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M L Garcia

Publications and source records attributed to M L Garcia.

At least 19 recordsLinked to original sources
PubMed2005

Evaluation of home allergen sampling devices.

BACKGROUND: Simple, inexpensive methods of sampling from allergen reservoirs are necessary for large-scale studies or low-cost householder-operated allergen measurement. METHODS: We tested two commercial devices: the Indoor Biotechnologies Mitest Dust Collector and the Drager Bio-Check Allergen Control; two devices of our own design: the Electrostatic Cloth Sampler (ECS) and the Press Tape Sampler (PTS); and a Vacuum Sampler as used in many allergen studies (our Reference Method). Devices were used to collect dust mite allergen samples from 16 domestic carpets. Results were examined for correlations between the sampling methods. RESULTS: With mite allergen concentration expressed as microg/g, the Mitest, the ECS and the PTS correlated with the Reference Method but not with each other. When mite allergen concentration was expressed as microg/m2 the Mitest and the ECS correlated with the Reference Method but the PTS did not. In the high allergen conditions of this study, the Drager Bio-Check did not relate to any methods. CONCLUSIONS: The Mitest Dust Collector, the ECS and the PTS show performance consistent with the Reference Method. Many techniques can be used to collect dust mite allergen samples. More investigation is needed to prove any method as superior for estimating allergen exposure.

Allergens↗
PubMed2005

[Values of GERMCIDE neuropsychological protocol in a sample of normal subjects].

INTRODUCTION: The diagnosis of patients with cognitive deterioration or dementia requires a global approach in which the neuropsychological examination is a key piece. As part of the GERMCIDE study (Group for the Study and Multicenter Registry of Incident Cases of Dementia in Spain), a protocol was designed that included an assessment of the different cognitive functions that are most frequently altered in dementias (memory, orientation, speech, praxis, abstraction capacity and executive function). METHODS: In order to obtain data in normal subjects, this neuropsychological protocol was applied to a group of persons over 50 years without cognitive deterioration or dementia. RESULTS: A total of 103 subjects whose ages ranged from 50 to 95 years (mean: 73.5; SD: 9.3 years); 39 (37.9%) men and 64 (62.1%) women were studied. The mean score on the Mini-Mental State Examination (MMSE) was 27/30 (SD: 2.0). In the speech and praxis tests, 90% of the subjects obtained the maximum value, while performances were more unequal in memory, reasoning and programming. Mean score, standard deviation and distribution in percentages for each subtest are presented. CONCLUSIONS: The values obtained in this sample of normal subjects and their distribution in percentages may be very helpful to facilitate the interpretation of the findings of the neuropsychological examination with the GERMCIDE protocol in the general neurology clinic visits and also in the specialized visits in dementia.

Aged↗
PubMed2003

Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice.

The most common inherited [correct] form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motor neurons, is caused by dominant mutations in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1). In chimeric mice that are mixtures of normal and SOD1 mutant-expressing cells, toxicity to motor neurons is shown to require damage from mutant SOD1 acting within nonneuronal cells. Normal motor neurons in SOD1 mutant chimeras develop aspects of ALS pathology. Most important, nonneuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant-expressing motor neurons.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels.

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

Amino Acid Sequence↗
PubMed2001

Binding of correolide to the K(v)1.3 potassium channel: characterization of the binding domain by site-directed mutagenesis.

Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [(3)H]Dihydrocorreolide (diTC) binds with high affinity to membranes expressing homotetrameric K(v)1.3 channels, and high affinity diTC binding can be conferred to the diTC-insensitive channel, K(v)3.2, after substitution of three nonconserved residues in S(5) and S(6) with the corresponding amino acids present in K(v)1.3 [Hanner et al. (1999) J. Biol. Chem. 274, 25237-25244]. Site-directed mutagenesis along S(5) and S(6) of K(v)1.3 was employed to identify those residues that contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX(1)A19Y/Y37F ([(125)I]HgTX(1)A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutant for both tetrameric channel formation and levels of channel expression. Substitutions at Leu(346) and Leu(353) in S(5), and Ala(413), Val(417), Ala(421), Pro(423), and Val(424) in S(6), cause the most dramatic effect on diTC binding to K(v)1.3. Some of the critical residues in S(6) appear to be present in a region of the protein that alters its conformation during channel gating. Molecular modeling of the S(5)-S(6) region of K(v)1.3 using the X-ray coordinates of the KcsA channel, and other experimental constraints, yield a template that can be used to dock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter to a hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predicted to be determined by the complementary shape between the bowl-shape of the cavity and the shape of the ligand. The conformational change that occurs in this region of the protein during channel gating may explain the state-dependent interaction of diTC with K(v)1.3.

Alanine↗
PubMed2001

Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells.

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.

Animals↗
PubMed2001

Going new places using an old MAP: tau, microtubules and human neurodegenerative disease.

The microtubule-associated protein tau was originally identified as a protein that co-purified with tubulin in vitro, stimulated assembly of tubulin into microtubules and strongly stabilized microtubules. Recognized now as one of the most abundant axonal microtubule-associated proteins, a convergence of evidence implicates an overlapping in vivo role of tau with other axonal microtubule-associated proteins (e.g. MAP1B) in establishing microtubule stability, axon elongation and axonal structure. Missense and splice-site mutations in the human tau gene are now known to be causes of inherited frontotemporal dementia and parkinsonism linked to chromosome 17, a cognitive disorder of aging. This has provided direct evidence for the hypothesis that aberrant, filamentous assembly of tau, a frequent hallmark of a series of human cognitive diseases, including Alzheimer's disease, can directly provoke neurodegeneration.

Animals↗
PubMed2001

Oxidative regulation of large conductance calcium-activated potassium channels.

Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca(2+)-activated K(+) channels (BK(Ca) or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca(2+), the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K(+) channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.

Animals↗
PubMed2001

A rat model of acute lung injury induced by cardiopulmonary bypass.

Impaired lung function is still a major complication after cardiac surgery with cardiopulmonary bypass. The purpose of the present study was to develop an experimental model of acute pulmonary injury induced by cardiopulmonary bypass in Wistar rats. Cardiopulmonary bypass was performed for 60 min using a non-pulsatile roller pump and a membrane oxygenator (n = 8 for cardiopulmonary bypass group and n = 7 for control rats). We measured tracheal pressure, airflow, and lung volume changes and obtained pulmonary resistance and dynamic elastance. After the cardiopulmonary bypass, lungs were submitted to a quick-freezing protocol and morphometric analysis was performed. There was a time-dependent increase in dynamic elastance, but not pulmonary resistance, only in the rats submitted to cardiopulmonary bypass (P = 0.005). Lungs from animals submitted to cardiopulmonary bypass showed significantly more alveolar hemorrhage (P = 0.025) and edema (P = 0.021), as well as perivascular edema (P = 0.003) when compared to control rats. In our experimental model, rats submitted to cardiopulmonary bypass developed acute pulmonary changes similar to the early phase of acute pulmonary distress syndrome. Cardiopulmonary bypass resulted in an increase in pulmonary elastance without changes in resistance. This experimental model is suitable for studies concerning the mechanisms of acute lung injury induced by cardiopulmonary bypass.

Animals↗
PubMed2001

Effects of formaldehyde on the frog's mucociliary epithelium as a surrogate to evaluate air pollution effects on the respiratory epithelium.

The increasing use of alcohol as an alternative fuel to gasoline or diesel can increase emission of formaldehyde, an organic gas that is irritant to the mucous membranes. The respiratory system is the major target of air pollutants and its major defense mechanism depends on the continuous activity of the cilia and the resulting constant transportation of mucous secretion. The present study was designed to evaluate the effects of formaldehyde on the ciliated epithelium through a relative large dose range around the threshold limit value adopted by the Brazilian legislation, namely 1.6 ppm (1.25 to 5 ppm). For this purpose, the isolated frog palate preparation was used as the target of toxic injury. Four groups of frog palates were exposed to diluted Ringer solution (control, N = 8) and formaldehyde diluted in Ringer solution at three different concentrations (1.25, 2.5 and 5.0 ppm, N = 10 for each group). Mucociliary clearance and ciliary beat frequency decreased significantly in contact with formaldehyde at the concentrations of 2.5 and 5.0 ppm after 60 min of exposure (P<0.05). We conclude that relatively low concentrations of formaldehyde, which is even below the Brazilian threshold limit value, are sufficient to cause short-term mucociliary impairment.

Air Pollutants↗
PubMed2000

Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit.

The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.

Animals↗
PubMed2000

beta subunit heterogeneity of L-type Ca(2+) channels in smooth muscle tissues.

Various beta subunit isoforms stabilize different gating properties of voltage-gated L-type Ca(2+) channels. We therefore investigated the expression of Ca(2+) channel beta subunit isoforms in different smooth muscle types on the protein level by immunoblotting and immunoprecipitation employing beta subunit-selective sequence-directed antibodies. From the four known beta subunit isoforms only beta2 and beta3 were detected in porcine uterus, bovine trachea and bovine aorta membranes. Multiple immunoreactive beta2 bands were detected in a tissue-selective manner indicating structural heterogeneity of beta2. Immunoprecipitation of (+)-[(3)H]isradipine-prelabeled channels revealed that beta2 and beta3 participate in Ca(2+) channel formation in uterus and trachea, and beta3 in aortic smooth muscle. We conclude that beta2 and beta3 subunits form L-type Ca(2+) channels in smooth muscle tissues. This subunit heterogeneity may be important to fine-tune channel function.

Animals↗
PubMed2000

Correolide, a nor-triterpenoid blocker of Shaker-type Kv1 channels elicits twitches in guinea-pig ileum by stimulating the enteric nervous system and enhancing neurotransmitter release.

Correolide (1 - 10 microM), a nortriterpene purified from Spachea correae and a selective blocker of Kv1 potassium channels, elicits repetitive twitching in guinea-pig ileum. This effect is not seen in guinea-pig duodenum, portal vein, urinary bladder or uterine strips, nor in rat or mouse ileum. The time course and amplitude of the correolide-induced twitches in guinea-pig ileum are similar to those elicited by electrical stimulation of the enteric nervous system. The correolide-induced twitching is not affected by pre-treatment with capsaicin (1 microM), but is facilitated by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl esther (L-NAME, 200 microM). The correolide-induced twitching is abolished by tetrodotoxin (1 microM) or hexamethonium (100 microM), and is markedly inhibited by nifedipine (0.3 microM) or atropine (0.2 microM). The atropine-resistant component is inhibited by selective antagonists of NK1 and NK2 tachykinin receptors, namely GR 82334 and GR 94800 (1 microM each). The former compound is more effective in inhibiting the correolide-induced, atropine-resistant activity. Correolide intensified the twitching of ileum segments exposed to saturating concentrations of margatoxin (MgTX), which suggests that Kv1 sub-types other than Kv1.1 (Kv1.4 or Kv1.5) are involved in the relatively greater degree of stimulation of the enteric nervous system by correolide, as compared to MgTX. We propose that blockade of Kv1 channels by correolide increases the excitability of intramural nerve plexuses promoting release of acetylcholine and tachykinins from excitatory motor neurons. This, in turn, leads to Ca(2+)-dependent action potentials and twitching of the muscle fibres.

Animals↗
PubMed1999

Plasma membrane calcium ATPases as critical regulators of calcium homeostasis during neuronal cell function.

The plasma membrane calcium ATPases (PMCAs) are ubiquitously expressed proteins that couple the extrusion of calcium across the plasma membrane with the hydrolysis of ATP. In mammals, four separate genes encode distinct PMCA isoforms. Complex patterns of alternative RNA splicing generate additional isoform variability. Functionally, the PMCAs were originally assigned the role of maintaining basal levels of intracellular calcium. Recent evidence, however, is expanding the role of the PMCAs as important participants in dynamic Ca2+ regulation and as crucial players of Ca2+ export during normal and pathological conditions. This review highlights recent advances made on the biology of the PMCAs within the context of neuronal development, cellular responses to external stimuli and cell survival. Particular emphasis is placed on the role of the PMCAs in vestibular and auditory functions, localized calcium signaling in photoreceptor synaptic terminals and calcium-mediated cell death.

Animals↗
PubMed1999

Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.

Amino Acid Sequence↗