart rate (p = 0.0002), cardiac output (p = 0.0002), oxygen delivery (DO2l, p = 0.0003), and mixed venous Po2 (p = 0.0006), with a maintained systemic oxygen consumption (p = 0.52). The mean pulmonary arterial pressure markedly increased (mean increment 8.8 mm Hg; p < 0.0001), but the pulmonary vascular resistance did not change (p = 0.32). Cardiac filling pressures increased (p < 0.001) and the systemic vascular resistance fell (p = 0.003). All these alterations were progressively attenuated in the course of the first 36 h, despite persisting hypercapnia. Plasma lactate suffered a progressive decrement along the early period in LPA but not in control patients (p < 0.0001). No hemodynamic consequences of LPA were noticed in the late period and renal function was preserved. A multivariate analysis suggested that these acute hyperdynamic effects were related to respiratory acidosis, with no depressant effects ascribed to high PEEP levels. In contrast, high plateau pressures were associated with cardiovascular depression. Thus, as long as sufficiently low distending pressures are concomitantly applied, the sudden installation of PHY plus PEEP(IDEAL) induces a transitory hyperdynamic state and pulmonary hypertension without harmful consequences to this young ARDS population.

Adult↗