Biomedical subjects

O Bachs

Publications and source records attributed to O Bachs.

70 records · Page 4Linked to original sources

PubMed1988

Rearrangement of nuclear calmodulin during proliferative liver cell activation.

Calmodulin increases about three-fold in rat liver nuclei after partial hepatectomy. The increase is maximal after 24 hours, when DNA synthesis is also maximal. During the same time re-distribution of calmodulin within the nuclear structure takes place, leading to its association with the nuclear matrix. Incubation of normal rat liver nuclei with Ca2+ induces association of calmodulin with the matrix, indicating that the re-distribution of calmodulin during the replicative period is related to the increase in nuclear Ca2+. The nuclear matrix contains several calmodulin binding proteins of which one, having Mr of 130 kDa, has been identified as myosin light chain kinase (MLCK). Three acceptor proteins, having Mr of 120, 65, and 60 kDa decrease 24 hours after partial hepatectomy, MLCK and a protein of Mr 150 kDa instead increase.

Animals↗

PubMed1988

Reduced levels of sialic acid in the plasma membrane during hepatocellular proliferation.

When rats were infused with a solution containing triiodothyronine, amino acids, glucagon and heparin (solution A) the hepatocytes increased DNA synthesis and decreased plasma membrane sialic acid. In order to study whether the reduced levels of sialic acid in the plasma membrane were associated with hepatocyte proliferation, different mixtures of three components of solution A were infused into rats and the DNA synthetic activity as well as the sialic acid content measured. Results reported here show a correlation between DNA synthetic activity and sialic acid reduction suggesting that the decrease in the plasma membrane sialic acid can be a pre-replicative step associated to cell proliferation.

Amino Acids↗

PubMed1988

New synthesis of cyclic AMP-dependent protein kinases during liver regeneration.

As has been previously reported one surge in cytosolic calmodulin is produced between 4 and 12 h after a partial hepatectomy. Moreover, a surge in cytosolic cyclic AMP and another in cyclic AMP-dependent protein kinase activity can be detected during the late period of the prereplicative phase of liver regeneration after a partial hepatectomy. It is known that these three surges are involved in triggering DNA synthesis. By kinetic studies and by injecting transcription (actinomycin D) and translation (cycloheximide) inhibitors into hepatectomized rats we have demonstrated that the cyclic AMP-dependent protein kinase surge is produced by new synthesis of the enzyme. In thyroparathyroidectomized rats subjected to a partial hepatectomy the calmodulin surge was similar to that observed in normal hepatectomized rats whereas the surge in cyclic AMP-dependent protein kinase activity was strongly decreased suggesting that the cyclic AMP-dependent protein kinase surge is not generated by the previous surge in calmodulin.

Calmodulin↗

PubMed1988

Possible cyclic AMP-dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells.

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.

Animals↗

PubMed1988

Nuclear growth and chromatin relaxation-condensation cycle in hepatocytes during the proliferative activation of rat liver.

In order to quantify the changes in nucleolar and nuclear volumes and in chromatin condensation produced during proliferative activation we have carried out morphometric studies on hepatocyte nuclei during rat liver regeneration using electron microscopy. To minimize the artefactual effects produced by fixation on subcellular structures we have fixed the livers by perfusion with glutaraldehyde. The mean values for the nucleolar and nuclear volumes were progressively increased until 28 h after 66% partial hepatectomy. The maximum values raised for the nuclei and nucleoli at this time were 3 and 4.28 times, respectively, those of controls. Later, nuclear and nucleolar volumes progressively declined. Two waves of diminution in nuclear electron-dense material were produced after hepatectomy. The first occurred between 0 and 12 h, with minimum values 1.34 times lower than those from control animals at 8 h. The second occurred between 12 and 28 h, with minimum values 2.56 times lower than those from control rats at 24 h. These two waves in chromatin relaxation correlate very well with the transcriptional changes described by other authors during the pre-replicative, replicative and mitotic phases of liver regeneration.

Animals↗

PubMed1987

Calmodulin and calmodulin-binding proteins in liver cell nuclei.

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.

Animals↗

PubMed1987

Calcium transport from blood into the bile in normal and regenerating rat liver.

We have studied calcium movement from blood into the bile by injecting 45Ca2+ intravenously and measuring the radioactivity appearing in the bile. 45Ca2+ started to appear in the bile at 3 min and maximum values were observed at 5 min after its administration. The amount of calcium secreted into the bile was proportional to the blood calcium concentration indicating that the main pathway involved in calcium movement behaved as a non-saturable system. We have also studied the 45Ca2+ circulation from blood into the bile in rats subjected to a partial hepatectomy. Thereafter, the calcium transported into the bile per gram of liver increased by about 50 per cent. Since bile flow behaved in a similar way, the biliar calcium concentration remained unmodified after hepatectomy. Determination of the activities of the Ca2+ transporting systems in isolated plasma membrane fractions from regenerating livers showed no modification in these activities suggesting that the elevation in calcium movement observed after hepatectomy is not due to an increase in the circulation of Ca2+ through the transhepatocyte pathway, an observation compatible with the absence of saturation in the transport.

Animals↗

PubMed1986

Calmodulin may decrease cell surface sialic acid and be involved in the expression of fibronectin during liver regeneration.

The decrease of sialic acid in plasma membrane glycoproteins and the expression of cell surface fibronectin were studied during the pre-replicative phase of liver regeneration. The aim of this study was to correlate these cell-surface events to the intracellular surge of calmodulin observed a few hours after partial hepatectomy. The fact that calmodulin decreased the specific activity of UDP-N-acetyl-D-glucosamine 2'-epimerase, a key regulatory enzyme in the biosynthesis of glycoprotein sialic acids, and that trifluoperazine prevented the desialylation indicates that the membrane desialylation is a calmodulin-dependent process. On the other hand, Western blotting using anti-rat fibronectin antibody in trifluoperazine-treated animals suggests that calmodulin may also be involved in the surface expression of fibronectin in regenerating hepatocytes.

Animals↗

PubMed1986

Changes in sinusoidal plasma membrane enzyme activities during the pre-replicative phase of liver regeneration.

Changes in a range of plasma membrane enzyme activities during the early period of liver regeneration are thought to be related to the initiation of DNA synthesis and the triggering of cellular activation. The sinusoidal plasma membrane was isolated from control and partially hepatectomized animals at various intervals during the pre-replicative phase. The specific activities of 5'-nucleotidase, (Na+ + K+)-ATPase, Ca2+-ATPase, Mg2+-ATPase showed that after partial hepatectomy changes in the enzyme activities at the sinusoidal plasma membrane region occur. These changes are probably related to the remodeling of the cell-surface that occurs before the division of hepatocytes.

5'-Nucleotidase↗

PubMed1986

[Changes in sialic acid content of the plasma membrane in hepatocellular proliferation].

The content of sialic acid bound to the sinusoidal region of plasma membrane during the prereplicative phase after the intravenous injection of a solution containing triiodothyronine, amino acids, glucagon and heparin (T.A.G.H. solution) has been measured. The results obtained show that an important decrease in sialic acid content is produced as it occurs in the hepatic cells of hepatectomized animals. In order to know if sialidase activity is involved in the decrease of sialic acid content during liver regeneration, the activity of sinusoidal plasma membrane sialidases during the prereplicative phase after the partial hepatectomy has been studied. No modifications of sialidase activity were detected during this period of time indicating that this decrease in sialic acid content has to be produced by other mechanisms such as diminution in the synthesis of precursor molecules. On the other hand due to the importance of Ca2+-calmodulin complexes in the activation of the hepatic cell proliferation the possible implication of this complex on the loss of sialic acid, observing the effect of trifluoperazine (inhibitor of Ca2+-calmodulin complexes) during the prereplicative phase of liver regeneration has been studied. The results show a delay in the decrease of the amount of sugar studied from 10 to 12 hours compared to the results obtained with the hepatectomized rats that have not received trifluoperazine.

Animals↗

PubMed1985

ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane.

Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.

Adenosine Triphosphate↗

PubMed1985

Induction of plasma membrane alkaline phosphatase in rat liver.

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.

Alkaline Phosphatase↗

PubMed1985

Effect of trifluoperazine on DNA synthesis during liver regeneration.

An intraperitoneal injection of the calcium-calmodulin blocker trifluoperazine into rats at 4 hr after a partial hepatectomy produced a strong inhibition of DNA synthesis observed at 24 hr after surgery; but when injection was administered at 20 hr after hepatectomy, it did not produce any effect on DNA replication. These observations indicate that trifluoperazine acted by blocking one or more events involved in triggering DNA replication but it did not affect on-going DNA synthesis. A more detailed study indicated that when trifluoperazine was injected at 4 hr after surgery, a 12 hr delay in the cytosolic calmodulin surge observed between 6 and 12 hr after partial hepatectomy (previous to initiation of DNA replication) and also in the starting of DNA synthesis was produced. These findings suggest that the pre-replicative surge of cytosolic calmodulin could be involved in triggering DNA synthesis observed after partial hepatectomy.

Animals↗

PubMed1984

Quantitative and qualitative changes of plasma membrane glycoproteins in the early period of liver regeneration.

Alterations of cell surface glycoconjugates have been observed in many developing systems and may be important in the physiological control of growth and differentiation. Liver regeneration after partial hepatectomy is a suitable model in which to study the regulatory mechanisms of cell proliferation in vivo. We have isolated the sinusoidal plasma membrane of hepatocytes at different times after partial hepatectomy. The sialic acid content and the SDS-polyacrylamide gel electrophoresis pattern of glycoproteins were determined. A decrease of periodic acid-Schiff-profiles, a change in the binding capacities of 125I-concanavalin A, a reduction of the sialic acid content and the appearance and disappearance of specific components have been observed during the pre-replicative phase of liver regeneration. These findings during this early period are consistent with the active involvement of the plasma membrane glycoproteins in the transition of cells to the proliferative state.

Animals↗

PubMed1983

Cerebral subcellular distribution of CDP-choline and/or its metabolites after oral administration of methyl-14C CDP-choline.

Distribution of radioactivity in subcellular components of a cerebral extract after oral administration of methyl-14C labelled cytidine diphosphate choline (CDP-choline, citicoline, Somazina) was analyzed. Animals were decapitated 24 h after the administration and the brain immediately removed and gently homogenized in a buffer solution. The homogenate was fractionated by differential centrifugation and the protein concentration and the radioactivity were measured in each precipitate as well as in the final supernatant. Radioactivity was found to be associated mainly with membranous microsomal precipitate and with cytoplasmic membrane precipitate. Alternatively, materials sedimenting between 500 X g and 20,000 X g were fractionated in a density gradient centrifugation. Fractions were screened for enzymatic activities as indicators of cytoplasmic membrane, mitochondria, lysosomes, microsomes and synaptosomes. Radioactivity was found to be associated with cytoplasmic and microsomal membrane fractions. Identification of some fractions as enriched in synaptosomes also accumulating radioactivity, as well as the possible incorporation of the radioactivity to choline and phosphorylcholine cerebral pools are discussed.

Administration, Oral↗

PubMedDate not supplied

New nuclear functions for calmodulin.

The data reported here summarize a series of results which reveal new functions for nuclear calmodulin (CaM). The addition of CaM inhibitors to cultures of proliferating NRK cells blocked the activity of the cyclin-dependent protein kinases 4 (cdk4) and 2 (cdk2), which are enzymes implicated in the progression of G1 and in the onset of DNA replication, respectively. CaM modulates the activity of cdk4 by regulating the nuclear location of both cdk4 and cyclin D, its associated regulatory subunit. By using CaM-affinity chromatography, we have recently identified two new nuclear CaM-binding proteins: (i) the protein La/SSB, which is an autoantigen implicated in several autoimmune diseases such as lupus erythematosus and Sjögren's syndrome (since La/SSB participates in the process of transcription mediated by RNA polymerase III, CaM could be involved in the regulation of this process); and (ii) the protein SAP145, a member of the spliceosome-associated proteins (SAPs) which is a subunit of the splicing factor SF3(b). This finding suggests the involvement of CaM in pre-mRNA splicing. Finally, a screening for new CaM-binding proteins in the fission yeast performed by using the phage display analysis, revealed that several nucleolar-ribosomal proteins associate to CaM, suggesting that CaM modulates ribosomal assembly and/or function.

Animals↗