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M C Lopes

Publications and source records attributed to M C Lopes.

At least 37 records · Page 2Linked to original sources
PubMed1999

Involvement of JAK2 and MAPK on type II nitric oxide synthase expression in skin-derived dendritic cells.

In this report, we demonstrate that a fetal mouse skin-derived dendritic cell line produces nitric oxide (NO) in response to the endotoxin [lipopolysaccharide (LPS)] and to cytokines [tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Expression of the inducible isoform of NO synthase (iNOS) was confirmed by immunofluorescence with an antibody against iNOS. The tyrosine kinase inhibitor genistein decreased LPS- and GM-CSF-induced nitrite (NO(-2)) production. The effect of LPS and cytokines on NO(-2) production was inhibited by the Janus kinase 2 (JAK2) inhibitor tyrphostin B42. The p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB-203580 also reduced the NO(-2) production evoked by LPS, TNF-alpha, or GM-CSF, but it was not as effective as tyrphostin B42. Inhibition of MAPK kinase with PD-098059 also slightly reduced the effect of TNF-alpha or GM-CSF on NO(-2) production. Immunocytochemistry studies revealed that the transcription factor nuclear factor-kappaB was translocated from the cytoplasm into the nuclei of fetal skin-derived dendritic cells (FSDC) stimulated with LPS, and this translocation was inhibited by tyrphostin B42. Our results show that JAK2 plays a major role in the induction of iNOS in FSDC.

Animals↗
PubMed1998

Calcium-dependent nitric oxide synthase activity in rat thymocytes.

We examined the conversion of L-[3H]arginine to L-[3H]citrulline in lysate from rat thymocytes, which was dependent on Ca2+ and cofactors (FAD, BH4, NADPH). Removal of Ca2+ of the medium, reduced the total L-[3H]citrulline formation by about 97%. The L-[3H]citrulline formation was completely inhibited by the NO synthase inhibitors, NG-nitro-L-arginine and NG-monomethyl-L-arginine, with values for IC50 of 1.2 microM and 19.4 microM, respectively. In intact thymocytes, the L-[3H]citrulline formation was dependent on the intracellular Ca2+ ([Ca2+]i) concentration. Increasing the extracellular free-Ca2+ concentration up to 1.5 mM, was accompanied by an increase in [Ca2+]i inside the thymocytes and there was a parallel increase in the intracellular L-[3H]citrulline formation, which reached a maximal value of 371.2 nM of [Ca2+]i. Addition of NG-nitro-L-arginine to the medium, completely inhibited the formation of L-[3H]citrulline. The immunolabeling study revealed that 15% of the thymocytes isolated from rat thymus constitutively expressed the endothelial isoform of NO synthase.

Animals↗
PubMed1998

Acute effects of 3,4-dichloroaniline on blood of male Wistar rats.

In this work we investigated the acute effects of 3,4-dichloroaniline (DCA) on the changes of specific biochemical and cellular blood parameters using male Wistar rats, 24 hours after the intraperitoneal administration of five different doses of DCA (0, 81, 162, 324, 486, and 568 mg/Kg of body weight). We also evaluated the dose-dependent toxicity of DCA on the ratio "organ weight:total body weight" for kidney, liver, spleen and thymus, and on the changes of morphological characteristics of the spleen. The LOEL (lowest observed effect level) value for spleen and thymus injury was 324 mg DCA/Kg. The cellular blood parameters affected by DCA include: methemoglobin concentration, the number of circulating leukocytes and the number and size of platelets. The relative sensitivity of all the analysis used to assess the acute toxicity of DCA was evaluated by comparing their ED50 values, which ranged from 224 to 837 mg DCA/Kg. We concluded that the most sensitive endpoint to measure the acute toxicity of DCA was the methemoglobin formation. Platelet counts, urea and creatinine showed ED50 values slightly higher than methemoglobin (329-367 mg/kg).

Aniline Compounds↗
PubMed1998

Correlation between whole blood cholinesterase activity and cerebral cortex cholinesterase activity in rats treated with parathion.

Organophosphate and carbamate insecticides are inhibitors of cholinesterases (ChE). The depression of blood ChE activity is frequently used as indicative of exposure to these chemicals. However, it is not known whether the inhibition of blood ChE activity reflects the inhibition of ChE in target tissues (e.g. brain and muscle). In this study we investigated the possibility of using whole blood ChE activity to predict frontal cerebral cortex ChE activity in rats treated with parathion. Twenty four hours after the intraperitoneal administration of several doses of parathion, the activity of ChE in whole blood and the activity of ChE in frontal cerebral cortex were determined in each animal. A high correlation between the two parameters was found (r = 0.96, p < 0.05) and the model of linear regression fitted to the data accounted for 93% of its variability. Thus, these results seem to indicate that 24 hours after the treatment with parathion the effects induced on whole blood ChE activity may be used to predict the effects caused on frontal cerebral cortex ChE activity.

Animals↗
PubMed1996

Changes in the subcellular distribution of the rat uterus oestrogen receptor as induced by oestradiol, tamoxifen and ZD 182,780.

The aim of this work was to compare the subcellular distribution of the oestrogen receptor from the uteri of rats treated with vehicle alone (control group), oestradiol or one of the anti-oestrogenic drugs tamoxifen and ZD 182,780. The nuclear, microsomal and cytosolic oestrogen receptor contents were evaluated by an immunoenzymatic method ("ER-EIA" kit from Abbott Laboratories) and the results in each fraction were expressed as a percentage of the total number of receptors. Parallel studies were performed to assess the uterotrophic effect of these drugs and to assess that they had reached the uterus. In the control group, we found that the oestrogen receptor was distributed mainly between the microsomal (29.1 +/- 1.3%) and cytosolic (68.1 +/- 0.9%) fractions, with only a small amount located in the nucleus (2.8 +/- 0.5%). When oestradiol was administered, the oestrogen receptor distribution was: nuclear 11.7 +/- 2.0, microsomal 15.5 +/- 1.3 and cytosolic 72.8 +/- 3.3% and, in the tamoxifen group, the results were: nuclear 18.5 +/- 1.5, microsomal 26.0 +/- 3.1 and cytosolic 55.5 +/- 3.4%, which shows a relative shift both to the control and the oestradiol-treated groups. In the uteri of rats treated with ZD 182,780 the results were very similar to those obtained in the control group. Our results indicate that the subcellular distribution of the oestrogen receptor varies according to the drug administered and that this receptor may not be located in a single subcellular compartment. Moreover, the nuclear uptake of the ZD 182,780-oestrogen receptor complex seems to be blocked, possibly due to impaired receptor dimerization. In the case of tamoxifen, the intracellular transport of the receptor also seems to be blocked, probably due to the nuclear retention of the receptor, thus suggesting that tamoxifen must impair the oestrogen receptor function on a step subsequent to the receptor dimerization.

Animals↗
PubMed1994

Amino acids differentially inhibit the L-[3H]arginine transport and nitric oxide synthase in rat brain synaptosomes.

The nitric oxide synthase (NOS) present in the cytosol obtained from rat cerebral cortex synaptosomes was inhibited by NG-nitro-L-arginine (L-NOArg) with an IC50 value of approximately 0.06 microM. This compound did not affect the transport of L-arginine (L-Arg) into synaptosomes at concentrations up to 100 microM but other potential inhibitors of NOS (NG-monomethyl-L-arginine, NG-amino-L-arginine and L-arginine methyl ester) inhibited L-Arg transport at a concentration < 5 microM. We showed that concentrations of L-NOArg (0.001-3 microM) that did not block the uptake of tritiated arginine (L-[3H]Arg) inhibited the catalytic activity of NOS in intact synaptosomes. L-NOArg at a concentration of 1 microM inhibited the cytosolic enzyme by 98.0 +/- 2.0% of the total NOS activity whereas the enzyme studied in the intact synaptosomes was only inhibited by 75 +/- 5% which suggested that the NOS in synaptosomes is not fully accessible to the external L-NOArg. On the other hand, L-Lysine did not inhibit the cytosolic NOS activity of ruptured synaptosomes but at a concentration that blocked 50.0 +/- 4.5% of L-[3H]Arg uptake it inhibited the NOS activity in intact synaptosomes by 12.6 +/- 3.6%, suggesting that the transport of L-Arg may be an important regulatory step in the pathway for nitric oxide generation.

Amino Acid Oxidoreductases↗
PubMed1994

Calcium channel blockers inhibit the (Ca2+ + Mg2+)-ATPase activity and the 125I-calmodulin binding in brain membranes.

Ca2+ channel blockers belonging to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes (Santos et al., J. Neurochem. 52, S49D, 1989). We now report that (-)-desmethoxyverapamil and flunarizine are the most potent inhibitors of the Ca(2+)-activated ATPase activity of synaptic plasma membranes, decreasing the Vmax by 41% and 37%, respectively, with no significant effects on the Km for Ca2+ (162.7 +/- 14.9 nM free [Ca2+]), while nitrendipine did not affect these parameters. Trifluoperazine was the most potent inhibitor of the Ca(2+)-activated ATPase of synaptic plasma membranes with an IC50 of 8-10 microM. To clarify whether the inhibitory effects of Ca2+ channel blockers and of trifluoperazine on the (Ca2+ + Mg2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125I-calmodulin to the membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer to nitrocellulose and autoradiography. The autoradiograms revealed Ca(2+)-dependent CaM binding proteins of about 140, 70 and 55 kDa. Trifluoperazine (30-40 microM) inhibited by 50-60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca2+ + Mg2+)-ATPase protein. Flunarizine and (-)-desmethoxyverapamil (100 microM) inhibited the 125I-calmodulin binding to the 140 kDa peptides by 100 and 90%, respectively, and they inhibited by 55 and 40%, respectively, the binding of 125I-calmodulin to the peptides in the 70-55 kDa range, whereas nitrendipine did not show any effect. The results suggest that the inhibitory effects of (-)-desmethoxyverapamil and flunarizine, as well as trifluoperazine, on the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.

Animals↗
PubMed1993

Is dilution of cows' milk formula necessary for dietary management of acute diarrhoea in infants aged less than 6 months?

There is concern that feeding full-strength animal milk to infants aged less than 6 months with diarrhoea may have adverse consequences. We assessed the effects on clinical course of two feeding regimens in 159 Guatemalan and Brazilian infants aged 2 weeks to 6 months who had had acute diarrhoea for 120 h or less, showed signs of mild to moderate dehydration, and had no complications. After correction of dehydration, infants were assigned randomly to receive continued full-strength milk feeding or initial feeding with diluted milk with regrading to full-strength milk over 48 h. There were no significant differences between feeding groups in rate of treatment failures (-1%, 95% Cl -14 to 12%) or mean (SD) total stool output (full-strength milk 335 [268] g/kg, diluted milk 338 [354] g/kg) and duration of diarrhoea (92 [50] vs 92 [44] h). A significant association was found between presence of reducing substances in stools and treatment failure (OR 4.3, 95% CI 1.1 to 16.8), but reducing substances in stools were common both in treatment successes (61%) and in failures (87%). Our study supports the conclusion that, for infants under 6 months of age with diarrhoea whose only food is animal milk or formula, the milk or formula normally given should be provided in full strength as soon as dehydration has been corrected.

Academies and Institutes↗
PubMed1991

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors.

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.

Binding, Competitive↗
PubMed1990

Action of antiestrogens on the (Ca2+ + Mg2+)-ATPase and Na+/Ca2+ exchange of brain cortex membranes.

The effect of tamoxifen (TAM) and other antiestrogens on the Ca2+ transport activity of synaptic plasma membranes (SPM) and microsomal membranes isolated from sheep brain cortex was investigated. The maximal (Ca2+ + Mg2+)-ATPase activity of SPM, which is reached at a pCa of about 6.0-6.5, is decreased by about 30% in the presence of 50 microM TAM, whereas the (Ca2+ + Mg2+)-ATPase activity of microsomes, which is maximal at a pCa of about 5.0, is decreased by about 90% by 50 microM TAM. In parallel experiments, we observed that the ATP-dependent Ca2+ uptake is also affected differently by TAM in the two membrane preparations. We found that 50 microM TAM inhibits SPM Ca2+ uptake by about 25-30%, whereas the ATP-dependent Ca2+ uptake by the microsomal fraction is inhibited by about 60%. No significant effect of TAM was observed on the Na+/Ca2+ exchange of either membrane system. The results indicate that TAM is a more potent inhibitor of the active, calmodulin-independent Ca2+ transport system of the intracellular membranes than of that of the plasma membranes, which is calmodulin-dependent. It appears that TAM inhibits calmodulin-mediated reactions, probably through its binding to calmodulin, as we showed previously. However, the Ca2+ transport system of microsomes, which does not depend on calmodulin, is also particularly sensitive to TAM.

Animals↗
PubMed1990

Ca2(+)-dependent binding of tamoxifen to calmodulin isolated from bovine brain.

The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [3H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca2(+)-dependent manner and that two components are involved in the binding, with apparent dissociation constants (Kd) of about 6 nM and 9 microM. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a Bmax value of 120 nmol/mg protein. The stimulatory effect of Ca2+ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg2+. In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity (nH = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca2(+) + Mg2+)-ATPase. The results indicate that Tx may alter Ca2(+)-dependent processes by interacting directly with CaM.

Animals↗
PubMed1983

Beta + thalassemia--Portuguese type: clinical, haematological and molecular studies of a newly defined form of beta thalassaemia.

We have characterized 14 patients in 10 families with a mild form of homozygous beta thalassemia which has not been previously well defined. As these patients originate from a small area of northern Portugal we propose to call this beta + thalassaemia--Portuguese type. Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and they are characterized by low levels of HbF, less than 20%, indicating only a mild deficit in beta globin production. Heterozygotes are indistinguishable from those with the more common types of beta thalassaemia as regards red cell morphology, haemoglobin analysis and globin chain synthesis studies. Globin gene mapping excluded the presence of alpha thalassaemia in these patients and demonstrated no abnormalities in the beta-like globin gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are identical on both chromosomes in all of the homozygotes, confirming their homogeneity.

Adolescent↗
PubMed1975

Detection of Bhanja virus in cell cultures by fluorescent antibody technique.

The multiplication of Bhanja virus in CV-1 cells was studied both by staining the viral antigens with the FA technique and measuring the light intensity emitted by the fluorescent cells with a photomultiplier and by the simultaneous titration of intracellular and extracellular virus. The fluorescence appeared at 3 hrs post adsorption in the form of very small granules in the cytoplasm of the infected cells. Percentage of cells containing viral antigens reached its maximum at 18 hrs post adsorption. Later on the fluorescence slowly decreased. The virus in infected cells was demonstrated 9 hrs post adsorption and maximum titre was reached 48 hrs post adsorption. The cell sheet remained apparently normal and no sign of cytopathic effect was observed until 36 hrs post adsorption.

Animals↗