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Biomedical subjects

J Sastre

Publications and source records attributed to J Sastre.

At least 127 records · Page 7Linked to original sources
PubMed1996

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Animals↗
PubMed1996

Clinical cross-reactivity between amoxicillin and cephadroxil in patients allergic to amoxicillin and with good tolerance of penicillin.

In recent years, patients allergic to amoxicillin (AX) but with good tolerance of penicillin G (PG) have been described. It has been suggested that the epitope implicated in this type of sensitization might be located on the side-chain of the AX molecule. Thus, cross-reactivity between AX and cephadroxil (CEPH), a cephalosporin which shares an identical side-chain with AX, is suspected. This study aimed to demonstrate clinical cross-reactivity between AX and CEPH in patients allergic to AX and showing good tolerance of PG. In 76 of 576 subjects with suspected allergic reaction to PG and/or AX, the diagnosis of allergy was confirmed. All of these had specific IgE to PG, penicillin V, or AX, and/or positive skin tests to PPL (penicilloyl-polylysine), or MDM (minor determinant mixture), or PG, or AX, and/or positive challenge tests with PG and/or AX. Sixteen subjects (21%) allergic to AX (11 with positive skin test and five with positive challenge test to AX) and good tolerance of PG (all with negative parenteral challenge test) were selected. These 16 patients were subsequently challenged with CEPH (up to 500 mg). Fourteen patients tolerated CEPH, and two (12%) had an immediate allergic reaction. Our study indicates that allergy to the side-chain of aminopenicillins seems to have little clinical relevance in patients with allergic reactions to aminopenicillins but with good tolerance of PG, as 88% of patients with this clinical characteristic tolerate a cephalosporin which shares an identical side-chain. It seems that IgE from most of these patients recognizes an epitope different from the side-chain and the beta-lactam ring.

Adolescent↗
PubMed1996

Identification of a cat eye syndrome using DNA sequence dosage analysis.

We report here the detection of a Cat Eye Syndrome (CES) in a woman who does not exhibit the related phenotype, due to intensive surgery. The analysis of her karyotype reveals a small supernumerary bisatellited chromosome likely to correspond to a fragment of chromosome 13, 15, 21 or 22 on banding analysis. Southern blot of genomic DNA of this patient and her parents hybridized with probes specific of these chromosomes, revealed a DNA amplification of the 22q11 region for the patient, likely to correspond to a CES.

Abnormalities, Multiple↗
PubMed1996

Exercise causes blood glutathione oxidation in chronic obstructive pulmonary disease: prevention by O2 therapy.

The aim of the present study was to determine whether glutathione oxidation occurs in chronic obstructive pulmonary disease (COPD) patients who perform exercise and whether this could be prevented. Blood glutathione red-ox ratio [oxidized-to-reduced glutathione (GSSG/ GSH)] was significantly increased when patients performed exercise for a short period of time until exhaustion. Their resting blood GSSG/GSH was 0.039 +/- 0.008 (SD) (n = 5), whereas after exercise it increased to 0.085 +/- 0.019, P < 0.01. Glutathione oxidation associated with exercise was partially prevented by oxygen therapy (resting value: 0.037 +/- 0.014, n = 5; after exercise: 0.047 +/- 0.016, n = 5, P < 0.01). We conclude that light exercise causes an oxidation of glutathione in COPD patients, which can be partially prevented by oxygen therapy.

Carbon Dioxide↗
PubMed1995

A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc- (50-60%) and the gamma-glutamyl cycle. Our results provide the first evidence linking gamma-GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress.

Adenosine Triphosphate↗
PubMed1995

L-cysteine and glutathione metabolism are impaired in premature infants due to cystathionase deficiency.

There are conflicting reports in the literature as to whether L-cysteine is an essential amino acid in premature infants as the result of the absence of hepatic cystathionase activity. To analyze the physiological importance of the cystathionase deficiency, we studied sulfur amino acid metabolism in human neonates of different gestational ages. Plasma cystathionine concentrations are higher in premature infants < or = 32 wk gestation (group 1) than in premature infants of 33-36 wk gestational age (group 2) or in full-term infants (group 3), whereas plasma cysteine concentrations are much lower in group 1 and 2 premature infants than in mature infants. Furthermore, erythrocytes from group 1 premature infants synthetize glutathione from L-methionine (a process dependent on the cystathionase pathway) at a much lower rate than do erythrocytes from group 2 premature or full-term infants. Thus, the metabolic flow through the transsulfuration pathway may be insufficient to meet the glutathione and cysteine requirements of very premature infants.

Amino Acids, Sulfur↗
PubMed1994

A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples.

A high-performance liquid chromatography method to determine oxidized glutathione (GSSG) in biological samples with ultraviolet-visible detection using N-ethylmaleimide to prevent reduced glutathione (GSH) oxidation is described. Previous methods based on high-performance liquid chromatography to quantitative GSH and GSSG are unsuitable for determining GSSG in biological samples. This is due to GSH oxidation during sample processing. N-Ethylmaleimide, but not iodacetic acid, prevents this oxidation. Blood GSH oxidation measured by the widely used method of Reed et al. (Anal. Biochem. 106, 55-62, 1980) can be as high as 24 +/- 6% (n = 6). When blood samples were assayed by our procedure, GSH oxidation was only 0.13 +/- 0.28% (n = 5). GSH can be determined enzymatically, i.e., with glutathione-S-transferase, but perchloric acid should not be used to deproteinize samples. Trichloroacetic acid (15% final concentration) may be used. This method allows an accurate calculation of the GSH/GSSG ratio, which is important for determining oxidative stress in tissues in various pathophysiological situations.

Animals↗
PubMed1994

Antioxidant administration to the mother prevents oxidative stress associated with birth in the neonatal rat.

In the fetal-to-neonatal transition, important circulatory and respiratory changes ensue which lead to oxidative stress evidenced by changes in glutathione status. Administration of N-Acetyl-Cysteine (NAC), a glutathione precursor, to the mother might be a rational approach to protect the fetus against oxidative stress. We have found that NAC administration to pregnant rats partially prevents the change in hepatic GSSG that occurs in the fetal-neonatal transition: GSSG increased 11-fold (from 1 to 12 nmol/g) in controls and less than two-fold (from 5 to 9 nmol/g) in animals exposed to NAC in utero. The GSH/GSSG ratio in liver of NAC-treated newborns was 411 +/- 216 and in liver of controls it was 283 +/- 176. Thus, the oxidative stress that occurs in the fetal-to-neonatal transition is partially prevented by oral NAC administration.

Acetylcysteine↗
PubMed1994

Influence of erythropoietin on paradoxical responses of growth hormone to thyrotropin-releasing hormone in uremic patients.

Several alterations in growth hormone (GH) secretion have been reported in patients with chronic renal failure. The aim of the present report has been to assess the effect of acutely administered recombinant human erythropoietin (rHuEPO) infusion on GH responses to thyrotropin-releasing hormone (TRH) in uremic patients. Twelve male patients (mean age 46.2 years, range 24 to 69) were studied. Seven of them were on continuous ambulatory peritoneal dialysis (CAPD), two on chronic hemodialysis (HD) and two in pre-dialysis (PreD). None had been treated before with rHuEPO. Each patient was tested with TRH (400 micrograms i.v. in bolus), and with TRH plus rHuEPO (40 U/kg in constant infusion for 30 min) on different days. TRH administration provoked a paradoxical response of GH (peak > 5 micrograms/liter) in nine (5 CAPD, 2 HD, 2 PreD) out of 12 patients. In this group of patients with anomalous GH responses, rHuEPO infusion produced an abolishment of the paradoxical responses (GH peak < 5 micrograms/liter) in eight patients and a marked decrease in a further one. On the contrary, in patients with no paradoxical GH response, stimulation with TRH plus rHuEPO did not induce any change in GH release compared with that observed after TRH alone. rHuEPO had no effect on TRH-induced thyrotropin release. These results suggest that the paradoxical GH response to TRH in patients with chronic renal failure is blocked by rHuEPO administration. This rHuEPO action might be mediated by an increased release of somatostatin or an inhibited GH-releasing hormone secretion.

Adult↗
PubMed1994

In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects.

The effect of in vitro glutathione (GSH) supplementation on mitogenic response, interleukin-1, interleukin-2 and prostaglandin E2 production, and cellular GSH level in peripheral blood mononuclear cells (PBMC) from healthy young and old human subjects was studied. In vitro addition of GSH increased cellular GSH level (P < 0.001). Glutathione supplementation at concentrations between 2 to 10 mmol/L enhanced lymphocyte proliferation but at low concentrations (0.5 and 1 mmol/L) decreased mitogenic response. Glutathione-induced enhancement of lymphocyte proliferation due to phytohemagglutinin or concanavalin A was greater in the PBMC from old subjects than in those from young subjects. At optimal concentration (5 mmol), GSH increased interleukin-2 production (P < 0.05) and decreased prostaglandin E2 and leukotriene B4 production (P < 0.01) in both age groups. Furthermore, decreased PBMC mitogenic response by in vitro addition of prostaglandin E2 was reversed by GSH supplementation. Glutathione did not have an effect on interleukin-1 production by PBMC from young subjects; however, GSH supplementation tended (P = 0.08) to increase interleukin-1 production by PBMC from old subjects. We conclude that GSH supplementation enhances T cell-mediated mitogenic response in young and old subjects. This effect is due at least in part to decreased eicosanoid production.

Adult↗
PubMed1994

Dose-response characteristics of galactose-induced cataract in the rat.

The onset and progression of cataract was investigated in weanling Sprague-Dawley rats fed 10, 15, 20 and 30% dietary galactose (groups 1-4) for 45-226 days. Cataracts were graded on a 0-5 scale. After 226 days, 9% of the rats fed 10% galactose developed lesions beyond the very early stage of cataractogenesis (grade 1). After 154 days 50% of the rats fed 15% galactose developed subcortical cataract (grade 3) with no nuclear cataract. In the rats fed 20% galactose, an initial rapid development by 31 days of a grade 3 cataract was observed in 50% of the eyes. Advancement to grade 4 and grade 5 cataract proceeded more slowly; by 207 days, 45% of the eyes had grade 5 cataract. In rats fed 30% galactose, rapid vacuolization and development of nuclear cataracts (grade 5) were observed by day 44. Dietary galactose at levels of 20 and 30% was associated with a significant reduction in weight gain at the early stage of dietary treatment. These observations demonstrate that 10-30% dietary galactose induces cataract in rat lens in a dose- and time-dependent fashion. These data serve to further establish this animal model as a useful model for studying the sequelae of cataractogenesis.

Animals↗
PubMed1994

The human hepatoma cell line, HepG2, secretes functional cholinesterase.

Confluent monolayers of the human hepatoblastoma-derived cell line, HepG2, were incubated in serum-free medium. This medium was harvested and concentrated. Catalytic activity and immunoblotting of concentrated medium revealed the presence of a functional cholinesterase. This cell line may be an useful model for investigating the regulation and physiological role of the enzyme secreted by liver.

Carcinoma, Hepatocellular↗
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↗
PubMed1994

Growth hormone and cortisol secretion before and after erythropoietin therapy in CAPD patients.

Correction of anemia with recombinant human erythropoietin (rHuEPO) in patients with end-stage renal disease (ESRD) has been associated with improvement of several endocrine abnormalities. However, long-term effects of this therapy on pituitary secretion in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) is not known. The aim of the present work was to assess the growth hormone (GH) and cortisol responses to insulin-induced hypoglycemia before and after the correction of anemia with rHuEPO therapy in CAPD patients. Five well-nourished and clinically stable patients were studied before and after 12-24 weeks of rHuEPO treatment. Seven normal volunteers were studied as controls. Insulin-induced hypoglycemia tests were performed prior to starting rHuEPO therapy and again after partial correction of anemia. Blood samples for GH, cortisol, and glucose were collected between -30 and 120 min after insulin (0.1 U/kg bw) administration. GH responses to hypoglycemic stress were characterized by marked differences in single patients when compared with the control group. However, the GH peak and the area under the secretory curves (AUC) of GH responses in CAPD patients (10.6 +/- 4.8 micrograms/L and 15.4 +/- 6.1 micrograms h/L, respectively) did not differ from those obtained in control subjects (14.3 +/- 4.1 micrograms/L and 19.4 +/- 3.5 micrograms h/L, respectively). The study, after correction of anemia, showed an evident decrease of GH values at each time point on the response curve in 4 patients, and no modification in 1 patient. Mean values of GH peak and AUC were 8.8 +/- 2.8 micrograms/L and 9.6 +/- 2.3 micrograms h/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[Self-induced Cushing's syndrome due to dexamethasone abuse in nasal spray: clinical and biochemical study].

We studied a 55-year-old woman with remarkable cushingoid features, which presented biochemical evidence of suppression of the hypothalamus-hypophysis-adrenal axis. In the detailed anamnesis, we discovered that the patient had used during more than two years a nasal spray containing dexamethasone. In the initial assessment of the suprarenal function, in addition to suppressed concentrations of cortisol and ACTH, we observed the absence of response to acute stimulus (insulinic hypoglycemia and short ACTH test), as well as a normal response to sustained ACTH stimulus. After discontinuing the intranasal administration of dexamethasone, the patient remained asymptomatic; the basal concentrations of ACTH and cortisol, as well as the levels of urinary free cortisol, were normal at 3 months and the responses of cortisol and ACTH to their stimulus were restored to normal levels at 6 months. We conclude that in the cases of Cushing's syndrome with suppression of the hypothalamus-hypophysis-adrenal axis, it is necessary to conduct a detailed investigation on the consumption of glucocorticoids through unusual ways. The presence of a normal response to the sustained stimulus with ACTH may be a quick recovery index of the suprarenal function.

Adrenocorticotropic Hormone↗