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O Bachs

Publications and source records attributed to O Bachs.

At least 19 recordsLinked to original sources
PubMed1996

Changes in the activity of nuclear protein kinase CK2 during rat liver regeneration.

Protein kinase CK2 has been found in two nuclear fractions obtained after treatment of purified rat liver nuclei with nucleases (S1 fraction) and subsequently with 1.6 M NaCl (S2 fraction). In both fractions three isoforms of the alpha subunit were identified. Two of them corresponded to the classical alpha and alpha' subunits, whereas the identity of the third one (alpha 3) remains unknown. In the S1 fraction two peaks of CK2 activity were detected at 6 h (5.5 fold) and 24 h (1.9 fold) after partial hepatectomy, whereas no significant changes were found in the S2 fraction. At 6 h after laparatomy a much lower increase of CK2 in S1 fraction was also detected (2.5 fold). The increases in CK2 activity found at 6 h after hepatectomy or laparatomy were accompanied with rises in the amount of the alpha subunit.

Animals↗
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

Cyclin/cdk2 complexes in the nucleus of HeLa cells.

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.

Blotting, Western↗
PubMed1994

Casein kinase 2 and protein substrates are released from rat liver cells nuclei by DNase or RNase digestion.

Casein kinase 2 was released from rat liver cells nuclei by digestion with DNase I plus RNase A. This treatment also released three major substrates of 50, 40-42, and 37 kDa. Casein kinase 2 and substrates were also extracted by DNase or RNase separately. However, in DNase extracts only the 37 kDa protein was phosphorylated by casein kinase 2, whereas in RNase extracts all three substrates were phosphorylated. When the DNase extracts were subsequently treated with RNase the 40-42 substrates were then phosphorylated, indicating that their interaction with RNA prevents their phosphorylation by casein kinase 2. The ratio of B: alpha subunits of casein kinase 2 present in the nuclease extracts was higher than that of the purified enzyme, which is assumed to be 1:1. A further analysis by sucrose gradient centrifugation revealed that under physiological salt conditions casein kinase 2 from nuclease extracts formed large aggregates (higher than 300 kDa) which were disrupted at 400 mM KCl. At the latter KCl concentration CK-2 activity was localized at a position corresponding to a M(r) of 230-250 kDa, which is still higher than the typical tetrameric form of the enzyme.

Animals↗
PubMed1994

Microsomal localization of cyclin A and cdk2 in proliferating rat liver cells.

The expression and intracellular localization of cyclin A and cdk2 have been analyzed in rat liver cells proliferatively activated in vivo by a partial hepatectomy. Western blot analysis revealed that cyclin A started to increase during G1 (at 6 h after hepatectomy) reaching maximal levels during S phase (at 18 h). Cdk2 began to increase during late G1 (at 12 h) peaking also at 18 h. At the latter time cyclin A was mainly localized in the microsomal fraction, although it was also present in cytosol, plasma membrane and nucleus. Active cyclin/cdks complexes containing cyclin A and cdk2 were obtained by precipitation with p13-Sepharose after solubilization of microsomes with triton X-100. The presence of active cyclin A/cdk2 complexes in microsomes was confirmed by immunoprecipitation experiments with anti-cdk2 antibodies. These results suggest a putative role of cyclin A/cdk2 during S phase which would be related with microsomal function.

Animals↗
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

Calmodulin expression during rat liver regeneration.

We have investigated the messenger RNAs expressed from the three calmodulin genes during rat liver regeneration. The results revealed that all the calmodulin transcripts increased from 8 hr after a partial hepatectomy, although differences in the timing and the level of expression from the three genes were observed. Calmodulin I transcripts peaked at 16 hr, whereas calmodulin II and calmodulin III progressively increased from 8 to 24 hr. At 24 hr after surgery, calmodulin I, calmodulin II and the 2.3 kb calmodulin III transcripts reached values of a 6-fold increase, whereas the 0.8 kb product of calmodulin III increased 25-fold. At 30 hr the levels of all the calmodulin transcripts were similar to those observed at 24 hr. The transcription rates of the three calmodulin genes augmented after hepatectomy (calmodulin I and calmodulin II twofold and calmodulin III fourfold), indicating that the elevation of the calmodulin transcripts could be, at least partially, the result of this increase in the transcription rates. The total calmodulin concentration also increased twofold at 24 hr after hepatectomy. We also report that the administration of the beta-adrenergic blocker, D,L-propranolol inhibited the accumulation of calmodulin protein without significantly affecting the increase of the messenger RNAs. These results indicate that the expression of calmodulin observed during liver regeneration could be regulated by cyclic AMP at the translational or posttranslational level.

Animals↗
PubMed1994

Expression of calmodulin and calmodulin binding proteins in lymphoblastoid cells.

Calmodulin is encoded in vertebrates by three different genes: CALM1, CALM2, and CALM3. We have examined the mRNAs expressed from these three genes in eight lines of human lymphoblastoid cells (Namalwa, Raji, Ramos, JY, Molt-4, Jurkat, CEM, and HPB-ALL). We found that all these cell lines (except Ramos) overexpressed CALM3 transcripts, which led to an increase of total CaM protein with respect to quiescent normal T lymphocytes. The nuclear concentration of calmodulin was measured in two of these lymphoblastoid cell lines (JY and HPB-ALL) and compared to quiescent and phytohemagglutinin-activated T lymphocytes. Activated lymphocytes showed a 2-fold increase of nuclear calmodulin with respect to quiescent cells, whereas in the two lymphoblastoid cell lines, nuclear calmodulin remained similar to that of quiescent cells. The levels of a calmodulin-binding protein of 150 kDa in the homogenates of the eight lymphoblastoid lines was found to be higher than those of quiescent and activated lymphocytes. Likewise, the amount of three calmodulin-binding proteins of 240, 200, and 170 kDa was also increased in several of the cell lines, but not in all of them. The 170-kDa protein was only expressed by activated lymphocytes and lymphoblastoid cells, suggesting that it could be specific for proliferating cells. In the nuclei of activated lymphocytes and lymphoblastoid cells, a decrease of a calmodulin-binding protein of 110 kDa and increases of three other of 240, 180 and 170 kDa were also detected.

Animals↗
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↗
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↗