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O Bachs

Publications and source records attributed to O Bachs.

At least 19 recordsLinked to original sources
PubMed1995

Differential distribution of heterogeneous nuclear ribonucleoproteins in rat tissues.

Heterogeneous nuclear ribonucleoproteins bind to RNA as long as it is transcribed. Since their binding can be sequence-specific, it has been suggested that their expression in different tissues could vary depending on the specific mRNA processing requirements. In order to better establish this possibility we studied the presence of the heterogeneous nuclear ribonucleoproteins A1, A2/B1, C and D in the cell nuclei of different rat tissues by one- and two-dimensional immunoblotting. We found that these proteins were heterogeneously distributed among tissues and that they were found in different proportions.

Alternative Splicing↗
PubMed1995

Calmodulin is involved in the induction of DNA polymerases alpha and delta activities in normal rat kidney cells activated to proliferate.

Normal rat kidney cells that reenter the cell cycle from quiescence start DNA synthesis at 12 h following serum addition and reach a maximum after 20 h. We have previously shown that the activation of DNA polymerase alpha, and the expression of the proliferating cell nuclear antigen were inhibited when the anti-calmodulin drug W13 is added to the cell cultures. Here we have analyzed the effect of W13 on the activity of DNA polymerase delta and on the expression of replication protein A. The results showed that the blockade of calmodulin by W13 produced an almost complete inhibition of DNA polymerase delta activity whereas the activity of DNA polymerase alpha was only partially inhibited. Finally, the expression of replication protein A was not affected after W13 treatment. Our data suggest that calmodulin might regulate DNA replication through the control of the activities of DNA polymerases alpha and delta and the expression of proliferating cell nuclear antigen.

Animals↗
PubMed1995

Addition of calmodulin antagonists to NRK cells during G1 inhibits proliferating cell nuclear antigen expression.

The mRNAs of most proteins involved in DNA synthesis show an S phase correlated expression when mammalian cells are stimulated to proliferate from G0. This is the case for proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase delta that is essential for the synthesis of the leading and lagging strands of DNA. Normal rat kidney cells re-entering the cell cycle from quiescence start DNA synthesis at 12 h and reach a maximum at 20 h. The expression of PCNA parallels the synthesis of DNA. Progression through the S phase was inhibited by addition of the anticalmodulin drug W13 to the cells during G1, 5 h after activation. W13 also inhibited the increase in both PCNA protein and mRNA indicating that calmodulin regulates its expression. Using TK-ts13 cells transfected with a plasmid containing the thymidine kinase gene under the control of the human 2.8 kb PCNA promoter, we demonstrated that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment with W13, indicating that the decrease in the mRNA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays revealed that control cells produced predominantly complete PCNA transcripts during S phase, while short incomplete transcripts were generated in W13-treated cells at the same time. These results indicate that calmodulin participates in a more direct or indirect way during G1 in the activation of PCNA expression. From data presented here it can be suggested that calmodulin activates the release of a transcriptional block leading to an increase in the amount of PCNA during S phase.

Animals↗
PubMed1995

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.

Adenosine Triphosphate↗
PubMed1994

Calmodulin regulates the expression of cdks, cyclins and replicative enzymes during proliferative activation of human T lymphocytes.

Cell cycle is regulated by the activation of complexes of cyclins and cyclin-dependent protein kinases at specific points. Quiescent cells lack both cyclins and cyclin-dependent kinases but their expression is induced after proliferative activation. Cyclin A/cdk2 complexes are involved in the onset of DNA replication whereas cyclin B/cdc2 trigger mitosis. We report here that Ca2+ and calmodulin regulate the expression of cdk2, cdc2, cyclin B and the proliferating cell nuclear antigen (a co-factor of DNA polymerase-delta) in human T lymphocytes. Likewise, the expression of cdk4, cyclin A and DNA polymerase-alpha is dependent of the synergistic effect of both the Ca2+/calmodulin and the protein kinase C pathways. Thus, calmodulin controls DNA synthesis by regulating the levels of cdk2 and proliferating cell nuclear antigen and mitosis entry by modulating the expression of cyclin B and cdc2.

Autoantigens↗
PubMed1994

Protein kinase C regulates calmodulin expression in NRK cells activated to proliferate from quiescence.

We have investigated the levels of calmodulin protein and calmodulin mRNA species during proliferative activation of NRK cells. Cells activated to proliferate from quiescence started to replicate DNA at 15 h, reaching a maximum at 20 h after serum addition. The maximum of mitosis was observed at 24 h. Quiescent cells showed a calmodulin concentration of 1.5 ng/micrograms of protein. At 10 h after serum addition the amount of calmodulin started to increase, reaching values of 3.0 ng/micrograms of protein at 24 h. NRK cells expressed predominantly 3 species of calmodulin transcripts: the 1.7 kb from CaM I, the 1.4 kb from CaM II and the 2.3 kb from CaM III. The amount of all the 3 transcripts was low in quiescent cells and 10 h after activation the levels were already high, reaching a maximum around 20 h. At the latter time the amount of the 3 calmodulin mRNAs was 5-10-fold higher than in serum starved cells. Run-on experiments showed that at 20 h after activation the transcription rates of the 3 calmodulin genes were higher than in quiescent cells. The addition of protein kinase C inhibitors to the cultures blocked the increase of the calmodulin transcripts while inhibitors of protein kinase A did not have any effect. Moreover, the addition of submitogenic doses of phorbol 12-tetradecanoate induced the increase of all 3 calmodulin transcripts. These results indicate that protein kinase C regulates calmodulin expression when NRK cells are activated to proliferate.

Alkaloids↗
PubMed1994

Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in interphasic and mitotic cells.

We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA-binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchromatin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over heterochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.

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↗