Search PubMed⌕ Search

Biomedical subjects

E Alonso

Publications and source records attributed to E Alonso.

At least 73 records · Page 4Linked to original sources
PubMed1995

Affinity cleavage of carbamoyl-phosphate synthetase I localizes regions of the enzyme interacting with the molecule of ATP that phosphorylates carbamate.

Two ATP molecules are used in the reaction catalyzed by carbamoyl-phosphate synthetase I. One molecule (ATPA) phosphorylates HCO3- and the other (ATPB) phosphorylates carbamate. Carbamoyl-phosphate synthetase I is a 160-kDa polypeptide consisting of a 40-kDa N-terminal moiety and a 120-kDa C-terminal moiety, the latter being composed of two similar halves of molecular mass 60 kDa. We showed [Alonso, E., Cervera, J., García-España, A., Bendala, E. & Rubio, V. (1992) J. Biol. Chem. 267, 4524-4532] that Fe.ATP bound at the site for ATPB catalyzes the oxidative inactivation of carbamoyl-phosphate synthetase I in a model oxidative system consisting of Fe3+, ascorbate, and O2, and we detected ATP-promoted oxidative cleavage of the enzyme. We now provide further evidence indicating that this cleavage is catalyzed by bound Fe.ATPB, and we demonstrate that the enzyme is cleaved at seven points, which we identify as residues 1002, 1064, 1083, 1128, 1200, 1242, and 1270. All these cleavage points are confined within and distributed throughout the more N-terminal 40-kDa region of the C-terminus of the 120-kDa moiety. Thus, this 40-kDa region contains the ATPB site, is folded as a globular domain with the polypeptide recurring several times towards the nucleotide, and appears to be a modular unit equivalent to carbamate kinase, with full responsibility for ATPB binding and carbamate phosphorylation. The present results and our previous demonstration [Rodríguez-Aparicio, L., Guadalajara, A.M. & Rubio, V. (1989) Biochemistry 28, 3070-3074] of the binding of N-acetyl-L-glutamate in the C-terminal 20-kDa region, strongly support the idea that each homologous half of the 120-kDa moiety of carbamoyl-phosphate synthetase I is composed of a 40-kDa ATP-binding domain and a 20-kDa domain that, in the carboxyl half, is the regulatory domain.

Adenosine Triphosphate↗
PubMed1995

Differential in vitro DNA binding activity to a promoter element of the gn1 beta-1,3-glucanase gene in hypersensitively reacting tobacco plants.

In a hypersensitive reaction to pathogen infection, expression of the beta-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5'-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the beta-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to -138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from -250 to -217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.

Base Sequence↗
PubMed1995

[Juvenile polyposis in 2 patients of the same family].

Two cases of juvenile familiar polyposis (one 40-year-old male and his 14-year-old daughter) are presented. The girl presented intermittent rectal bleeding since the age of 10 with growth retardation and chronic anemia. The child was treated by colectomy with more than 100 juvenile polyps without adenomatous alterations being observed in the surgical specimen. Juvenile gastric polyps were also endoscopically observed. The father underwent sigmoidectomy for a adenomatous polyp of this localization. Later review of the surgical specimen demonstrated a juvenile polyp with zones of adenomatous alterations. The diagnostic difficulties of this rare entity and the therapeutic options available are discussed.

Adenomatous Polyposis Coli↗
PubMed1995

Clinico-pathological correlation in dementias.

The object of this study is to investigate whether or not there are clinical signs and symptoms in patients with dementia that, by themselves or jointly, can be associated with the pathological diagnosis of Alzheimer's disease. Twelve patients with dementia were studied, in whom the clinical diagnosis of Alzheimer's disease was made according to established criteria. A sample of leptomeninges, cortex and subcortical white matter was obtained from each patient and was processed for light and electron microscopy. In the cases in whom neuritic plaques and neurofibrilary tangles were present, pathological changes were quantified. The diagnosis of Alzheimer's disease was confirmed in 5 cases, whereas in 3 patients spongiform encephalopathy was present. In the remaining patients, the number of neuritic plaques was within normal limits for the age of the subjects. Comparison of the data in Alzheimer (n = 5) and non-Alzheimer (n = 7) groups showed an increased, statistically significant incidence of acalculia, abnormalities of judgment, impairment of abstraction and primitive reflexes in the former. Although good fitting models were obtained, none achieved perfect discrimination. The model that included alterations of judgment and acalculia gave the best fit.

Aged↗
PubMed1994

Effect of theophylline on airway responsiveness to methacholine and on exercise-induced bronchoconstriction.

The aim of this study was to compare the relative effect of (1) a single dose of a rapid release theophylline, (2) treatment for 1 week with a sustained release theophylline, and (3) placebo after a washout period of at least 2 weeks on bronchial hyperresponsiveness to methacholine and on exercise-induced bronchoconstriction in 14 mildly asthmatic children. A dose of rapid release theophylline increased geometric mean (GM) PC20 (mg/mL) for methacholine from 0.25 to 0.85 (P < .001) at a mean (+/- SD) serum theophylline concentration of 9.5 +/- 2.5 mg/L (52.3 +/- 14.24 mumol/L). After 1 week on a slow release theophylline, GM PC20 increased to 0.41 (P < .01) at a mean serum theophylline concentration of 13.63 +/- 4.7 mg/L (74.9 +/- 25.8 mumol/L), (P < .01) compared with rapid release theophylline. The difference in PC20 between both theophylline treatments was statistically significant (P < .05). No significant changes in PC20 were noted after placebo compared with baseline values. During baseline and after placebo mean maximum FEV1 fall (MMFF) after exercise was 40 +/- 19.9% and 31.7 +/- 15.5%, respectively (n.s.). After a dose of rapid release theophylline, MMFF on exercise test was 8.7 +/- 8.2% (mean serum theophylline concentration 10 +/- 3.2 mg/L (55.4 +/- 17.82 mumol/L)), P < .001 compared with baseline and placebo. After 1 week of treatment with slow release theophylline, MMFF was 26.2% +/- 18.3 (mean serum theophylline concentration 12.2 +/- 3.9 mg/L (67.43 +/- 21.5 mumol/L)) (P = .01 and P = .30 compared with baseline and placebo, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Determination of thiabendazole residues in waters by solid-phase spectrofluorometry.

A new, sensitive and simple spectrofluorometric method for the determination of thiabendazole (TBZ) residues was studied. TBZ on Sephadex G-15 gel, pH 4.70 acetate buffer, is fixed to give fluorescence. The fluorescence of the gel-TBZ system, packed in a 1-mm quartz cell was measured directly using a solid-surface attachment. The applicable concentration range was 1.5-10.0 ng.mL-1 with a relative standard deviation of 1.4% and a detection limit of 0.1 ng.mL-1. The method was applied to the determination of thiabendazole in natural, waste, sea, and residual waters (98-100% recovery). The method is more sensitive and selective than other methods described in the literature. The presence of other fluorescent pesticides (dichlone, carbaryl, morestan, o-phenylphenol) with similar spectral features (lambda exc and lambda em neighboring to excitation and emission wavelengths of the TBZ) did not cause interference.

Hydrogen-Ion Concentration↗
PubMed1993

Rapid karyotyping in ectopic pregnancies.

The aim of this study was to determine the feasibility of karyotyping ectopic Fallopian tube pregnancies utilizing dividing cytotrophoblast cells. Villi from 78 ectopic conceptuses were processed by the direct chromosome technique and cytogenetic diagnosis was successful in 60 cases (76.9 per cent). The amount of villi obtained, as well as villus morphology, was correlated with cytogenetic success rate. Histological examination of the Fallopian tube was also carried out. A total of 47 cases were chromosomally abnormal (78.3 per cent), which is the highest frequency of cytogenetic abnormalities reported to date.

Chorionic Villi Sampling↗
PubMed1993

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses.

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3' non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3' non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed.

Amino Acid Sequence↗
PubMed1993

[Transitional carcinoma of the bladder. Evaluation of prognostic factors].

This study evaluates prognostic factors in 25 patients with transitional cell carcinoma (TCC) of the bladder with a minimum follow up of two years. Histologically there were 12 patients with grade I tumors according to the Mostofi's classification, 10 patients with grade II lesions and 3 grade III. The course of the grade I and II tumors was similar and the grade III worse. According to stage, there were 11 patients stage 0, 8 stage A, 3 stage B and 3 stage C. Patients with all stages recurred but only those with stage B and C died of their disease. In 18 patients the expression of the beta subunit of HCG and of the blood group isoantigens was demonstrated by immunohistochemistry. There were 8 negative cases and 10 positive for the beta subunit of HCG and 10 negative and 8 positive for the blood group isoantigens. DNA analysis was done with an image analyzer on cytological smears of the urine in the 25 cases after decolorization of the smears and Feulgen staining. There were 5 diploid and 25 aneuploid cases. The DNA malignancy grade was determined with a range between 0.100 and 2.689 and the number of events more than 5c (5N). The presence of HCG, aneuploid tumors, a malignancy grade higher than 0.500 and the presence of events more than 5c were statistically significant unfavorable prognostic factors. The presence of blood group isoantigens was associated with a better evolution of the tumor but did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell↗
PubMed1992

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Adenosine Triphosphate↗
PubMed1992

Allergens from rye pollen (Secale cereale). I. Study of protein release by rye pollen during a 19-hour extraction process. Allergen identification.

We have studied the proteins and allergens released by rye pollen in the course of a 19-h pollen incubation process. Nearly 40% of the total extracted proteins were collected during the first 5 min, and most of them had a molecular weight less than 28 kDa. Between 5 and 30 min, 15% of the proteins from total extract were released, showing in the SDS-PAGE analysis an increase in which components moved close to 30 kDa standard. From 30 min to 19 h several extracts were collected. Electrophoretical profile of components from these extracts reveals that bands moving below 28 kDa were practically absent and those of 28 and 23 kDa became very intense. At the end of the process there was a rise of 67 kDa proteins. Dot-immunobinding and immunoblotting techniques reveal that allergens leave the rye pollen, for the most part, after 5 min incubation and are proteins with 28 kDa, 33 kDa, 48 kDa and 67 kDa molecular weights.

Allergens↗
PubMed1992

Allergens from rye pollen (Secale cereale). II. Characterization and partial purification.

Rye pollen was incubated for 30 min and proteins extracted at this time were collected as extract A (EA). The same pollen grains were resuspended in buffer and incubated for 18.5 h. Proteins extracted in this period were designated extract B (EB). Both extracts were subfractionated by DEAE ion-exchange chromatography and allergen presence in peaks detected by the dot-immunobinding technique. The results reveal that unretained proteins (peaks 1 and 2) and proteins eluted at 0.2 M NaCl from extract B contain the highest proportion of allergens. SDS-PAGE of chromatographic peaks showed that peak 2 from extract B contains a highly purified 28 kDa band. On the skin of allergic patients this band gave a stronger positive prick test than for the crude extract.

Allergens↗
PubMed1991

Influence of anions on the activation of carbamoyl phosphate synthetase (ammonia) by acetylglutamate: implications for the activation of the enzyme in the mitochondria.

Rat liver carbamoyl phosphate synthetase is shown to be inhibited by anions competitively with acetylglutamate (the allosteric activator of the enzyme) with a potency decreasing in the order NO3- greater than SO4(2-) greater than Cl- approximately HCO3-. Inhibition by chloride accounts for most of the inhibition reported [Lund, P., and Wiggins, D. (1987) Biochem. J. 243, 273-276] in Tris buffer. Mes, acetate, and isethionate give little or no inhibition and phosphate inhibits noncompetitively. Plots of the KA value for acetylglutamate versus the concentration of chloride or nitrate are curved upward and binding assays demonstrate that the inhibitory anions displace acetylglutamate from the enzyme. Thus, the anions may compete with the carboxyls of acetylglutamate for positive charges at the binding site. Of the organic anions found in the mitochondrial matrix, alpha-ketoglutarate, malate, succinate, and citrate increase substantially the KA for acetylglutamate. Changes in the concentrations of ATP, HCO3-, NH4+, and Mg2+, and high concentrations of protein (60 mg/ml serum albumin) influence the KA value. Changes in the concentration of the enzyme have no effect. Under assay conditions approaching the ionic, buffer, and substrate concentrations expected to occur in the mitochondrial matrix, the KA value for acetylglutamate is 27 microM and the Vmax is decreased about 50%. These results indicate that physiological changes in the level of acetylglutamate significantly influence the degree of activation of carbamoyl phosphate synthetase in vivo.

Animals↗
PubMed1991

N-acetyl-L-glutamate in brain: assay, levels, and regional and subcellular distribution.

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200-300 times lower than the levels of N-acetyl-L-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 micrograms/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-L-aspartyl-L-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.

Animals↗