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Biomedical subjects

A Fernandez

Publications and source records attributed to A Fernandez.

At least 199 records · Page 11Linked to original sources
PubMed1990

Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction.

Attention has focused on the regulation of the eucaryotic cell division cycle since the protein kinase p34cdc2 was identified as a key enzyme in mitotic induction. The level of this kinase remains constant throughout the cell cycle but its activity alters, particularly before M phase. Although the factors regulating cdc2 activity are still unknown, there is increasing evidence that it is influenced by p34cdc2 dephosphorylation. Protein phosphatase inhibitor-2 (I2) is a specific inhibitor of phosphatase type-1, which with type-2A is one of the two principal Ser(P) and Thr(P) phosphatases. Here we show that the level of I2, assayed by immunofluorescence staining, activity measurements, western immunoblotting and metabolic labelling, oscillates during the cell cycle in rat fibroblasts, peaking at S phase and mitosis. Moreover, when we inhibited I2 in vivo by microinjection of anti-I2 antibodies in S-phase cells, the pseudo-mitotic cellular response to injected p34cdc2 was restored, indicating that I2 might have a role in the modulation of p34cdc2 activity.

Animals↗
PubMed1990

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts.

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.

Actin Cytoskeleton↗
PubMed1990

ras-induced c-fos expression and proliferation in living rat fibroblasts involves C-kinase activation and the serum response element pathway.

We have examined the early events involved in the proliferative activation of quiescent rat embryo fibroblasts by microinjection of oncogenic ras protein. Cells injected with ras show a transient expression of c-fos after 30-60 min visualized by immunofluorescence in the nucleus. This c-fos expression can be specifically suppressed by coinjection of a double-stranded oligonucleotide which corresponds to the serum response element (SRE) present in the c-fos promoter, implying that ras utilizes a pathway which activates the binding of serum response factor(s) (SRF) to SRE to induce c-fos transcription. Inhibition of this pathway also abolished ras-induced DNA synthesis indicating that the proliferative induction by ras requires expression of SRE-regulated genes. Both c-fos induction and DNA synthesis were prevented when ras oncoprotein was injected into quiescent cells together with either antibodies against calcium phospholipid-dependent protein kinase (C-kinase) or a synthetic peptide that specifically inhibits C-kinase. These data demonstrate the involvement of both functional C-kinase and the SRE pathway in the activation of quiescent cells by ras and suggest a potential relationship in their mechanism of action.

Animals↗
PubMed1990

Psychiatric disorders of abused women at a shelter.

Thirty physically abused women were randomly selected from the population of a local women's shelter and evaluated by psychiatric interview and psychiatric rating scales. High prevalences of major depression disorder (37%) and PTSD (47%) were determined. Furthermore, these disorders were found to be positively associated. These results suggest the need for immediate availability of psychiatric services at such shelters along with further study of their populations and possible intervention strategies.

Adult↗
PubMed1990

Dietary modulation of small intestinal glutamine transport in intestinal brush border membrane vesicles of rats.

The effects of a glutamine-enriched diet on the transport of glutamine across brush border membrane vesicles (BBMV) from the rat jejunum were studied to gain further insight into the effects of diet on regulating gut glutamine utilization. Following fasting, rats were randomized to one of three nutritionally complete elemental diets supplemented with glutamine, glutamate, or glycine (control). Brush border membrane vesicles were prepared by a Mg2+ aggregation/differential centrifugation technique and uptake of radioactive [3H]glutamine by the BBMV was studied using a rapid mixing/filtration technique. BBMVs from all test diet groups were enriched in alkaline phosphatase 14-fold. [3H]Glutamine uptake courses for all groups demonstrated sodium dependency, overshoots, and similar 2-hr equilibrium values. Vesicles from animals fed the glutamine-enriched diet had a 75% increase in glutamine uptake compared to those of the control diet and a 250% increase compared to those of the glutamate-enriched diet (P less than 0.05). alpha-Methylamino isobutyric acid and glycine did not significantly inhibit total [3H]glutamine uptake, whereas asparagine and glutamine inhibited total [3H]glutamine uptake compared to the mannitol control. The brush border appears to possess the glutamine selective System N transporter, the activity of which can be stimulated by providing dietary glutamine.

Amino Acids↗
PubMed1990

PAF-induced airway responses in sheep: effects of a PAF antagonist and nedocromil sodium.

Platelet-activating factor (PAF) is a potent inflammatory mediator that can cause bronchoconstriction and airway hyperresponsiveness in selected human subjects and animals. The mechanism by which PAF induces these changes is not clearly understood. We therefore studied the effects of intratracheal instillation of PAF (30 micrograms/kg) on airway resistance and airway responsiveness in allergic sheep (n = 7) and attempted to modulate these effects with the specific PAF antagonist, WEB-2086, and the antiasthmatic agent, nedocromil sodium (NED). Specific lung resistance (SRL) was measured to assess bronchial responses to PAF, and airway responsiveness was determined by deriving a provocative dose of carbachol in breath units causing an increase in SRL to 4 L times centimeters of H2O per liters per second (PD4) from carbachol dose-response curves. PAF instillation increased mean +/- SD SRL to 228 +/- 134% above baseline. Two to 4 hours after PAF instillation, PD4 decreased by 55 +/- 9% from a baseline of 39 +/- 9 breath units (p less than 0.05). Airway responsiveness remained increased at 24 hours but returned to baseline by 48 hours. Pretreatment with WEB-2086 (3 mg/kg, intravenously) or NED (1 mg/kg, nebulized) blocked (p less than 0.05) PAF-induced bronchoconstriction and PAF-induced airway hyperresponsiveness. Instillation of lyso-PAF (30 micrograms/kg) did not cause bronchoconstriction or airway hyperresponsiveness. Thus, instilled PAF causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by a receptor-mediated mechanism that likely involves the release of secondary mediators, the latter process being sensitive to NED.

Aerosols↗
PubMed1990

Changes in the phospholipid and fatty acid composition in normal erythrocytes from sheep of different ages. Aminophospholipid organization in the membrane bilayer.

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.

Aging↗
PubMed1990

Immunohistological detection of bovine viral diarrhoea virus antigen in the central nervous system of persistently infected cattle using monoclonal antibodies.

In a total of 25 cattle persistently infected with bovine viral diarrhoea virus (BVDV) the distribution of viral antigens in the central nervous system was studied. Using a panel of monoclonal antibodies (anti pestivirus C16; anti cytophathic BVDV C38; anti cytopathic and non-cytopathic BVDV C42; anti gp53 BVDV CA-1 and CA-3) and the indirect immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus. Morphological cellular alterations were not seen. Reactive perivascular lymphocytic infiltrations were occasional findings.

Animals↗
PubMed1990

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-135 degrees C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10(-19) J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na(+)-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na(+)-dependent cotransporter activity Jmax without affecting apparent Km. The size of the transporting functional unit was 290 +/- 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 +/- 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.

Animals↗
PubMed1990

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.

Actin Cytoskeleton↗
PubMed1990

Bronchodilators in patients with chronic obstructive pulmonary disease on mechanical ventilation. Utilization of metered-dose inhalers.

Bronchodilators are used widely in the acute exacerbations of chronic obstructive pulmonary disease (COPD), although their effectiveness is not clearly established. We used three bronchodilators in 20 patients with COPD who were being mechanically ventilated. Two of the bronchodilators, ipratropium bromide and salbutamol, were administered from metered-dose inhalers (MDI) through an adapter to the endotracheal tube, and the third, aminophylline, was administered in the form of intravenous infusion. Before administering each drug, peak airway pressure, end-inspiratory pressure, resistive pressure, and auto positive end-expiratory pressure (auto-PEEP) were measured, and inspiratory resistance (Rins) and compliance were calculated. Heart rate (HR) and blood pressure were also recorded, and arterial pH and blood gas determinations were made. These measurements were repeated 60 min after the administration of aminophylline, 15 and 60 min after administering salbutamol, and 30 and 60 min after administering ipratropium bromide. With these three drugs, airway pressures were reduced, as well as auto-PEEP and Rins, with respect to basal values (p less than 0.05). The changes in compliance were only significant with salbutamol (p less than 0.05). HR was only significantly modified with aminophylline (p less than 0.05). No blood gas change was observed with any of the three drugs. It can be concluded that: (1) the three drugs used in this study were equally effective in producing significant bronchodilation in patients on mechanical ventilation for severe acute exacerbation of COPD; (2) the administration of bronchodilators by MDI in intubated patients through a special adapter was as effective as the intravenous administration of aminophylline.

Aged↗
PubMed1990

Long-term treatment with Madopar HBS in parkinsonians with fluctuations.

1. Of 22 parkinsonians with fluctuations under long-dating dopatherapy in whom standard Madopar was substituted by the HBS form, 16 who performed the trial longer than 1 year were particularly studied to evaluate some parameters of this long-term follow-up (30-36 months). 2. The outstanding beneficial effects were an enhancement of the antiparkinsonian response, improvement or disappearance of motor oscillations, longer "on" periods, less severe "off" periods, and a more sustained nocturnal antiparkinsonian effect with a reduction of dystonias and pain at night and decreased or absent early morning parkinsonism. 3. The decrease or disappearance of dystonia was observed since the early stages of the trial and can be explained by the more sustained dopaminergic effect. 4. Surprisingly, dyskinesias also decreased in spite of the higher dopaminergic effect. The avoidance of sharp and repeated plasmatic peaks and the lower levels of L-dopa under HBS could explain this phenomenon. 5. The negative aspects of Madopar HBS are a lower bioavailability that means a dosage increase and a longer latency for the therapeutic response in the morning. 6. The dosage increase went up by 80% to 100% in relation to standard Madopar during the long-term treatment. 7. As Madopar HBS is a sustained release preparation, we had to increase the initially reduced the number of intakes, again in order to obtain better results. In the most severe cases with poor or absent response, benefits were achieved only when administering the HBS intake every hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1990

Bakers' asthma: prevalence and evaluation of immunotherapy with a wheat flour extract.

One hundred thirty-nine bakers and pastry cooks were included in a prevalence study of IgE-mediated hypersensitivity to wheat flour demonstrated by skin tests, specific IgE to wheat flour (RAST), and inhalation challenge. From the sensitized workers, we selected 30 asthmatic patients. Twenty patients were treated with a standardized wheat flour extract, and ten with a placebo in a double-blind clinical trial. Before and after immunotherapy we performed tests in vivo (skin tests with wheat flour and methacholine tests), and in vitro (total IgE and specific IgE to wheat flour). We found substantial prevalence of wheat flour allergy (25.17% of workers), and a significant decrease (P less than .001) in hyperresponsiveness to methacholine, skin sensitivity (P = .002), and specific IgE (P less than .005) to wheat flour after 20 months of immunotherapy. There was also significant subjective improvement (P less than .001). The placebo group showed no changes in these variables.

Adolescent↗
PubMed1989

Inhibition of the Na+/glucose cotransporter functional lysyl residues by 4-(4-methoxy-3-sulfophenyl)-2,6-bis(4-sulfophenyl)pyryl ium.

Pyrylium salts react exclusively with the epsilon-amino group of lysyl moieties and display a degree of selectivity exceeding all other known amino group reagents. This study represents the first use of a pyrylium salt in positively identifying the functional role of lysyl residues in a biologically active molecule, the Na+/glucose cotransporter protein. We synthesized a hydrophilic pyrylium salt, 4-(4-methoxy-3-sulfophenyl)-2,6-bis(4-sulfophenyl)pyryliu m perchlorate, which formed a stable pyridinium cation upon reaction with N alpha-acetyl-L-lysine methyl ester. This pyrylium inhibited Na+/glucose cotransporter activity in purified brush border membranes, in a concentration-dependent manner. The data unequivocally establish that critical lysyl residues are responsible for the activity of the membrane-bound glucose cotransporter protein.

Animals↗