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J Martins-Silva

Publications and source records attributed to J Martins-Silva.

At least 19 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↗
PubMed2006

Effects of low-fat milk enriched with phytosterols on plasma cholesterol concentrations and hemorheological parameters of Wistar rats.

Clinical and experimental studies have shown that the use of phytosterol esters as a food ingredient reduces the plasma concentrations of cholesterol and LDL cholesterol, not affecting the HDL cholesterol levels. Based on the use of phytosterols as a food ingredient, we have conducted a 30-day feeding study with Wistar rats, drinking low-fat milk containing phytosterols, in order to evaluate the plasma cholesterol concentrations and the hemorheological parameters. Throughout the study, clinical observations, body weights and food and milk consumption were measured and at the end of the feeding period, blood samples were collected for biochemical and hemorheological determinations. There were no clinical changes, alterations in growth, food or milk consumption. In the plasma cholesterol and HDL concentrations there were no significant differences, but LDL levels decreased about 70%. In the hemorheological parameters, significant changes were observed in plasma viscosity and in membrane fluidity in all experimental groups. The blood viscosity and the erythrocyte deformability show significant improvements with the ingestion of the phytosterols enriched milk. With these results we conclude that phytosterols maintain their cholesterol lowering properties when incorporated in milk and can be considered a hypolipemic food component.

Animals↗
PubMed2006

Phytosterols in milk as a depressor of plasma cholesterol levels: experimental evidence with hypercholesterolemic Portuguese subjects.

Plant sterols have been reported to decrease plasma concentrations of cholesterol without any side effects. To evaluate the effects on plasma cholesterol concentrations and the hemorheological parameters, we performed a study with hypercholesterolemic patients (n = 19) treated with phytosterol-enriched milk (2 g/day). Hypercholesterolemic patients (n = 15) of matched age drinking equal type of milk but without phytosterols were used as control group. Concentrations of total cholesterol, HDL-C, LDL-C and hemorheological parameters were measured in the beginning, after 15 and 30 days of milk intake. After 15 days of beverage intake, hypercholesterolemic subjects treated with phytosterol-enriched milk showed a significant decrease in plasma concentrations of total cholesterol and LDL-C by 9.62% (p < 0.05) and 12.20% (p < 0.05), respectively. After 30 days, a little increase in the total cholesterol and LDL-C concentrations were observed. In the hypercholesterolemic control group there were nonsignificant changes between plasma concentrations of total cholesterol, HDL-C and LDL-C during the study. The evaluation of plasma viscosity and erythrocyte aggregation shows no changes statistically significant during the study for both groups studied. The results obtained during the study show a positive effect with the phytosterol-enriched milk as plasma cholesterol-lowering as combined treatment for hypercholesterolemia.

Adult↗
PubMed2006

The relation between the erythrocyte nitric oxide and hemorheological parameters.

We stimulated human erythrocytes obtained from patients with hypercholesterolemia (HC; n = 42), renal transplantation (RT; n = 18) and hypertension (HT; n = 10) with acetylcholine (ACh 10 microM) and measured the amperometric NO production, comparing with the NO levels achieved on erythrocytes of healthy persons (n = 27). We also measured the hemoglobin, hematocrit, erythrocyte aggregation, erythrocyte deformability, plasma viscosity and fibrinogen concentration from human blood samples. The erythrocytes NO levels were of 2.5 +/- 0.7 nM (P = 0.038, HC), 2.4 +/- 1.1 nM (RT) and 2.2 +/- 0.8 nM (HT) against the 2.0 +/- 0.8 nM for the control groups. For each group and at each shear stress value, the erythrocytes deformability decreases with the increase of the NO concentration after ACh stimulation. We observed a significant increase of the control values on the erythrocyte aggregation results on each patient group. Besides the lower erythrocyte deformability obtained on HC, RT and HT blood samples, the erythrocytes produced higher NO levels after ACh stimulation than the healthy ones. The power of erythrocyte hemorheological behaviour could be compensated by the NO production at the presence of acetylcholine. We can hypothesises that cholinergic drugs could be used as co-adjuvants of specific therapeutics compounds on these studied diseases.

Acetylcholine↗
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↗
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↗
PubMed2002

Fluorescent probes DPH, TMA-DPH and C17-HC induce erythrocyte exovesiculation.

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.

Cell Membrane↗
PubMed2002

Biochemical, biophysical and haemorheological effects of dimethylsulphoxide on human erythrocyte calcium loading.

The studies using dimethylsulphoxide (DMSO) and/or the 4-bromo-calcium ionophore A23187 (Br-A23187) often neglect the precise knowledge of some of their biochemical, biophysical and haemorheological effects. The aim of the present study was to evaluate these effects on erythrocytes after whole blood incubations with DMSO or Br-A23187 dissolved in DMSO. There were no significant differences between the different aliquots in the values of P(50), pH, erythrocyte deformability, erythrocyte membrane fluidity, haemoglobin and intracellular Ca(2+) concentrations ([Ca(2+)](i)). Aliquots with DMSO (independently of the presence of Br-A23187 or added Ca(2+)) had lower erythrocyte aggregation indexes and higher plasma concentrations of K(+)], Na(+)] and Ca(2+) than the aliquots without DMSO (independently of the presence of added Ca(2+)). Aliquots with added calcium (without the presence of Br-A23187 in DMSO) had a significantly higher erythrocyte acetylcholinesterase activity. Our data shows that calcium loading, the usual objective of Br-A23187 incubations, cannot be fulfilled with the studied experimental conditions. The coherence between our results and those obtained by other authors with different biological systems and different modulators of the rise on [Ca(2+)](i) suggests a non-specific effect of DMSO, disabling the action of the modulator. It can be reasoned that the decreased erythrocyte aggregation (without significant changes on the deformability or membrane fluidity) can result either from the decrease of the hydrogen bonding contribution to erythrocyte aggregation or the increased ionic strength influence on the erythrocyte membrane surface.

Acetylcholinesterase↗
PubMed2001

Hemorheological effects of sodium fluorescein in rats.

Sodium fluorescein is widely used in clinical practice for the study of the retinal circulation by angiography. It has been reported several hemorheological and microvascular abnormalities induced by this compound. The aim of this work was to analyse the hemorheological effects of intravenous sodium fluorescein in an animal model. Twenty male 10-16 weeks-old Wistar rats were used, under systemic anaesthesia. The animals were divided in 2 groups of 10 each: (1) intravenous injection of sodium fluorescein (14 mg/kg of body weight)--Group NaF, (2) controls (injection of NaCl 0.9%)--Group CTRL. A blood sample was drawn by aortic puncture after 60 minutes and hemorheological parameters determined: hematocrit, hemoglobin, metahemoglobin, carboxyhemoglobin, plasma viscosity, erythrocyte deformability, membrane fluidity and acetylcholinesterase activity. In the Group NaF there was a 20% reduction of the AChE activity (p<0.05) and an increase in PV (p<0.05). Concerning hemoglobin status, a three-fold increase in COHb (p<0.001) was shown. In conclusion, the NaF injection in the animal model produces hemorheological abnormalities similar to those reported in the human.

Animals↗
PubMed2001

Effects of acetylcholine and spermineNONOate on erythrocyte hemorheologic and oxygen carrying properties.

PURPOSE OF THE STUDY: To determine the effects of acetylcholine and spermineNONOate--a NO donor--on RBC membrane and oxygen carrying properties. MATERIAL AND METHODS: Aliquots of venous blood from eleven healthy subjects were incubated with ACh 10(-3) M, ACh 10(-5) M, spermineNONOate 10(-5) M and spermineNONOate 10(-4) M. The following parameters were determined: erythrocyte aggregation and deformability, hematocrit, plasma pH, osmolality, K(+), Na(+), Ca(2+) concentrations, hemoglobin, oxyhemoglobin, carboxyhemoglobin and methemoglobin concentrations, oxygen saturation of hemoglobin, oxygen and carbon dioxide partial pressures and p50. RESULTS: In presence of ACh there is an increase of erythrocyte deformability, decrease of erythrocyte aggregation, plasma pH, K(+) and Na(+) concentration, increase of Ca(2+) concentration and p50. In presence of spermineNONOate there is an increase of erythrocyte deformability, plasma pH, decrease of Na(+) and Ca(2+) concentration, increase of metHb concentration and decrease of p50. CONCLUSION: Acetylcholine and spermineNONOate are able to induce changes on RBC membrane and oxygen carrying properties.

Acetylcholine↗
PubMed1999

Sodium fluorescein influence on the hemorheological profile of non-insulin dependent diabetes mellitus patients.

Sodium fluorescein angiography is a widely used technology in ophthalmology, which allows us to visualise the chorioretinal microcirculation. Previous reports showed a prolongation of the retinal circulation time along with erythrocyte hyperaggregation and a decrease of erythrocyte acetylcholinesterase activity and a possible interference with the erythrocyte's membrane fluidity. The aim of the present work is to investigate the influence of sodium fluorescein on the hemorheological profile of a group of 23 non-insulin dependent diabetes mellitus (NIDDM) patients undergoing routine retinal angiography. Thirty minutes after the endovenous administration of the fluorescein there was: (I) an increase of whole blood viscosity (p = 0.015), erythrocyte elongation index (EEI, p < 0.05), whole blood pH (p < 0.001), methemoglobin (p < 0.001) and carboxyhemoglobin (p < 0.001) concentrations; (II) no variation of plasma osmolality and erythrocyte aggregation index (EAI); (III) a decrease of the erythrocyte acetylcholinesterase activity, p < 001; (IV) no variation in membrane lipid fluidity, although 1,6-diphenyl-1,3,5-hexatriene (DPH) correlated directly with the EEI, while 1,4-trimethyl-phenyl-1,3,5-hexatriene (TMA-DPH) and EAI correlated inversely, suggesting that the decreasing EEI (lower erythrocyte deformablity) might be associated with an increased rigidity of the external polar region and fluidification of the hydrophobic region of the erythrocyte membrane, with an increasing EAI. In conclusion, the endovenous administration of sodium fluorescein in NIDDM patients during the retinal angiography procedure interferes with the erythrocyte membrane and possibly with the microcirculatory blood flow.

Angiography↗
PubMed1999

Impairment of the erythrocyte membrane fluidity in survivors of acute myocardial infarction. A prospective study.

Erythrocytes have to constantly adapt themselves to the varying circulatory system shear stress forces and capillaries diameter. Membrane lipid and protein content have an important role in determining the erythrocyte shape and are main determinants of the membrane solid and fluid behaviour which enables the erythrocyte to respond to the outer environment modifications. Membrane fluidity is an inverse index of membrane microviscosity. The aim of the present work is to evaluate prospectively in three periods of time (discharge, after 6 months and one year later) in survivors of an acute myocardial infarction (AMI) the erythrocyte membrane fluidity (outer and inner bilayer) and establish a relation with the cardiovascular events or need of coronary revascularization during a two year clinical follow up. Sixty survivors of acute myocardial infarction were recruited during 1994-96 and were prospectively studied in three periods (discharge, 6 months and after one year), and were compared with a control group (n = 36). Membrane lipid fluidity was determined by means of fluorescence polarisation with two probes: 1,6-diphenyl-1,2,5-hexatriene (DPH) and 1,4-trimethylamine 6-phenyl hexa-1,3,5-triene (TMA-DPH), for the characterisation of the hydrophobic and external polar region, respectively. The hydrophobic region was more rigidified (p < 0.01) in the erythrocytes from AMI patients, in relation to the control group. During the time of the study there was a progressive erythrocyte membrane rigidification (DPH p < 0.001; TMA-DPH p < 0.001). We found no relation between erythrocyte membrane fluidity and the coronary risk factors, cardiovascular events or the need of coronary revascularization during the clinical follow-up. In conclusion, after the myocardial infarction erythrocyte membrane of AMI survivors becomes more rigid with time, which could contribute to the decreased erythrocyte deformability and the increased blood viscosity previously described in this group of patients.

Aged↗