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Biomedical subjects

L Boscá

Publications and source records attributed to L Boscá.

At least 91 records · Page 5Linked to original sources
PubMed1993

Rat liver messenger ribonucleic acid and enzyme activity of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase impairment during the late period of pregnancy.

Fructose 2,6-bisphosphate concentration, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase2) activity, and messenger RNA decreased in maternal rat liver during the last days of gestation, and the recovery started after delivery. Phospho(enol)pyruvate carboxykinase activity and messenger RNA increased in contrast to PFK-2 changes. Measurement of the glycolytic capacity in isolated hepatocytes prepared from rats 1 h after parturition showed a low glucose consumption and an impaired capacity to metabolize glucose. These results stress the relevance of the PFK-2/fructose 2,6-bisphosphate system in the control of the glycolytic flux in liver, and these changes are intended to prevent glucose consumption by maternal liver and contribute to allow gluconeogenesis to proceed at the end of gestation. The physiological basis of this adaptation may lay on the diversion of glucose from maternal to fetal metabolism.

Animals↗
PubMed1992

Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide.

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Amino Acid Oxidoreductases↗
PubMed1992

Characterization of 6-phosphofructo-2-kinase from foetal-rat liver.

Foetal and adult liver 6-phosphofructo-2-kinase (PFK-2) were purified by identical protocols. The native molecular masses of both enzymes were determined by gel filtration and were 89.1 and 100.0 kDa respectively. No differences were found in SDS/PAGE in 10%-acrylamide gel (55 kDa per subunit). The kinetic properties displayed by both enzymes were similar, except for the sensitivity to inhibition by sn-glycerol 3-phosphate. Foetal PFK-2 was a good substrate for phosphorylation by cyclic AMP-dependent protein kinase and protein kinase C, whereas the adult enzyme was phosphorylated only by cyclic AMP-dependent protein kinase. However, the phosphorylation affected only the kinetic properties of the adult enzyme, suggesting the presence in both enzymes of different sites of phosphorylation by cyclic AMP-dependent protein kinase. These differences in primary structure were consistent with the distinct chromatographic profiles of the phosphopeptides after digestion of the protein with CNBr. Western-blot analysis with antibodies specific for the N-terminal region of the liver-type PFK-2 poorly recognized the foetal enzyme, suggesting that both enzymes differ at least in the N-terminal sequence.

Aging↗
PubMed1992

Isoenzymes of carbohydrate metabolism in primary cultures of hepatocytes from thioacetamide-induced rat liver necrosis: responses to growth factors.

Hepatocytes isolated from the liver of rats after a necrotizing dose of thioacetamide (6.6 mmol/kg) were used to study the postnecrotic process of liver regeneration. Flow cytometry analysis revealed populations of dedifferentiated hepatocytes exhibiting physical properties (size and fluorescence emission at 530 nm) similar to those found in fetal (22 days old) liver cells. The percentage of these cells increased progressively from 24 to 48 and 72 hr after thioacetamide administration. In primary cultures of hepatocytes the effects of phorbol 12-myristate 13-acetate, bombesin and insulin were investigated on the 6-phosphofructo 2-kinase/fructose 2,6 bisphosphate system. Bombesin and insulin stimulated 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate content both in control and in thioacetamide-treated hepatocytes. However, phorbol 12-myristate 13-acetate stimulated 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphate concentration in thioacetamide-treated liver cells, whereas no similar response was found in hepatocytes from control rats. The response of postnecrotic thioacetamide-treated hepatocytes to phorbol 12-myristate 13-acetate was similar to that obtained from 22-day-old fetal liver cells, which reveals that different methods might control fructose 2,6-bisphosphate content and therefore the mechanisms of glycolysis and gluconeogenesis at this regulatory step. The lack of response to glucagon of glycogen phosphorylase a and 6-phosphofructo 2-kinase from thioacetamide-treated hepatocytes may indicate that the expression of specific enzymes of carbohydrate metabolism undergoes transitions to less-differentiated isoenzymatic forms. Moreover, the isoenzyme pattern of hexokinases elicits a complete disturbance in glucokinase and hexokinases activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Oleic acid promotes changes in the subcellular distribution of protein kinase C in isolated hepatocytes.

The effect of oleate on the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes and in perfused rat liver in the presence of physiological concentrations of serum albumin. A time- and dose-dependent translocation of PKC from the cytosol towards the membranes was observed at oleate concentrations that fell within the range of concentrations reached under several physiological conditions. Analysis of the membrane-bound isoenzymes of PKC by hydroxylapatite chromatography revealed that the beta isoenzyme was preferentially translocated to this compartment in hepatocytes incubated with oleate. Activation of PKC after incubation of hepatocytes with oleate involved at least three different effectors of the enzyme: the fatty acid itself, the diacylglycerol synthesized from oleate, and the rise in the cytosolic calcium concentration elicited by oleate. As a result of PKC activation, protein phosphorylation of intact hepatocytes in response to oleate exhibited an enhancement in the phosphate content of a protein of 82 kDa, similar to that phosphorylated in the presence of phorbol dibutyrate.

Animals↗
PubMed1991

B cell triggering by bacterial lipopeptide involves both translocation and activation of the membrane-bound form of protein kinase C.

B cell activation by the lipopeptide trispalmitoyl-cysteinyl-alanyl-glycine (TPP), the biologically active moiety of bacterial lipoprotein, results in protein kinase C (PKC) translocation from the cytosol to the plasma membrane, as well as a significant increase in the activity of membrane-associated PKC that can be observed by in vitro incubation with TPP of partially purified PKC at calcium concentrations in the range of those prevailing in unstimulated B cells. TPP does not affect either the phosphoinositide turnover or the cytosolic-free calcium concentration, but promotes an increase in the intracellular pH that can be blocked by the PKC-inhibitors staurosporine or H-7. Moreover, incubation of B cells with staurosporine suppressed the proliferative response promoted by TPP at a half-maximum effective dose of 16 nM. Activation by TPP of PKC isoenzymes resolved after hydroxylapatite chromatography revealed that the resulting beta I/beta II isoenzyme was more sensitive than the alpha isoenzyme. These results suggest that TPP might mediate B cell activation via interaction with the membrane-associated fraction of PKC.

Animals↗
PubMed1991

Protein kinase C mobilization in B lymphocytes. Differential isoenzyme translocation upon activation.

The subcellular distribution of protein kinase C has been analyzed in murine B lymphocytes exposed to LPS, anti-IgM antibodies and phorbol dibutyrate. An accurate determination of the enzyme mobilized from the soluble to the particulate fractions by these activators, has been made possible by the use of B cells in which the major part of the activity was present in the cytosol. Upon stimulation, we have analyzed the isoenzymatic forms translocated to the B cell membrane, showing a differential pattern of isoenzyme mobilization between LPS and anti-IgM antibodies. These data, together with the different Ca2+ requirements for the activation of the translocated protein kinase C isoenzymes, might help to unravel the mechanism responsible for the clonal expansion and differentiation of B lymphocytes, induced by the two ligands.

Animals↗
PubMed1990

Lack of translocation of protein kinase C from the cytosol to the membranes in vasopressin-stimulated hepatocytes.

The ability of Ca2(+)-mobilizing hormones to promote changes in the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes. In recently isolated cells the distribution of PKC between the soluble and particulate fractions was 47 and 53% respectively. Exposure of the hepatocytes to 100 nM-vasopressin produced an increased phosphoinositide turnover, as reflected by the changes in the concentrations of inositol trisphosphate and Ca2+, and in glycogen phosphorylase a activity. However, the distribution of both PKC activity and [3H]phorbol dibutyrate binding between the cytosol and the membranes remained unchanged under these conditions. To determine the threshold values of the concentrations of Ca2+ and diacylglycerol required to produce a redistribution of PKC, the hepatocytes were treated with the Ca2+ ionophore ionomycin, and with permeant diacylglycerol derivatives. Hepatocytes incubated in the presence of 100 nM-vasopressin required concentrations of Ca2+ 2.5 times those produced physiologically by the hormone to produce translocation of PKC from the cytosol to the membranes. These studies suggest that, at least in hepatocytes, activation of PKC in response to Ca2(+)-mobilizing hormones involves only the pre-existent membrane-bound enzyme without affecting the soluble enzyme.

Animals↗
PubMed1990

Substrate-dependent inhibition of protein kinase C by specific inhibitors.

Protein kinase C (PKC) and its proteolysis-derived protein kinase independent of Ca2+ and phospholipids (PKM), were purified from rat brain. By using histone H1 and protamine as substrates, we assayed the effect of several inhibitors of PKC and PKM. The inhibition turned out to be dependent on both the nature of the kinase and the type of substrate assayed. These results may help to interpret the different responses elicited by PKC inhibitors in vivo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Identification of an anti-A and anti-B blood group glycosyltransferase antibody after incompatible bone marrow transplant.

The occurrence of a potent antibody against plasmatic A and B glycosyltransferase activities has been characterized in a patient (blood group A1) transplanted with a bone marrow from a blood group O donor. A and B glycosyltransferases were purified to near homogeneity from plasma of A1 and B blood-group individuals. The half-maximal inhibition of both enzymes was obtained at 1 to 2 micrograms/mL of the post-transplant IgG fraction, prepared by protein A-sepharose chromatography. A and B glycosyltransferases were also recognized by the post-transplant IgG fraction after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer to nitrocellulose membranes.

ABO Blood-Group System↗
PubMed1989

Oleate-induced translocation of protein kinase C to hepatic microsomal membranes.

The incubation of rat liver homogenates in the presence of oleate induces the translocation of protein kinase C from the cytosol to the endoplasmic reticulum membranes. The half-maximal effect was obtained at 0.3 mM oleate. The redistribution of this enzyme induced by oleate was also obtained with purified protein kinase C and hepatic microsomal membranes. This effect seems to be mediated by long-chain fatty acids since translocation was not obtained with esterified derivatives.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Glucagon-induced changes in fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase in cultured rat foetal hepatocytes.

The sensitivity of 6-phosphofructo-2-kinase to glucagon and cyclic AMP was studied during the perinatal period. In liver homogenates from foetal and neonatal rats, incubation with cyclic AMP produced inactivation of 6-phosphofructo-2-kinase 3 h after birth. The maximal effect was obtained 12 h after birth. In primary cultures of hepatocytes from 22-day-old foetuses, glucogon induced an inhibition of 6-phosphofructo-2-kinase that required 45 min to reach the half-maximal effect. Cycloheximide prevented the glucagon-induced changes in this activity from cultured foetal hepatocytes. These results suggest that the adult form of 6-phosphofructo-2-kinase is rapidly induced after birth, probably by the hormonal changes that occur in this period.

Animals↗
PubMed1989

Lack of correlation between translocation and biological effects mediated by protein kinase C: an appraisal.

Protein kinase C is involved in the mechanism of action of hormones, growth factors, mitogens and tumour promoters. The correlation between the extent of the biological effects mediated by protein kinase C and its fractional activation shows cell type specific patterns of behaviour. The discrepancy between enzyme activity and biological effects elicited by protein kinase C is particularly relevant to lymphoblastic cells. In B cells, the full expression of some biological responses mediated by protein kinase C may be achieved by activation of less than 5% of the enzyme activity.

Animals↗
PubMed1989

Translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in thioacetamide-induced liver tumours in rats.

The translocation of phosphatidate phosphohydrolase induced by oleate was higher (two-fold) in liver homogenates obtained from long-term thioacetamide-treated rats than from control rats. These differences between thioacetamide-treated and control livers were noticeably higher (four-fold) in the presence of physiological concentrations of salt (0.15 M KCl). In homogenates from control rats, there was a lack of response when physiological concentrations of the salt were present. The enhanced response to translocate phosphatidate phosphohydrolase activity in liver homogenates from thioacetamide-treated rats was due to an increased binding ability of microsomal membranes.

Acetamides↗
PubMed1988

Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver.

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.

Aging↗
PubMed1988

Activation of protein kinase C from B lymphocytes by lipid A.

The effect of lipid A, a residue of the lipopolysaccharide molecule, on protein kinase C from B lymphocytes has been studied. Lipid A activates and promotes the translocation of protein kinase C from the soluble to the particulate membrane fraction in a cell-free system reconstituted with purified enzyme and membranes isolated from B lymphocytes. These results demonstrate that the activating effect of lipopolysaccharide on protein kinase C from B cells is due to the lipid moieties of this molecule.

Animals↗
PubMed1988

Presence of antibody to A- and B-transferases in minor incompatible bone marrow transplant.

The contribution of the bone marrow to plasma A- or B-transferase activities has been studied in patients who underwent incompatible bone marrow transplantation (BMT). As deduced from major incompatibility (group O recipient/A donor), the contribution of the marrow to these plasma activities was c. 5-10% of the total activity. In cases of minor incompatible transplants (A recipient/O donor), normal plasma activity was present in two patients, while no activity was found in a further two in whom a potent antitransferase was detected. The antibody inhibited both A- and B-transferase activities to a high titre. The patients in whom this antibody arose exhibited severe graft-versus-host disease.

ABO Blood-Group System↗