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Biomedical subjects

M A García

Publications and source records attributed to M A García.

At least 19 recordsLinked to original sources
PubMed2001

Study of retention in micellar liquid chromatography on a C8 column by the use of linear solvation energy relationships.

Linear solvation energy relationships (LSERs) are used to investigate the fundamental chemical interactions governing the micellar liquid chromatographic retention of 22 aromatic compounds (11 benzene derivatives and 11 aromatic polycyclic hydrocarbons) in 80 mobile phases on a C8 column. The systems studied involve combinations of 0.050 to 0.140 M sodium dodecyl sulfate or cetyltrimethylammonium bromide, with 0 to 10% methanol, n-propanol, and n-butanol as mobile phase modifiers. The ability of the LSERs to account for the chemical interactions underlying solute retention is shown. A comparison of predicted and experimental retention factors suggests that LSER formalism is able to reproduce adequately the experimental retention factors of the solutes studied in the different experimental conditions investigated.

Benzene Derivatives↗
PubMed2001

Analysis of Origanum vulgare volatiles by direct thermal desorption coupled to gas chromatography-mass spectrometry.

Volatile components of samples of a population of Spanish Origanum vulgare have been analyzed by direct thermal desorption coupled to GC-MS. The method is fast and reliable and requires a low amount of sample, allowing analysis of leaves and flowers from a single individual plant. Volatile yield is highly variable among individual plants and concentration also presents a high variation for most Origanum volatile compounds, linalool being the main component in most samples. Statistical analyses are applied in order to find patterns in composition data.

Adsorption↗
PubMed2001

The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-kappaB activation.

The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-kappaB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-kappaB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-kappaB induction. In PKR(0/0) cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-kappaB. An inactive PKR mutant (K296R), was unable to induce NF-kappaB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-kappaB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2alpha phosphorylation and NF-kappaB activation. However, PKR mutants unable to activate NF-kappaB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-kappaB.

Animals↗
PubMed2000

Identification of amino acid residues of transcription factor AP-2 involved in DNA binding.

AP-2 is a cell-type specific, developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identified, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. Here, we describe the identification of a region of AP-2 which was protected by an oligonucleotide probe containing its binding site from trypsin digestion, monitored by peptide mapping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependent APOE promoter, as well as the ability to inhibit the effect of the wild-type protein of each one of a set of single-site substitution AP-2 mutants spanning the identified region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential for its DNA-binding activity. Particularly, the individual substitution in any of the residues 253, 254, 255, 257 or 260 is sufficient for completely abolishing the interaction with DNA and the stimulation of APOE promoter activity. These results indicate a crucial role of this region in the formation of an active DNA-binding domain and strongly suggest that these residues provide direct contacts with the DNA structure at the AP-2 binding site.

Amino Acid Sequence↗
PubMed2000

Separation of a group of N-phenylpyrazole derivatives by micellar electrokinetic chromatography: application to the determination of solute-micelle association constants and estimation of the hydrophobicity.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of a group of N-phenylpyrazole derivatives. Sodium dodecyl sulfate (SDS) as micellar system and 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) as separation buffer (pH 10) were employed in the absence and presence of different percentages of medium chain alcohols (n-propanol or n-butanol). The separation of multicomponent mixtures of the solutes studied enabled the rapid determination of their retention factors which, in turn, allowed the study of the separation selectivity of compounds and the determination of their solute-micelle association constants (from the linear variation of the retention factors as a function of the total surfactant concentration in the separation buffer). Separation selectivity was studied according to the elution range and number of solutes separated in all the electrolyte solutions employed (45 micellar phases). The effect of the buffer concentration (0.05, 0.08 and 0.10 M), the alcohol nature (n-propanol or n-butanol) and the alcohol percentage (1, 3 or 5%) of the values obtained for the solute-micelle association constants was also studied. The best separation (12 solutes) was performed when a 0.08 M CHES buffer, pH 10, 0.02 M SDS modified by 5% n-butanol was used. The possibilities of using MEKC for evaluating the hydrophobicity of compounds was investigated through the study of the correlation between the logarithm of the retention factors of N-phenylpyrazole derivatives and their logarithm of the octanol-water distribution coefficients estimated by high performance liquid chromatography (HPLC).

Alcohols↗
PubMed2000

Accumulation of mercury in edible macrofungi: influence of some factors.

The levels of mercury in terrestrial ecosystems are generally very low, but the strong accumulation in some species of macrofungi is an exception from this rule. Mercury contents of 41 samples of edible mushrooms fruiting bodies representing eight species (six wild and two cultivated) were determined by an anodic stripping voltammetric technique (ASV) using a gold disc as the working electrode. Wild fungi were collected in unpolluted and polluted areas in the Province of Lugo (NW Spain). Influence of some factors (ecology, species, traffic pollution, and morphological portion) and the importance of mushrooms as a dietary source of mercury have been studied. Wild saprophytic species showed higher levels (ppm DW) than mycorrhizals species, with the exception of Boletus pinicola. There were significant differences according to the species analyzed (p < 0.001), the highest average content of mercury was found in Boletus pinicola (7.37 ppm DW), and the range was 0.35-33.07 ppm DW for hymenophore and 0.18-20.30 ppm DW for the rest of the fruit body. The cultivated species accumulated lower than wild species because the mean life is shorter. The traffic pollution factor did not show significant differences, so mushrooms are not realiable bioindicators of traffic pollution by mercury. Hymenophore was always the morphological portion that contained the highest mercury levels (p < 0.05), and the mean ratio of hymenophore/rest of the fruit body was 2.13. The mercury concentrations were compared to literature data and levels set by legislation, and the contribution of mushrooms to the weekly intake of mercury per person was evaluated. The possible health risk for people is pointed out.

Agaricales↗
PubMed2000

Organochlorine and heavy metal residues in the water/sediment system of the Southeast Regional Park in Madrid, Spain.

A study into levels of contamination by organochlorine compounds (insecticides and PCBs) and heavy metals (Cd and Pb) in the water/sediment system of the Southeast Regional Park (SERP) in Madrid, Spain, has been carried out. Residue levels of xenobiotics were determined in surface and underground waters and sediments from selected sites throughout the protected area. The results showed these contaminants to be widespread throughout the studied area. p,p'-DDT concentration levels were consistently higher than its metabolite p,p'-DDE, indicating a recent use of this organochlorine insecticide in the area. PCB levels exceeded, in the majority of the cases, the levels taken as the maximum (100 ng/microl) for highly polluted waters. Cd and Pb levels found in water samples were under the detection limits of the methodology used. Pb levels found in sediment samples were higher than Cd.

Benzophenones↗
PubMed2000

[Client categorization: a tool to assess nursing workload].

BACKGROUND: Client categorization is a management tool that allows an objective and structured assessment of the care demands imposed by patients and nursing workloads. AIM: To characterize the need for direct nursing care of patients admitted to a university hospital. PATIENTS AND METHODS: During two months, all patients admitted to intensive, intermediate care units and general services were categorized, their need for nursing care and the time invested by nurses in their care was registered. All patients were classified as maximal, high, median or low risk and as independent, totally or partially dependent on nursing care. Considering four degrees of risk and three degrees of dependency, 12 categories of patients were defined. RESULTS: Patients admitted to intensive care units were of maximal risk and totally dependent and required 1 nurse per 2.2 patients. Those admitted in intermediate care units required 1 nurse per 3.8 patients and those in general services, 1 nurse per 11.5 patients. CONCLUSIONS: Client categorization is a reproducible method that determines a standard measuring unit to define nursing needs. This allows the comparison of workloads between different services within a hospital or between hospitals.

Humans↗
PubMed1999

Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation.

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP-2 [Garcia et al. (1996) J. Neurosci. 16, 7550-7556]. Here, we study whether cAMP-induced phosphorylation modulates the activity of AP-2. Recombinant AP-2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP-2 by PKA, but not the DNA binding activity of AP-2. Cotransfection studies showed that PKA stimulated the effect of AP-2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP-2 activity by PKA-induced phosphorylation of this factor.

Amino Acid Substitution↗
PubMed1999

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical fitting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting configuration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of 'in-gel' protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modifications such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.

Animals↗
PubMed1999

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.

Animals↗
PubMed1998

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides -1017 to +406. Upon a population study, three new polymorphic sites (-491, -427 and -219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites -491 and -219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Adolescent↗
PubMed1998

Lead content in edible wild mushrooms in northwest Spain as indicator of environmental contamination.

Lead content was determined in wild growing mushrooms collected from two different areas in the Province of Lugo (NW Spain). It has been analyzed by graphite furnace atomic absorption spectrometry in 95 samples of 13 species (7 mycorrhizals and 6 saprophites). In an assessment of lead concentrations, the following factors have been considered: species and ecology, morphological portion, and traffic pollution. The average lead concentration of the samples was 1 ppm dry weight (dw). Saprophite species presented higher levels than mycorrhizal ones (< 1 ppm), Coprinus comatus reaching the maximum mean concentration with 2.06 and 2.79 ppm of dw in the hymenophore and the rest of the fruit body. Morphological portion, statistically, did not show significant difference between the two portions; however, Macrolepiota procera always presented lead high levels in the hymenophore in all samples. The effect due to traffic pollution has been specially observed in Coprinus comatus, presenting the highest concentration with values of 6.51 and 10.43 ppm, respectively, in samples collected in the city center. This species, as other researchers have indicated, could be considered as an indicator by lead contamination. The contribution of mushrooms to the weekly intake of lead was calculated and the possible health risk for the consumer is pointed out. These data are of great importance in view of toxicology and partly environmental protection.

Basidiomycota↗
PubMed1998

A polymorphism in the regulatory region of APOE associated with risk for Alzheimer's dementia.

The epsilon4 allele of the apolipoprotein E gene (APOE) has been associated with an increased risk of developing Alzheimer's disease (AD; refs 1,2). However, it is apparent that the APOEepsilon4 allele alone is neither necessary nor sufficient to cause the disease. We have recently found three new polymorphisms within the APOE transcriptional regulatory region (M.J.A. et al., manuscript submitted) and now establish an association between one of these polymorphisms (-491A/T) and dementia as observed in Alzheimer's disease, in two independent clinical populations. The results suggest that homozygosity of a common variant (-491A) is associated with increased risk for AD, and that this association is independent of APOEepsilon4 status. In vitro studies suggest that the -491A/T polymorphism may increase risk for AD by altering the level of ApoE protein expression.

Alleles↗
PubMed1998

Influence of some factors in toxicity and accumulation of cadmium from edible wild macrofungi in nw Spain.

Cadmium content of 97 samples of some edible wild mushrooms, corresponding to 13 different species, was determined by graphite furnace atomic absorption spectrophotometry. Influence of some factors (species and ecology, morphological portion, and traffic pollution) and the importance of mushrooms as a dietary source of this heavy metal have been studied. Saprophite species showed higher levels than mycorrhizal ones (p < 0.001), with some exceptions such as Boletus and Amanita. The hymenophore was always the morphological portion which contained the highest cadmium levels (p < 0.001). Traffic pollution was not a significant factor for the cadmium accumulation in fungi. The samples of Agaricus macrosporus showed the highest mean levels (68.96 and 36.84 ppm dry matter (d. m.) for hymenophore and the rest of the fruit-body, respectively). The average cadmium content of the samples, with the exception of A. macrosporus, was 0.96 for hymenophore and 0.53 ppm d.m. for the rest of the fruit-body; and the range was between 0.18-4.32 and 0.10-1.91 ppm d.m. for hymenophore and the rest of the fruit-body. The concentration levels were compared to literature data and levels set by legislation, and the contribution of mushrooms to the weekly intake of cadmium per person was calculated. The possible health risk for the consumer is pointed out.

Basidiomycota↗