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Carlota Saldanha

Publications and source records attributed to Carlota Saldanha.

17 recordsLinked to original sources
PubMed2006

Fluorescence spectroscopy evaluation of fibrinogen-beta-estradiol binding.

Fluorescence spectroscopy experiments were performed in order to study conformational changes induced by the binding of beta-estradiol to fibrinogen at different ligand concentrations. The association constant (Ka) obtained for the fibrinogen-beta-estradiol binding was 6.47x10(6)M(-1), indicating a high affinity interaction. Fluorescence quenching experiments showed that approximately 30% of the tryptophan residues in the protein quaternary structure are accessible to ionic quenchers. The extent of quenching in the absence and presence of beta-estradiol was maximum for cesium ions and minimum for iodide, suggesting the presence of negatively charged residues in the vicinity of the tryptophan residues. The quenching parameters obtained at different beta-estradiol concentrations show alterations that confirm a conformational change, possibly due to a discrete reorganization of tryptophan residues during fibrinogen-beta-estradiol binding. This binding may be responsible for the effects of beta-estradiol on the decrease of erythrocyte aggregation and on cardiovascular risk reduction.

Databases, Protein↗
PubMed2006

Fibrinogen-beta-estradiol binding studied by fluorescence spectroscopy: denaturation and pH effects.

Fibrinogen is a blood plasma protein that plays a crucial role in hemostasis. It is known that erythrocyte aggregation increases in the presence of fibrinogen, and that beta-estradiol decreases erythrocyte aggregation with a constant fibrinogen concentration. In this work, we have used intrinsic tryptophan fluorescence to obtain information on the conformational changes of fibrinogen upon the recently proposed interaction with beta-estradiol. To evaluate the effect on the conformational changes during fibrinogen-beta-estradiol binding, fluorescence experiments were performed using guanidine hydrochloride (0-6 M) as denaturant, at different pH values. The results obtained for pH 6.5 and 8.0 showed no effect during the binding. The main differences were observed between pH 4.2 and 7.4, in the absence and in the presence of two different denaturant concentrations (1 and 5 M). A red shift of the fluorescence emission from 344 to 354 nm is observed when denaturant concentration is above 3 M for all studied pH values. This phenomenon may be explained by the loss of compact structure of the protein in the presence of denaturant, with tryptophan residues exposure to the aqueous environment and alteration of fibrinogen-beta-estradiol binding. These results demonstrate that the binding sites of fibrinogen are strongly dependent on the conformational state of the protein.

Binding Sites↗
PubMed2006

Changes in blood cell membrane properties in HIV type-1-infected patients.

To evaluate the possible HIV-1 infection-induced changes in cell membrane properties and in calcium signaling, membrane fluidity, acetylcholinesterase (AChE, a glycosylphosphatidylinositol-anchored protein) activity, and intracellular calcium concentration ([Ca2(+)](int)) were evaluated in lymphocytes and erythrocytes of infected individuals, previous to their engagement in antiretroviral therapy. Membrane fluidity was assessed by fluorescence spectroscopy measurements, using the fluorescence probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). AChE activity was determined by the colorimetric Ellman's method and [Ca2(+)](int) using the fluorescent fura-2 acetoxymethyl ester. When compared with the control group, lymphocytes of infected patients presented significantly decreased membrane fluidity, decreased AChE activity, and increased [Ca2(+)](int). Erythrocytes from HIV-infected patients presented decreased [Ca2(+)](int) when compared with the control group and decreased membrane fluidity near the lipid/water interface. Our data show that HIV-1 infection leads to biochemical and biophysical changes in the membrane itself and in membrane protein activity in lymphocytes (average of infected and noninfected subpopulations) and even in erythrocytes. The present observations are in agreement with a process of facilitated propagation of the infection to new cells, stimulation of virion production, and maintenance of a reservoir of erythrocyte-bound infectious virus.

Acetylcholinesterase↗
PubMed2005

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase.

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

Acetylation↗
PubMed2005

Long-term prognostic value of protein C activity, erythrocyte aggregation and membrane fluidity in transmural myocardial infarction.

The objective of this study was to evaluate the long-term predictive value of the haemostatic, inflammatory and haemorheologic disturbances in transmural myocardial infarction (MI). Sixty-four (59 male) consecutive survivors of a MI, with a mean age of 58.3 +/- 12.0 years, were followed over a period of 36 months. Eighteen patients had a cardiovascular event defined as the composite of death, non-fatal MI, unstable angina and stroke. The haemostatic (protein C activity-PtC, antithrombin III, plasminogen activator inhibitor-1), haemorheologic (blood fluidity and components, erythrocyte membrane fluidity) and inflammatory (polymorphonuclear elastase, leukocyte count) profiles were determined at hospital discharge, using standard methodology. Our results can be summarized as follow: (i) at hospital discharge, the subgroup of patients with events had higher leukoactivity, leukocyte count, membrane fluidity, prognosis cyte count (7833.0 +/- 1696.0 vs. 10294.0 +/- 3129.0; p = 0.011), lower PtC (100.65 +/- 19.08 vs.81.25 +/- 19.95; p = 0.002), and lower erythrocyte aggregation (14.26 +/- 5.94 vs. 11.47 +/- 3.45; p = 0.031) in relation to the ones without events; (ii) By Cox regression the protein C activity lower tertile (OR 0.169; 0.045-0.628; p = 0.008); erythrocyte membrane outer layer fluidity upper tertile (OR 0.067; 95% CI 0.011 - 0.393; p = 0.003); and erythrocyte aggregation lower tertile (OR 0.182; 0.038 - 0.876; p = 0.034) were independent predictors of the composite endpoint. We can conclude that some haemostatic, haemorheologic and inflammatory disturbances, at hospital discharge, are long-term independent predictors of recurrent cardiovascular events in transmural myocardial infarction survivors.

Adult↗
PubMed2005

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation.

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.

Cholesterol↗
PubMed2004

Amperometric measurements of nitric oxide in erythrocytes.

In the recent years, there has been an increase in the development of new biosensors that could be helpful in the study of various physiological processes. In this study, we report the development of a new in vitro experimental design for real-time nitric oxide (NO) amperometric measurements in erythrocyte suspensions. To achieve this, we employed human erythrocyte suspensions in sodium chloride 0.9%, pH 7 (haematocrit 0.05%). The production of NO by erythrocytes was measured with a commercial NO sensor during stimulation by L-arginine, acetylcholine, choline, atropine and velnacrine maleate (10 microM of final concentrations). We also measured the nitrite and nitrate concentrations produced by erythrocyte suspensions stimulated with the above effectors by means of the Griess reaction method. We observed that there was a direct relation between the electric current produced by the NO sensor, and the NO standard concentrations, thereby leading to a good calibration curve. The in vitro erythrocytes produced significant amperometric NO values in response to a wide range of effectors and these results have the same variation profile of the nitrites and nitrates results achieved with the Griess method. In conclusion, the amperometric NO sensor constitutes a reliable method for direct, and real-time measurement in vitro of the NO production of erythrocyte suspensions, As such, it offers a potential diagnostic technique for the evaluation of diseases, and the therapeutic progression of diseases, related to intracellular NO metabolism.

Adult↗
PubMed2003

Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects.

DMSO is an amphipathic molecule with a highly polar domain and two apolar methyl groups, making it soluble in both aqueous and organic media. It is one of the most common solvents for the in vivo administration of several water-insoluble substances. Despite being frequently used as a solvent in biological studies and as a vehicle for drug therapy, the side-effects of DMSO (undesirable for these purposes) are apparent from its utilization in the laboratory (both in vivo and in vitro) and in clinical settings. DMSO is a hydrogen-bound disrupter, cell-differentiating agent, hydroxyl radical scavenger, intercellular electrical uncoupler, intracellular low-density lipoprotein-derived cholesterol mobilizing agent, cryoprotectant, solubilizing agent used in sample preparation for electron microscopy, antidote to the extravasation of vesicant anticancer agents, and topical analgesic. Additionally, it is used in the treatment of brain edema, amyloidosis, interstitial cystitis, and schizophrenia. Several systemic side-effects from the use of DMSO have been reported, namely nausea, vomiting, diarrhea, hemolysis, rashes, renal failure, hypertension, bradycardia, heart block, pulmonary edema, cardiac arrest, and bronchospasm. Looking at the multitude of effects of DMSO brought to light by these studies, it is easily understood how many researchers working with DMSO (or studying one of its specific effects) might not be fully aware of the experiences of other groups who are working with it but in a different context.

Amyloidosis↗
PubMed2003

Evaluation of lipopolysaccharide aggregation by light scattering spectroscopy.

Lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria. These molecules behave as bacterial endotoxins and their release into the bloodstream is a determinant of the development of a wide range of pathologies. These amphipathic molecules can self-aggregate into supramolecular structures with different shapes and sizes. The formation of these structures occurs when the LPS concentration is higher than the apparent critical micelle concentration (CMC(a)). Light scattering spectroscopy (both static and dynamic) was used to directly characterize the aggregation process of LPS from Escherichia coli serotype 026:B6. The results point to a CMC(a) value of 14 microg mL(-1) and the existence of premicelle LPS oligomers below this concentration. Both structures were characterized in terms of molecular weight (5.5 x 10(6) and 16 x 10(6) g mol(-1) below and above the CMC(a), respectively), interaction with the aqueous environment, gyration radius (56 and 105 nm), hydrodynamic radius, (60 and 95 nm) and geometry of the supramolecular structures (nearly spherical). Our data indicates that future in vitro experiments should be carried out both below and above the CMC(a). The search for drugs that interact with the aggregates, and thus change the CMC(a) and condition LPS interactions in the bloodstream, could be a new way to prevent certain bacterial-endotoxin-related pathologies.

Algorithms↗
PubMed2003

An in vitro study of adrenaline effect on human erythrocyte properties in both gender.

The possibility that erythrocytes may function as a reservoir for noradrenaline and adrenaline and as a modulator of circulating catecholamine concentrations had been suggested. The aim of this work was to study the adrenaline effect on erythrocyte membrane fluidity, acetylcholinesterase (AChE) enzyme activity, P(50) and erythrocyte deformability and also to verify if the role of adrenaline on erythrocyte properties is sex-dependent. Blood samples from 42 healthy donors were obtained, and its aliquots incubated 30 min without (control) and with 10(-5) M concentrations of adrenaline alone (A(1)) and adrenaline with an alpha and an beta-blocker (A(2)). Results demonstrate that initial AChE values in female are higher (p<or=0.01) than male values. In female, adrenaline decreases AChE activity either when alpha and beta-adrenergic receptors are blocked (p<or=0.01) or when they are not. In male, adrenaline increases AChE activity when none of adrenergic receptors are blocked. Control values of male and female erythrocyte membrane fluidity are very similar but behaviour became differently (p<or=0.05) when adrenaline is present because it decreases male and increases female values. Gender differences in erythrocyte deformability are verified at high shear stress values (p<or=0.02). In female we have also registered the existence of an inverse significant correlation (r=-0.62) between membrane rigidity and AChE activity in A(2) values. Adrenaline increases p(50) values (p<or=0.03) in both sexes. Peripheral blood film has shown echinocytes when adrenaline 10(-5) M is present. We conclude that in this in vitro study sex-related differences in erythrocyte acetylcholinesterase enzyme activity, membrane fluidity and erythrocyte deformability under adrenaline influence were found.

Acetylcholinesterase↗
PubMed2003

Evidence of prolonged disturbances in the haemostatic, hemorheologic and inflammatory profiles in transmural myocardial infarction survivors.

Haemostatic, hemorheologic and inflammatory disturbances have been associated with acute coronary syndromes. Most knowledge is reported in cross sectional studies and are without time dependent evolution of these profiles. The aim of this study was to evaluate, during the first year, the evolution of the haemostatic, hemorheologic and inflammatory profiles determined at hospital discharge in survivors with transmural myocardial infarction (MI). Eighty eight (79 male; 9 female) mean age of 58 +/- 11 years, survivors of a transmural MI were prospectively studied at discharge, 6 months and one year after the event. Haemostatic (protein C, antithrombin III and plasminogen activator inhibitor 1), hemorheologic (blood fluidity and components) and inflammatory profiles (polymorphonuclear elastase and leukocyte count) were determined using standard methodology. The results of the study can be summarized as follows: (1) Protein C decreased (p < 0.05) over time while PAI-1 only varied significantly until 6(th) month. (2) Plasma viscosity and fibrinogen (p < 0.001) decrease over time, while erythrocyte aggregation (p < 0.001) and haematocrit increased. Whole blood viscosity did not vary. (3) Leukocyte decreased (p < 0.001) and elastase did not (4). Those patients with cardiovascular events (n = 7) had higher PAI-1 concentration (p < 0.05) and leukocyte count (p < 0.01), at discharge (5) Left ventricle ejection fraction correlated significantly with plasma viscosity (r = 0.35 p < 0.05). The results of this longitudinal study show dynamic modifications of the haemostatic, hemorheologic and inflammatory profiles during the first year of a transmural myocardial infarction. In addition, there are interrelations between them and the clinical profile that could help to explain the clinical evolution of this group of patients.

Aged↗
PubMed2003

Effects of choline on hemorheological properties and NO metabolism of human erythrocytes.

PURPOSE OF THE STUDY: To determine the effects of choline on red blood cell membrane properties and NO metabolism. MATERIAL AND METHODS: Aliquots of venous blood from eleven healthy subjects were incubated in vitro with choline concentrations 10(-10) to 10(-3) M. The following parameters were determined: erythrocyte deformability, aggregation and membrane lipid fluidity, plasma K+, Na+, Ca2+, total blood haemoglobin and methemoglobin concentrations. Additionally, plasma and intra-erythrocyte nitrites concentrations were measured. RESULTS: Choline increases erythrocyte deformability at lower shear stresses, decreases erythrocyte aggregation, increases membrane lipid fluidity, and decreases of Na+ plasma concentrations. We also find an increase of nitrites concentration both in the plasma and in the intra-erythrocyte compartment. CONCLUSION: Choline induces changes on erythrocyte membrane properties, Na+ plasma concentration, and NO metabolites concentrations.

Adult↗
PubMed2002

[Biohemorheologic factors and cardiovascular events curve in survivors of transmural acute myocardial infarct--24-month follow-up].

INTRODUCTION: Previous reports have shown several biohemorheological disturbances in acute myocardial infarction (AMI), either in the acute phase and after hospital discharge. There is no clearly established relationship between these parameters and the patients' clinical outcome. OBJECTIVE: To evaluate in transmural AMI survivors, a relationship between biohemorheological parameters and the cardiovascular events curve during a 24 month follow-up period. METHODS: Sixty-four consecutive patients (58.0 +/- 12.0, 59 men), transmural AMI survivors (30 anterior and 34 inferior) were included in the study. Clinical follow-up was 24 months (at 6, 12 and 24 months). The following cardiovascular events (CVE) were collected: cardiovascular death, stroke, AMI, unstable angina, embolism. We determined, at hospital discharge, these biohemorheological parameters: plasma viscosity, fibrinogen, PAI-1 inhibitor, leukocyte count, C protein (C Pt), erythrocyte aggregation (EA). For each parameter we determined the 25, 50 and 75 percentiles and other significant cut-off point, grouping patients according to these values. STATISTICS: Group t test, Kaplan-Meier survival curve (with the log rank test), and Cox logistic regression. RESULTS: (1) Patients with CVE (n = 19) during the 24 months of clinical follow-up had at hospital discharge higher leukocyte count (p < 0.001), lower C Pt (p < 0.01) and lower EA (p < 0.05). (2) The higher the percentile of the leukocyte count higher the probability for a CVE. Patients with leukocyte count above the 50 percentile had 6 times more CVE (p < 0.01); (3) The higher the C Pt lower the risk for a CVE. Patients with C PT lower than the 50 percentile had 9 times more risk for a CVE (p < 0.01), and those above the 75 percentile had no CVE (p < 0.01). (4) For the EA we identified a cutoff point (= 14.5), independent of the percentiles values. Patients with EA below 14.5 had six times more CVE. By multivariate analyses, we identified leukocyte count and C Pt as independent risk predictive factors (p < 0.05). CONCLUSION: In this group of transmural MI survivors a relationship was established between some biohemorheological (leukocyte count, C Pt, EA) and the CVE curve during 24 months of clinical follow-up.

Blood Viscosity↗
PubMed2002

Nitric oxide effects on human erythrocytes structural and functional properties--an in vitro study.

NO is present in the blood at 10(-7) M under physiological conditions, but at concentrations higher than 10(-6) M during inflammatory disease states. The aim of this study was to characterize what are the effects of these different NO concentrations on erythrocyte structural and functional properties. Blood was collected from eleven healthy men and incubated with SpermineNONOate in order to expose it during incubation time to NO concentrations between 10(-7) M and 10(-3) M. We measured erythrocyte aggregation and deformability, membrane lipid peroxidation and fluidity, p50, hemoglobin, oxyhemoglobin, methemoglobin concentrations and plasma pH, pO(2), pCO(2), Na(+), K(+) and Ca(2+). When blood was exposed to NO 10(-7) M erythrocyte deformability increase and p50 decrease. In presence of NO 10(-5) M lipid fluidity and p50 decrease. When blood was exposed to NO 10(-3) M methemoglobin concentration increase and erythrocyte deformability and p50 decrease but membrane fluidity and lipid peroxidation were similar to control. In conclusion, dependent of NO concentrations there is different effects on erythrocytes structural and functional properties.

Adult↗
PubMed2002

Long-term prognostic value of the hemorheological profile in transmural myocardial infarction survivors: 60-month clinical follow-up.

UNLABELLED: Previous reports have shown several hemorheological and hemostatic abnormalities in acute coronary syndrome survivors. Some of these abnormalities were related to cardiovascular events during a 24-month follow-up. The aim of the present work is to evaluate, in transmural myocardial infarction survivors, the long-term (60 months) prognostic value of the biohemorheological profile determined at hospital discharge. Sixty-four patients (59 men), mean age of 58 +/- 12.0 years, transmural myocardial infarction survivors, were prospectively studied for 60 months (32.0 +/- 17 months, median 33 months). The following cardiovascular events (CVE) were analyzed: death, non-fatal infarction, unstable angina, and stroke. Twenty-nine patients had a CVE (nine died). The following parameters were determined at hospital discharge: plasma viscosity, whole blood viscosity, erythrocyte membrane fluidity, erythrocyte aggregation, protein C, plasminogen inhibitor type I (PAI-1), leukocyte count and elastase. The quartiles were determined for each parameter, grouping patients according to these values. STATISTICS: Group-t-test, Kaplan-Meier survival curve (with log rank test), and Cox logistic regression. RESULTS: 1) Leukocyte count (p < 0.01), protein C activity (p < 0.05) and erythrocyte membrane fluidity (p < 0.05) were predictors of the CVE curve; 2) The higher the value of the leukocyte count quartile, the higher the risk for a CVE (p < 0.05). Patients with a leukocyte count above the median had 4 times more risk for a CVE; 3) The lower the protein C activity, the higher the risk for a CVE. Those with protein C activity lower than the lowest quartile had double the risk; 4) The higher the membrane polarization value (membrane rigidity), the higher the risk of a CVE; 5) By multivariate analysis the 3 parameters were independent predictors of a CVE. CONCLUSION: In the present group of transmural myocardial infarction survivors a close relationship was established between hemorheologic, hemostatic and inflammatory factors and the cardiovascular events curve during long-term follow-up.

Female↗
PubMedDate not supplied

Acetylcholine and choline effects on erythrocyte nitrite and nitrate levels.

Acetylcholine has been detected in human blood. Acetylcholine receptors and acetylcholinesterase are present in erythrocyte membranes. We tested the acetylcholine and choline effects on nitric oxide metabolites (NOx), namely nitrites and nitrates, and observed if they are dependent on interactions with muscarinic receptors and acetylcholinesterase. Human erythrocyte suspensions were incubated with acetylcholine and choline in the absence or presence of 10 microM atropine or 10 microM velnacrine maleate. The nitrite and nitrate concentrations were determined by the Griess method. Acetylcholine or choline increased NOx control concentrations (P <0.001). The nitrite concentrations decreased in the presence of atropine or velnacrine maleate (P <0.03). The nitrate concentrations only decreased when velnacrine maleate was incubated with acetylcholine or choline (10 microM, P <0.03). These results demonstrated that acetylcholine and choline modulate nitric oxide metabolites on erythrocytes and this effect is mediated by interactions with erythrocyte membrane muscarinic receptors and membrane enzyme acetylcholinesterase. A hypothesis for the signal transduction mechanism has been discussed for acetylcholinesterase and muscarinic receptor (M1) participation.

Acetylcholine↗