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O Bachs

Publications and source records attributed to O Bachs.

At least 55 records · Page 3Linked to original sources
PubMed1993

Calmodulin can modulate protein phosphorylation in rat liver cells nuclei.

This report describes the immunological identification of a 60-kDa calmodulin-binding protein, previously detected in the nuclei of rat liver cells (Bachs, O., Lanini, L., Serratosa, J., Coll, M.J., Bastos, R., Aligué, R., Rius, E., and Carafoli, E. (1990) J. Biol. Chem. 265, 18595-18600), as the calmodulin-dependent protein phosphatase calcineurin. Calcineurin could be extracted from the nuclei by incubation with DNase and RNase, indicating that it is associated with nuclear structures sensitive to the action of nucleases (chromatin or/and ribonucleoproteins). The presence of calcineurin in the nuclei of rat liver cells indicates that calmodulin may modulate the phosphorylation level of nuclear proteins by promoting their dephosphorylation. This report also shows that calmodulin inhibits the activity of casein kinase-2 in the nuclear fractions obtained by nuclease extraction. Phosphorylation experiments indicate that casein kinase-2 phosphorylates three major substrates of 100, 42-44, and 37 kDa as well as other minor proteins in the nuclease extracts. Calmodulin reduces the phosphorylation level of the two latter major proteins and of a minor band of 50 kDa. Thus, nuclear calmodulin in rat liver cells could regulate phosphorylation of nuclear proteins by at least two mechanisms: 1) activation of calcineurin and 2) inhibition of casein kinase-2.

Animals↗
PubMed1993

Effect of alpha 1-adrenergic blockade on nucleolar growth, chromatin relaxation, and histone H1(0) content in regenerating liver.

alpha 1-Adrenergic agonists are known to be involved in the regulation of hepatocyte proliferation after a partial hepatectomy. The blockade of alpha 1-adrenergic receptors with the specific antagonist prazosin inhibits DNA synthesis which peaks at 24 h after surgery. In this report we have studied the effects of prazosin administration on several events occurring during liver regeneration. The results show that the nuclear volume and nucleolar volume density of hepatocytes were increased and that the relative amount of heterochromatin decreased at 24 h. The increase in hepatocyte nucleolar volume density and the decrease in the relative amount of heterochromatin were partially abolished by prazosin administration while the increase in the nuclear volume was not affected. The relative amount of the histone H1 variant H1(0) was reduced in 24-h regenerating liver and prazosin treatment prevented this reduction.

Animals↗
PubMed1993

Calmodulin expression during proliferative activation of human T lymphocytes.

We have investigated the levels of calmodulin mRNA species and calmodulin protein during proliferation of human T lymphocytes. Quiescent lymphocytes expressed the 1.7 kb transcript of CaM I, the 1.4 kb of CaM II and the 2.3 kb of CaM III. Phytohaemagglutinin added to peripheral blood lymphocytes induced DNA replication which started at 48 h and reached a maximum at 72 h after activation. All the species of calmodulin mRNAs, including the 4.0 kb transcript of CaM I and the 0.8 kb of CaM III which were not detected in quiescent cells, increased during lymphocyte proliferation. At 72 h after activation, the increase of CaM I and CaM II transcripts were found to be 2-fold whereas CaM III mRNAs increased 9-fold. The cellular content of calmodulin protein was also found to increase during proliferation and calmodulin accumulations in cytosol and nuclei of activated cells were observed. Two calmodulin binding proteins of 180 and 170 kD were found to increase in the nuclei of proliferating lymphocytes, whereas on the contrary 3 other calmodulin binding proteins of 110, 62 and 60 kD decreased during proliferation.

Antibodies, Monoclonal↗
PubMed1993

Nuclear calmodulin-binding proteins in rat neurons.

By using a 125I-calmodulin overlay assay, three major high-affinity calmodulin-binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60- and 50-kDa proteins have been previously identified as subunits of the calmodulin-dependent protein kinase II. We report here the immunoblot identification of the 135-kDa calmodulin-binding protein as myosin light chain kinase. We also show that the calmodulin-dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.

Animals↗
PubMed1992

The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum.

The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.

Animals↗
PubMed1992

Calmodulin regulates DNA polymerase alpha activity during proliferative activation of NRK cells.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.

Animals↗
PubMed1992

Increase of cytokeratin D during liver regeneration: association with the nuclear matrix.

An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.

Amino Acid Sequence↗
PubMed1991

Regulation of DNA polymerase alpha activity by the alpha 1-adrenergic receptors in proliferatively activated rat liver cells.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.

Animals↗
PubMed1991

Presence of calmodulin and calmodulin-binding proteins in the nuclei of brain cells.

The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 micrograms/mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of alpha-spectrin and a 62-kDa calmodulin-binding protein (p62) in the nuclei of neurons and glial cells by immunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by immunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.

Animals↗
PubMed1990

Decrease of calmodulin and actin in the plasma membrane of rat liver cells during proliferative activation.

After proliferative activation of rat liver cells in vivo by a partial hepatectomy a decrease of the calmodulin content in the three plasma membrane domains (blood sinusoidal, canalicular and lateral) was observed. At 24 hours after partial hepatectomy calmodulin was found to be 3 fold lower in the sinusoidal and lateral fractions whereas a 2 fold decrease was detected in the canalicular domain. Decreases on the actin levels have been also detected at 24 hours after a partial hepatectomy. Since at this time after surgery increases on nuclear actin and calmodulin have been reported, these results suggest the possibility that the actin and calmodulin dissociated from the plasma membrane after a partial hepatectomy could subsequently be translocated into the nuclei.

Actins↗
PubMed1990

Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells.

alpha-Spectrin, myosin light chain kinase (MLCK), and caldesmon have been detected in the nuclei of rat liver cells by 125I-calmodulin overlay, immunoblotting, and immunocytochemical methods. alpha-Spectrin is localized in the nuclear matrix, nuclear envelope, and nuclear pores. It has also been detected inside the nuclei in the form of small aggregates. MLCK is present in the nuclear matrix, envelope, nucleoli, and in a nuclease extract (S1 subfraction) but not in the nuclear pores. Caldesmon shows a diffuse distribution pattern inside the nuclei but it is not present in the nucleoli. Since all these proteins are components of the actin-myosin motility systems the presence of actin in the different nuclear subfractions has also been investigated: actin is present in the nuclear matrix, nuclear envelope, nucleoli, and nuclear pores. Proliferative activation of rat liver cells in vivo by partial hepatectomy induces the increase of alpha-spectrin, MLCK, and actin in different nuclear subfractions. This, together with the increase of nuclear calmodulin at the same time after hepatectomy (Pujol, M. J., Soriano, M., Aligúe, R., Carafoli, E., and Bachs, O. (1989) J. Biol. Chem. 264, 18863-18865), indicates that nuclear calmodulin could activate a nuclear contractile system during proliferative activation. A 62-kDa protein (p62) which binds to calmodulin columns and shows immunological similarities to caldesmon is specifically located in the region surrounding the nuclear envelope and is associated with the heterochromatin.

Animals↗
PubMed1990

Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane.

A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.

Amino Acid Sequence↗
PubMed1990

Isolation of rat liver spectrin and identification of functional domains.

Immunohistochemical studies carried out with liver sections show that spectrin is uniformly distributed along the whole plasma membrane of hepatocytes. The bilecanalicular spectrin is released during the purification of liver subplasma membrane fractions, whereas most of the basolateral spectrin remains tightly bound to the membrane. Spectrin associated with the basolateral membranes has been purified and its subunits isolated. The alpha-subunit retains the ability to bind both calmodulin and actin. Fragments have been obtained either by chemical or by proteolytical digestion of the 240 kDa alpha-subunit. Treatment with CNBr yields fragments of about 30 kDa which bind actin and calmodulin. Digestion with Staphylococcus aureus V-8 proteinase yields a calmodulin-binding fragment of 27 kDa and an actin-binding fragment of 31 kDa.

Actins↗
PubMed1990

Increase in a 55-kDa keratin-like protein in the nuclear matrix of rat liver cells during proliferative activation.

We have identified a protein (p55) with a molecular weight of 55 kDa and a pI of 6.2, which was strongly increased in the nuclear matrix of rat liver cells during proliferative activation. This protein is highly insoluble since it could not be solubilized either by detergents or by alkaline extraction. We have obtained three partial amino acid sequences which revealed that p55 has a high homology with cytokeratins. Polyclonal antibodies raised against p55 were used to carry out Western blot and immunocytochemical studies which indicated that p55 was localized only in the nuclei, specifically in the nuclear matrix. Autoradiographic experiments revealed that not all the cells presenting an increase in p55 incorporated [3H]thymidine, indicating that this protein is not related to DNA replication. Immunocytochemical studies also revealed that during mitosis p55 is localized surrounding the chromosomes and associated with the mitotic apparatus, suggesting that p55 is involved in the separation of chromosomes during cell division.

Amino Acid Sequence↗
PubMed1989

Effect of alpha-adrenergic blockers on calmodulin association with the nuclear matrix of rat liver cells during proliferative activation.

A transient peak of cytosolic calmodulin (CaM) was produced during the prereplicative phase of rat liver cell proliferation following partial hepatectomy. After accumulating in the cytosol, CaM apparently translocated into the nuclei, associating with the nuclear matrix. The administration of alpha 1-adrenergic blockers to hepatectomized rats prevented the association of CaM with the nuclear matrix without affecting the increase in the total nuclear CaM. The inhibitory effect of the alpha 1-antagonists was reversed by the simultaneous injection of the alpha-agonist noradrenaline. Since the activation of alpha 1-adrenergic receptors results in the release of Ca2+ from endoplasmic reticulum stores, the results suggest that the association of CaM with the nuclear matrix during proliferative activation is mediated by Ca2+ released from endoplasmic reticulum and show that the association with the matrix is independent of its intranuclear accumulation.

Adrenergic alpha-Antagonists↗
PubMed1988

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.

Animals↗
PubMed1988

Increased calmodulin synthesis in the pre-replicative phase of rat liver regeneration.

A cytosolic calmodulin wave has been described in the pre-replicative phase of rat liver regeneration. Here we demonstrate that this calmodulin increase is reduced by injection of cycloheximide (translation inhibitor) and actinomycin D (transcription inhibitor). In addition, we found that calmodulin mRNA levels are increased during the early pre-replicative period of liver regeneration, and this increase is affected by actinomycin D. Together, these results strongly indicate that the calmodulin wave observed in the pre-replicative phase of liver regeneration is due to de novo synthesis of this protein.

Animals↗