Search PubMed⌕ Search

Biomedical subjects

M Villa

Publications and source records attributed to M Villa.

At least 91 records · Page 5Linked to original sources
PubMed1995

Agonist-induced down-regulation of NMDA receptors in cerebellar granule cells in culture.

In contrast to the acute toxic effect of NMDA on mature cerebellar granule cells, chronic treatment with NMDA (140 microM from 1 to 9 days in vitro) did not compromise cell survival. Such treatment markedly suppressed NMDA receptor activity: at 8 days in vitro NMDA-induced 45Ca2+ influx was reduced by approximately 60% and acute exposure to NMDA (highest concentration tested, 1 mM) at 9 days in vitro did not cause detectable toxicity. The reduction in NMDA receptor activity was accompanied by a significant decrease (approximately 80% at 9 days in vitro) in the level of the NR1 and the NR2A NMDA receptor subunit protein, detected using the selective photoaffinity ligand [125I]CGP55802A. It seems, therefore, that the agonist-induced decrease in NMDA receptor activity is due to receptor down-regulation. In contrast to the marked influence of chronic NMDA exposure on the cellular content of the NMDA receptor subunit proteins, mRNA levels of the different subunits (NR1, NR2A, NR2B and NR2C) were not significantly affected. It seems, therefore, that agonist-induced down-regulation of the NMDA receptor involves critically mRNA translation and/or post-translational regulation.

Animals↗
PubMed1995

Prenatal diagnosis of fragile X syndrome: (CGG)n expansion and methylation of chorionic villus samples.

Fragile X syndrome is the most common form of inherited mental retardation, due to an expansion of the (CGG)n trinucleotide repeat in the FMR-1 gene and hypermethylation of its 5' upstream CpG island. Two major problems remain to be resolved for fragile X prenatal diagnosis: the abnormal methylation patterns of chorionic villus samples (CVS) and the inability to predict the mental status of females with the full mutation. We present here the results of ten prenatal diagnoses of fragile X syndrome using Southern blotting and polymerase chain reaction (PCR) amplification, and the analysis of 50 further CVS to test the methylation status of the CpG island of the FMR-1 gene. In the ten 'at-risk' CVS, eight normal (five males and three females) and two affected male fetuses were detected. Absence of methylation in the CVS was observed in two cases, which was not found upon subsequent examination of the newborn or of fetal tissues. In the 50 CVS not 'at risk' for fragile X syndrome, abnormal fragment patterns for probe StB12.3 were detected in 32 per cent for female and 24 per cent for male fetuses. This abnormal pattern could be due to absent or partial methylation of the CpG island of the FMR-1 gene in chorionic villus tissues.

Blotting, Southern↗
PubMed1995

Interaction of the insecticidal crystal protein CryIA from Bacillus thuringiensis with amino acid transport into brush border membranes from Bombyx mori larval midgut.

The activities of three related Bacillus thuringiensis delta-endotoxins, designated CryIA(a), CryIA(b), and CryIA(c), as inhibitors of K(+)-dependent amino acid transport into membrane vesicles prepared from the anterior and posterior portions of Bombyx mori larval midgut were measured. Under experimental conditions similar to those occurring in vivo (membrane potential approximately -100 mV, inside negative; pH 7.2in/8.8out; an inwardly directed K+ gradient) CryIA(a) toxin produced a clear dose-dependent inhibitory effect on leucine uptake by both the anterior and the posterior gut membrane vesicles, giving half-maximal inhibition constants (IC50) of 2.6 +/- 0.3 and 2.1 +/- 0.2 microgram toxin/mg membrane protein, respectively. The other two delta-endotoxins were practically inactive. A dose-dependent inhibition of amino acid transport by CryIA(a) toxin was also observed on the carrier-mediated K(+)-independent component, i.e., the leucine-only form. This result strongly indicates that the activity of the K+/amino acid cotransporter is directly affected after binding of delta-endotoxin to the brush border membrane. When the extravesicular pH was lowered to pH 7.2, the interaction of CryIA(a) toxin with the brush border appeared more complex, as suggested by the Hill coefficient of the dose-response curves higher than 1. In conclusion, our data indicate that (i) CryIA(a) toxin specifically inhibited K+/leucine symport along the length of the midgut; (ii) the interaction between cotransporters and toxin is affected by the pH of the medium; and (iii) the K+/leucine cotransporter or a strictly associated protein may serve as membrane receptor for CryIA(a) delta-endotoxin in the B. mori larval midgut.

Animals↗
PubMed1995

Regulation of the expression of NMDA receptor subunits in rat cerebellar granule cells: effect of chronic K(+)-induced depolarization and NMDA exposure.

The influence of K(+)-induced membrane depolarization and NMDA treatment on the regulation of NMDA receptor subunit (NR) expression was investigated during the development of granule cells in culture, as a follow-up of previous work on NMDA receptor activity. In spite of the increase in NMDA receptor activity elicited by these treatments (K25 or K10 + NMDA cultures), the main developmental changes in receptor mRNA levels were similar to those in untreated cells (K10) (a threefold increase in total NMDA receptor mRNA, quantitative dominance of NR1 mRNA, late expression of NR2C, and virtual absence of NR2D. However, high K+ and NMDA treatment resulted in a greater increase of NR2A mRNA levels and a retardation in the developmental changes in the relative amounts of NR2B and NR2C mRNAs. The correspondence between NMDA receptor activity and the amount of NR1 and NR2A subunit proteins was excellent, the rank order being K25 > K10 + NMDA > K10 at 9 days in vitro. Because the increase in subunit mRNA was not always paralleled by an increase in subunit protein, the control of NMDA receptor expression involves critically, in addition to gene transcription, regulation of translational and/or posttranslational events.

Affinity Labels↗
PubMed1994

Effect of arginine modification on K(+)-dependent leucine uptake in brush-border membrane vesicles from the midgut of Philosamia cynthia larvae.

The effect of phenylglyoxylation on the midgut K(+)-dependent leucine transport was studied using lepidopteran brush-border membrane vesicles. The inhibition of leucine uptake by phenylglyoxal (PGO) showed a biphasic inactivation pattern. The second-order rate constant for the slow and fast phases were 0.0020 mM-1 min-1 and 0.0091 mM-1 min-1, respectively. However, substitution of borate buffer for Hepes-Tris buffer produced a mono-exponential inactivation pattern, suggesting modification of a single arginine group. The effect of PGO was dose-dependent and the concentration causing half-maximal inhibition of leucine uptake was 5.1 +/- 0.3 mM. Leucine transport was significantly inhibited also in the absence of a potassium electrochemical gradient (i.e., [K+]in = [K+]out = 100 mM), suggesting that inhibition was not related to a decrease in the driving force. Moreover, intravesicular volume remained unchanged after preincubation with PGO. Kinetic analysis of the interaction of PGO with the leucine cotransporter revealed that (i) inhibition was related to a decrease in the Vmax value and (ii) neither leucine nor K+ were able to prevent the inhibition. Our results suggest an important role for arginine residues in the molecular mechanism of K+/leucine cotransport in lepidopteran larvae midgut.

Animals↗
PubMed1993

Region-specific expression of multiple lactase-phlorizin hydrolase genes in intestine of rabbit.

We have identified a total of 4 sequences coding for lactase-phlorizin hydrolase (LPH) in the rabbit. Individual rabbits each yielded three different LPH cDNA sequences, or three chromosomal segments containing exon 1 of the LPH gene, representing either three genes or allelic variants of two genes. The three sequences were found in differing proportions in intestinal mRNA depending on the position along the small intestine from which the RNA was isolated. If all these mRNAs are translated, posttranslational mechanisms previously implicated in the regulation of LPH in the rabbit will be acting on different enzyme species in different parts of the intestine. However, we find no evidence for more than one LPH gene in the rat, and have previously shown that humans have only one LPH gene.

Animals↗
PubMed1993

Body composition in children and Tanner's stages: a study with dual-energy x-ray absorptiometry.

Total body bone mineral content (TBBM), body fat content (BF), and fat-free mass (FFM) were measured in 154 children using dual-energy x-ray absorptiometry (DEXA). Total body calcium level (TBCa) was calculated from TBBM. Children were divided into groups according to Tanner's stages 1, 2, 4, and 5. Children in stage 3 were not included in the study in order to better differentiate between prepubertal and postpubertal individuals. We did not find differences in TBBM, TBCa, BF, and FFM between Tanner's stages 1 and 2 or between sexes. TBBM and TBCa in stages 4 and 5 were lower in girls than in boys (P < .001 and P < .01, respectively); no differences were observed between girls of both groups, although boys showed significant differences (P < .05). FFM for both sexes was lower in stages 1 and 2 than in stages 4 and 5. Girls showed lower FFM (P < .001) than boys in stages 4 and 5; FFM was higher in boys in stage 5 than in those in stage 4 (P < .005), and the same was true for girls (P < .002). Boys in stage 4 had less BF than girls (P < .005), and the same was true for stage 5 (P < .001). Girls in stages 4 and 5 had greater BF than those in stages 1 and 2 (P < .001). These differences suggest that as boys go through puberty, both TBBM and FFM continue to increase, while in girls only BF and FFM increase. These data indicate clear sex differences in somatic postpubertal development.

Absorptiometry, Photon↗
PubMed1993

A rapid and sensitive in vitro assay for the activity of Bacillus thuringiensis delta-endotoxins.

1. Activated delta-endotoxins from Bacillus thuringiensis strains toxic to lepidopteran larvae inhibit K(+)-dependent accumulation of amino acids into brush border membrane vesicles (BBMV) from the midgut of the susceptible species Bombyx mori. 2. The activated toxins interfere with the K(+)-dependent uptake of histidine into BBMV only if they are effectively active in vivo. 3. To calculate IC50 values (the toxin concentration which determines 50% of the effect), dose-response curves were performed for each toxin. The values obtained correlate well with the LD50 determined by bioassay. 4. This amino acid inhibition test could represent a rapid (3-6 hr, compared to 3-4 days for bioassay) and sensitive method for the screening of larvicidal activity of known or new recombinant delta-endotoxins.

Animals↗
PubMed1993

Cellular immune factors associated with mother-to-infant transmission of HIV.

OBJECTIVE: To study a possible correlate of protection in mother-to-infant transmission of HIV infection. In particular, to determine whether lack of HIV-specific T-helper (TH) function as indicated by HIV and non-HIV antigen-stimulated interleukin (IL)-2 production of mother and/or newborn peripheral blood leukocytes (PBL) is associated with mother-to-infant transmission of HIV. METHODS: PBL from 21 HIV-seropositive pregnant women and 23 cord blood leukocytes (CBL) from their offspring were studied for in vitro TH function by IL-2 production in response to HIV and non-HIV antigens. Polymerase chain reaction (PCR) and viral culture assays were performed to determine HIV infection of the infants. RESULTS: PBL from 10 out of 21 (48%) mothers and from eight out of 23 (35%) CBL samples responded to two or more out of five synthetic gp 160 envelope (env) peptides. Three of the 23 (13%) offspring were shown to be HIV-infected by PCR and/or viral culture on follow-up. All three infected infants were from a subset whose CBL did not exhibit env-specific TH immunity. CONCLUSION: Our results demonstrate that fetal T cells can be primed to HIV env determinants in utero, suggest that HIV-specific TH immunity may be protective in newborns, and provide a possible means for identifying newborns who are at risk for HIV infection.

Adolescent↗
PubMed1992

Kinetics of leucine transport in brush border membrane vesicles from lepidopteran larvae midgut.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.

Animals↗
PubMed1992

The expression of lactase enzymatic activity and mRNA in human fetal jejunum. Effect of organ culture and of treatment with hydrocortisone.

Very sensitive procedures were developed for the parallel determination of intestinal lactase (LPH) activity and the cognate mRNA. Between 14 and 20 weeks of gestation, lactase activity is low and varies only slightly; at 37 weeks, a relatively high level of activity is observed. The amounts of LPH mRNA correlates with the enzymatic activity (r = 0.64). Culture of fetal jejunal explants for 5 days induces by itself a 2-fold increase in LPH mRNA, without any significant change in lactase enzymatic activity. This increase may reflect the loss of a negative transcriptional regulation operative in vivo, and suggests an additional post-transcriptional regulatory component. The addition of hydrocortisone (50 ng/ml) during culture induces a doubling of lactase activity without variation in LHP mRNA, indicating a post-transcriptional modulation by hydrocortisone. The intestinal lysosomal acid beta-galactosidase activity was shown to be unaffected by hydrocortisone treatment. This observation clearly illustrates that the two intestinal beta-galactosidases are regulated differently. Our results suggests a complex developmental regulation of human intestinal lactase and that the perinatal increase in lactase activity could be modulated at a post-transcriptional level by hydrocortisone.

Gene Expression Regulation, Enzymologic↗
PubMed1992

Identification of the major degradation products of 4-methoxy-2-(3-phenyl-2-propynyl)phenol formed by exposure to air and light.

Exposure of 4-methoxy-2-(3-phenyl-2-propynyl)phenol (CO/1828) to air and light (accelerated by temperature) yields 1-(2-hydroxy-5- methoxyphenyl)-3-phenylpropynone as the major degradation product. With extraction, this product rapidly degrades to 5-methoxyaurone and 6-methoxyflavone. In addition, a mixture of dimeric and heterodimeric compounds that are not fully identified was observed. These results indicate the formation of a reactive ortho-quinone methide as an unstable intermediate. This hypothesis is supported by evidence that the aurone slowly isomerizes into the flavone in control samples. Identification of compounds was accomplished with mass spectrometry, nuclear magnetic resonance spectrometry, UV-high-performance liquid chromatography, and comparison with authentic samples.

Administration, Topical↗
PubMed1991

The gamma 3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands.

The gamma 3-subunit of the GABAA-receptor in rat brain has been identified by molecular cloning. When co-expressed with the alpha 5- and beta 2-subunits in transfected cells a high potency for GABA (Ka = 4.9 +/- 1.2 microM) and a strong cooperativity in gating the channel (H = 1.9 +/- 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam and reduced by beta CCM. An analogous bi-directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations alpha 1 beta 2 gamma 3 and alpha 3 beta 2 gamma 3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the gamma 3-subunit, as tested with the alpha 3 beta 2- and alpha 5 beta 2-subunit combinations, the gamma 3-subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABAA-receptors. The gamma 3-subunit could functionally replace the gamma 2-subunit with regard to the bi-directional allosteric drug modulation.

Amino Acid Sequence↗
PubMed1991

Increased Na pump activity in the kidney cortex of the Milan hypertensive rat strain.

The (Na+,K+)-ATPase activity from the kidney cortex of the Milan hypertensive rat strain (MHS) and the corresponding normotensive control (MNS) was measured both in active solubilized enzyme preparations and in isolated basolateral membrane vesicles. Kinetic analysis of the purified enzyme showed that the Vmax value was significantly higher in MHS rats. The difference between MHS and MNS was not linked to a different number of sodium pumps, but was related to the molecular activity of the enzyme. Using basolateral membrane vesicles, an increased ATP-dependent ouabain-sensitive sodium transport was also demonstrated in MHS rats. These results support the hypothesis that a higher tubular sodium reabsorption may be involved in the pathogenesis of hypertension in this rat strain.

Animals↗
PubMed1991

Biological effects of magnetic fields.

The literature about the biological effects of magnetic fields is reviewed. We begin by discussing the weak and/or time variable fields, responsible for subtle changes in the circadian rhythms of superior animals, which are believed to be induced by same sort of "resonant mechanism". The safety issues related with the strong magnetic fields and gradients generated by clinical NMR magnets are then considered. The last portion summarizes the debate about the biological effects of strong and uniform magnetic fields.

Animals↗