Biomedical subjects

V Ferreira

Publications and source records attributed to V Ferreira.

At least 19 recordsLinked to original sources

PubMed2006

Multidimensional chromatographic approach applied to the identification of novel aroma compounds in wine. Identification of ethyl cyclohexanoate, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate.

A multidimensional chromatographic strategy has been developed and optimized with the purpose of identifying different odorants potentially relevant to the aroma and flavor of aged wines from Madeira or Sherry. Different techniques of extraction and fractionation were studied in order to get clear olfactometric and spectrometric signals from the target odorants. The best results were obtained with a dynamic headspace extraction followed by a fractionation on a normal phase medium pressure liquid chromatography on a silicagel column. Large volumes (50 microl) of the concentrated fractions were further analyzed in a dual gas chromatography-mass spectrometric system (GC-MS) equipped with two olfactometric ports. The strategy made it possible to identify in wine by first time the presence of the powerful strawberry-smelling compound, ethyl cyclohexanoate, and of two other novel fruity esters, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate. Some other unidentified odorants could be isolated and their mass spectra are given.

Butyrates↗

PubMed2005

Highly selective solid-phase extraction and large volume injection for the robust gas chromatography-mass spectrometric analysis of TCA and TBA in wines.

A reliable solid-phase extraction (SPE) method for the simultaneous determination of 2,4,6-trichloroanisole (TCA) and 2,4,6-tribromoanisole (TBA) in wines has been developed. In the proposed procedure 50 mL of wine are extracted in a 1 mL cartridge filled with 50 mg of LiChrolut EN resins. Most wine volatiles are washed up with 12.5 mL of a water:methanol solution (70%, v/v) containing 1% of NaHCO3. Analytes are further eluted with 0.6 mL of dichloromethane. A 40 microL aliquot of this extract is directly injected into a PTV injector operated in the solvent split mode, and analysed by gas chromatography (GC)-ion trap mass spectrometry using the selected ion storage mode. The solid-phase extraction, including sample volume and rinsing and elution solvents, and the large volume GC injection have been carefully evaluated and optimized. The resulting method is precise (RSD (%) < 6% at 100 ng L(-1)), sensitive (LOD were 0.2 and 0.4 ng/L for TCA and TBA, respectively), robust (the absolute recoveries of both analytes are higher than 80% and consistent wine to wine) and friendly to the GC-MS system (the extract is clean, simple and free from non-volatiles).

Anisoles↗

PubMed2004

Characterization of the aroma of a wine from maccabeo. Key role played by compounds with low odor activity values.

An extract from a dry young wine from Maccabeo was studied by aroma extract dilution analysis (AEDA), quantitative gas chromatography, and different sensory studies. In a first study, 53 different aroma compounds were quantified and used to prepare aroma models. 2-Methyl-3-furanthiol (FD = 16) and 4-methyl-4-mercaptopentan-2-one (FD = 2), could not be quantified and were not included in those models, which were not very similar to the original wine. Omission tests did not show the existence of impact compounds. In another set of experiments, selected aroma chemicals were added to the original wine, but in only in two cases (isoamyl acetate and gamma-nonalactone) was a positive effect noted, on banana and citric notes, respectively. After these discouraging results, 4-methyl-4-mercaptopentan-2-one and 2-methyl-3-furanthiol were quantified and included in the models. The concentration of the former was as low as 5 ng x L(-)(1) (odor threshold = 0.8 ng x L(-)(1)); however, its inclusion in the synthetic mixture had a significant effect, making it very close to the original wine. Its role was confirmed by omission tests. Results are briefly discussed.

Adult↗

PubMed2001

Use of solid-liquid distribution coefficients to determine retention properties of Porapak-Q resins. Determination of optimal conditions to isolate alkyl-methoxypyrazines and beta-damascenone from wine.

The solid-liquid distribution coefficients of different analytes--all of which are important aroma compounds--between hydroalcoholic solutions or wines and different sorbents have been determined by measuring the amount of analyte removed by a given mass of sorbent in equilibrium with a given volume of standard solution. These data have shown that the best extraction conditions for non-polar compounds from wine are the use of Porapak-Q resins and 6% (v/v) alcoholic solutions. Phase ratio, hold-up volumes and number of plates for Porapak-Q beds have been measured in different experiments. With all these data it has been possible to calculate breakthrough volumes in good agreement with experimental results. The Lövkvist-Jönsson model is more appropriate for estimating breakthrough volumes of a 2-cm Porapak-Q bed. The model estimates that a 5-cm bed is needed to achieve a quantitative recovery of 3-alkyl-2-methoxypyrazines and beta-damascenone from 500 ml of wine (diluted to 1000 ml with water). Experimental results confirm the predictions of the model and show that in a single isolation step detection limits below 10 ng/l can be reached for these compounds using GC-MS detection.

Chromatography, Gas↗

PubMed2001

Fast analysis of important wine volatile compounds development and validation of a new method based on gas chromatographic-flame ionisation detection analysis of dichloromethane microextracts.

A method for the simultaneous determination of major (10-200 mg/l) and minor (0.1-10 mg/l) volatile compounds from wine has been optimised and validated. A 3-ml volume of wine is diluted with water (7 ml), salted with 4.5 g of ammonium sulfate and extracted with 0.2 ml of dichloromethane. The extract is injected in the split mode in a GC system, separated on a Carbowax 20M capillary column and detected by flame ionisation detection. Volatiles from wine are divided into four groups according to their behaviour in the extraction, and a specific internal standard has been selected for each group. The method allows satisfactory determination of more than 30 volatile compounds of wine. Compounds analysed include acetaldehyde, diacetyl, acetoine (3-hydroxy butanone), fusel alcohols and their acetates, and fatty acids and their ethyl esters. The linear dynamic range of the method covers the normal range of occurrence of analytes in wine and extends from at least one magnitude order to more than two, with typical r2 between 0.9938 and 0.9998. Reproducibility ranges from 3.1 to 10% (as RSD) with 5.5% as the average. The analysis of spiked samples has shown that matrix effects do not significantly affect method performance.

Chromatography, Gas↗

PubMed2001

Identification and quantification of impact odorants of aged red wines from Rioja. GC-olfactometry, quantitative GC-MS, and odor evaluation of HPLC fractions.

An XAD-4 extract from a 5-year-old wine from Rioja (Spain) was analyzed by aroma extract dilution analysis. Most of the odorants were quantified by GC-MS. A second extract was fractionated in an HPLC system with a C-18 semipreparative column. Fifty fractions were recovered, their alcoholic degree and pH were further adjusted to those of the wine, and those fractions that showed strong odor characteristics were further re-extracted and analyzed by GC-O and GC-MS. Reconstitution experiments were carried out to confirm the role of the odorants detected in the fractions. Fifty-eight odorants were found in the Rioja wine, 52 of which could be identified. Methyl benzoate was found to be a wine aroma constituent for the first time. The most important odorants are 4-ethylguaiacol, (E)-whiskey lactone, 4-ethylphenol, beta-damascenone, fusel alcohols, isovaleric and hexanoic acids, eugenol, fatty acid ethyl esters, and ethyl esters of isoacids, Furaneol, phenylacetic acid, and (E)-2-hexenal. Comparison among the three techniques shows good agreement and demonstrates that they are complementary.

Chromatography, Gas↗

PubMed2001

Quantitative gas chromatography-olfactometry carried out at different dilutions of an extract. Key differences in the odor profiles of four high-quality Spanish aged red wines.

Four Spanish aged red wines made in different wine-making areas have been extracted, and the extracts and their 1:5, 1:50, and 1:500 dilutions have been analyzed by a gas chromatography-olfactometry (GC-O) approach in which three judges evaluated odor intensity on a four-point scale. Sixty-nine different odor regions were detected in the GC-O profiles of wines, 63 of which could be identified. GC-O data have been processed to calculate averaged flavor dilution factors (FD). Different ANOVA strategies have been further applied on FD and on intensity data to check for significant differences among wines and to assess the effects of dilution and the judge. Data show that FD and the average intensity of the odorants are strongly correlated (r(2) = 0.892). However, the measurement of intensity represents a quantitative advantage in terms of detecting differences. For some odorants, dilution exerts a critical role in the detection of differences. Significant differences among wines have been found in 30 of the 69 odorants detected in the experiment. Most of these differences are introduced by grape compounds such as methyl benzoate and terpenols, by compounds released by the wood, such as furfural, (Z)-whiskey lactone, Furaneol, 4-propylguaiacol, eugenol, 4-ethylphenol, 2,6-dimethoxyphenol, isoeugenol, and ethyl vanillate, by compounds formed by lactic acid bacteria, such as 2,3-butanedione and acetoine, or by compounds formed during the oxidative storage of wines, such as methional, sotolon, o-aminoacetophenone, and phenylacetic acid. The most important differences from a quantitative point of view are due to 2-methyl-3-mercaptofuran, 4-propylguaiacol, 2,6-dimethoxyphenol, and isoeugenol.

Chromatography, Gas↗

PubMed2001

Daily production of human tumor necrosis factor in lipopolysaccharide (LPS)-stimulated ex vivo blood culture assays.

Tumor necrosis factor (TNF) is a pleiotropic cytokine with immunological and neuroendocrine activities. A useful tool for studying TNF is the measurement of its in vitro and/or ex vivo over-expression, induced by a variety of stimuli on isolated peripheral mononuclear cells or whole blood, respectively. The capacity to over-express TNF, in ex vivo LPS-stimulated whole blood from 18 normal individuals, showed inter-individual variations ranging from high (3 ng/ml) to low (0.7 ng/ml) producers. Although at a lower level, a similar situation was observed in the spontaneous production of the cytokine. In order to detect cyclic effects in these variations, blood samples were taken at 08:00, 12:00, 16:00 and 20:00 hours, from nine healthy volunteers, and cultured in the ex vivo system. TNF and cortisol were measured by immunometric assays. Both, LPS-stimulated whole blood and plasma showed important, individual variations in TNF levels. Although cortisol levels presented a normal circadian cycle, these individual patterns in TNF production were basically conserved during the day (p > 0.05), and no correlation was observed between the levels of the hormone and those of the cytokine. When total TNF levels were determined at 20:00 hours, a moderate, temporary variation pattern of the cytokine production was found. These results suggest that cortisol does not play a predominant role in determining the ex vivo capacity of blood to produce TNF. Presumably, the variable capacity to produce the cytokine may have a strong genetic component.

Humans↗

PubMed2000

Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress.

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.

Animals↗

PubMed2000

Clues about the role of methional as character impact odorant of some oxidized wines.

A young white wine that had undergone spontaneous oxidation and showed a strong off-flavor reminiscent of cooked vegetables was demixed by salting out to obtain an ethanolic extract that retained the off-flavor of the wine, as was demonstrated by sensory analysis. This extract, together with a second one obtained from a non-oxidized wine sample, were chromatographed into a reversed-phase HPLC column using a water/ethanol gradient. The column effluents were collected in 14 different fractions that were evaluated for smell. Four fractions were found to differ between oxidized and non-oxidized wine, but only one showed a clear cooked vegetables off-flavor and, when added to a non-oxidized wine, made it significantly more similar to the oxidized wine. The fraction was analyzed by GC-MS-olfactometry, which made it possible to identify methional as the single important odorant of the fraction. The odor threshold of methional in a synthetic wine was found to be 0.5 microg L(-)(1). The analytical determination of methional through GC-FPD in several oxidized and non-oxidized wines showed that, in the former, methional can reach more than 200 Odor Units, whereas in the non-oxidized samples, it was not possible to detect methional. The methional concentration was found to increase in wines spiked with both methionol or methionine, which suggests that it can be formed from direct peroxidation of methionol or via Strecker degradation of methionine mediated, probably, by o-quinones formed during wine oxidation.

Aldehydes↗

PubMed2000

Molecular cloning of a truncated p62Dok1 isoform, p22Dok(del).

The p21Ras GTPase activating protein-associated 62-kDa protein, p62Dok1, is an early substrate of various tyrosine phosphorylation pathways. Its recent cloning in human myeloid cells and in murine pre-B cells revealed an N-terminal pleckstrin-homology domain and a tyrosine- and proline-rich C-terminal tail in its sequence. Here, we characterized a new 1261-bp cDNA identical to that of p62Dok1, but with a central 185-bp deletion (bp 456-640). This induced a frameshift leading to a premature stop codon. The deduced protein, designated p22Dok(del), corresponded to a truncated p62Dok1 isoform of 177 amino acids that can be expressed both in vitro and in vivo with an apparent molecular mass of 22 kDa. This newly identified molecule was composed of the N-terminal PH domain of p62Dok1 followed by a new 25-amino acid C-terminal sequence containing a typical class II proline-rich motif, suggesting a specific role for p22Dok(del) in signal transduction pathways.

Amino Acid Sequence↗

PubMed2000

A comparative study of the ability of different solvents and adsorbents to extract aroma compounds from alcoholic beverages.

Seven liquid solvent systems--dichloromethane, dichloromethane-pentane (1:1), freon 113, diethyl ether-pentane (1:1 and 1:9), ethyl acetate-pentane (with and without an additional salting-out effect) (1:3 and 1:20), and seven solid-phase extraction (SPE) systems (Amberlite XAD-2, 4, 7, and 16; Porapak Q; C8; and C18)--are comparatively studied. The distribution coefficients between the extraction system and a hydroalcoholic solution (12% v/v in ethanol, pH = 3.2) of 14 selected volatile compounds belonging to different chemical families and polarities are calculated. The results are processed by factor analysis and cluster analysis, and the following conclusions are reached. First, the efficiency of extraction decreases in this order: polymeric sorbents > silica-based sorbents > liquid-liquid systems with salting-out effect approximately dichloromethane > rest of liquid solvents. Second, the addition of salt mainly increases the recovery of compounds with Lewis acid properties. Third, the efficiency of the extraction of a liquid solvent depends not only on its polarity but also on its solubility in water. Fourth, in regards to the selectivity of the SPE systems, Porapak Q is the best to extract nonpolar compounds, Amberlite XAD 4 and 16 provide the least selective extraction profiles, and C8 and C18 have a special ability to extract compounds with a Brønstedt-Lowry character. Results indicate that in all cases liquid solvents can be replaced satisfactorily by SPE systems.

Adsorption↗

PubMed2000

Defective p56Lck activity in T cells from an adult patient with idiopathic CD4+ lymphocytopenia.

Idiopathic CD4(+) lymphocytopenia (ICL) is defined by a stable loss of CD4(+) T cells in the absence of any known cause of immune deficiency. This syndrome is still of undetermined origin. It affects adult patients, some of them displaying opportunistic infections similar to HIV-infected subjects. The hypothesis that the cellular immune defect may be due to biochemical failures of the CD3-TCR pathway is investigated here in a patient associating a severe selective CD4(+) lymphocytopenia with an increased CD8(+) T cell count discovered in the course of a cryptococcal meningitidis. A 40% reduction of T cell proliferation to CD3-TCR stimulation is observed only in the CD4(+) subpopulation. The early CD3-induced protein tyrosine phosphorylations are conserved in both CD4(+) and CD8(+) subsets, and the levels of the T cell protein tyrosine kinases p56(Lck), p59(Fyn) and ZAP-70 are normal. However, we find a 50% reduction of p56(Lck) kinase activity in the patient's T cells compared to a healthy control donor. p59(Fyn) activity does not appear to be altered. Nevertheless, we do not find any genetic abnormality of p56(Lck). These results thus suggest that a defect of an unknown protein regulating p56(Lck) activity takes place in this patient's T cells. Taken together, these findings reveal p56(Lck) alteration in ICL and confirm the critical role of this kinase in the maintenance of the peripheral CD4(+) T cell subpopulation.

Adult↗

PubMed2000

Paradigms for subdural grids' implantation in patients with refractory epilepsy.

RATIONALE: The need for invasive monitoring in patients with refractory epilepsy has been greatly reduced by the introduction of new technologies such as PET, SPECT and MRI in the clinical practice. On the other hand, 10 to 30% of the patients with refractory epilepsy have non-localizatory non-invasive preoperative work-up results. This paper reports on the paradigms for subdural electrodes implantation in patients with different refractory epileptic syndromes. METHODS: Twenty-nine adult refractory epileptic patients were studied. Patients were divided into five different epileptic syndromes that represented the majority of the patients who needed invasive recordings: bitemporal (Group I; n=16 ), bi-frontal-mesial (Group II, n=5), hemispheric (Group III; n=2), anterior quadrant (Group IV; n=3) and posterior quadrant (Group V; n=3). All of them were submitted to extensive subdural electrodes' implantation (from 64 to 160 contacts) covering all the cortical surface potentially involved in epileptogenesis under general anesthesia. RESULTS: All patients tolerated well the procedure. There was no sign or symptom of intracranial hypertension except for headache in 22 patients. In all except one Group II patient, prolonged electrocorticographic monitoring using the described subdural cortical coverage patterns was able to define a focal area amenable for resection. In all Groups II-V patients cortical stimulation was able to adequately map the rolandic and speach areas as necessary. CONCLUSION: Despite recent technological advances invasive neurophysiological studies are still necessary in some patients with refractory epilepsy. The standardization of the paradigms for subdural implantation coupled to the study of homogeneous patients' populations as defined by MRI will certainly lead to a better understanding of the pathophysiology involved in such cases and an improved surgical outcome.

Adult↗

PubMed1999

Semipreparative reversed-phase liquid chromatographic fractionation of aroma extracts from wine and other alcoholic beverages.

The suitability of reversed-phase HPLC for the semi-preparative fractionation of aroma extracts from wine and other alcoholic beverages has been explored. Aroma extracts are separated in a 250x10-mm Kromasil-C18 column using a water-ethanol gradient system as mobile phase. It has been demonstrated that the chromatographic separation does not induce any chemical change in the sample components. The maximum volume that can be injected without altering efficiency is as high as 2 ml if ethanolic extracts are injected, and slightly less in the case of less polar extracts. Aroma extracts are injected directly without the need of any pretreatment. As major compounds elute first, it is possible to fractionate all the volatiles contained in a 1-1.5-1 sample without peak distortion or mass overload problems. The usefulness of the method has been demonstrated by fractionating an extract from a Chardonnay wine to get 15 fractions that showed different aromas. The GC analysis with olfactometric and MS detection of those fractions has allowed us to identify more than 70 aroma compounds and to signal some of them as potential key aromas of Chardonnay wine.

Adult↗

PubMed1999

Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice.

Multiple antigen peptides (MAPs) containing epitopes of the major surface protein of the malaria sporozoite, the circumsporozoite (CS) protein, have been shown in previous studies to elicit antibody-mediated protection against sporozoite challenge in experimental murine and simian hosts. For the preparation for a phase I trial of a P. falciparum (T1B)4 MAP, which contains T and B cell epitopes from the CS repeat region, pre-clinical immunogenicity and adjuvant formulation studies were carried out in mice and Aotus monkeys. The (T1B)4 MAP was found to be immunogenic in three different species of owl monkeys, Aotus nancymae, A. vociferans and A. nigriceps. Optimal antibody responses were obtained in A. nancymae immunized s.c. with (T1B)4 MAP emulsified in Freund's, in which peak titers of over 10(6) were obtained in individual monkeys. MAP immunized A. vociferans also developed high levels of anti-sporozoite antibodies, although the kinetics and the magnitude of the response differed from A. nancymae. (T1B)4 MAP adsorbed to alum (aluminum hydroxide), a formulation that is acceptable for human use, was less immunogenic in naive A. nancymae, as well as A. nigriceps. The injection of MAPs/alum, however, significantly enhanced antibody responses in sporozoite-primed monkeys, suggesting that the administration of the MAP vaccine may be an effective means to increase the low levels of antibody present in individuals living in malaria endemic areas. The addition of a co-adjuvant QS-21, a purified saponin, significantly increased the immunogenicity of the alum-adsorbed MAP in both mice and monkeys, providing a vaccine formulation suitable for phase I trials in human volunteers.

Alum Compounds↗

PubMed1999

In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis.

To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression.

Animals↗

PubMed1998

Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells.

In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine.

Apoptosis↗