). 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., -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. Calmodulin (CaM) is a ubiquitous Ca 2ϩ-binding protein which has been highly conserved during evolution. The binding of Ca 2ϩ to CaM induces a conformational change which permits the interaction of CaM with specific target proteins, i.e., CaM-binding proteins. During the last few years, compelling evidence indicating that CaM is present in the cell nucleus (4) and that it has a role in several nuclear functions, such as gene transcription (21), DNA replication, DNA repair, and mitosis (see references 3, 4, and 36 for reviews), has accumulated.One of the ways to understand the molecular mechanisms by which CaM regulates these nuclear functions has been the identification of its nuclear protein targets. At least three groups of CaM-binding proteins have been found in the nuclei of different cell types: (i) proteins related to the actin motility systems, e.g., ␣-spectrin, myosin light-chain kinase, and caldesmon (6, 51, 57); (ii) transcription factors belonging to the basic helix-loop-helix structural group, e.g., human SEF-1, mouse E2A, and Max (21); and (iii) proteins involved in the phosphorylation or dephosphorylation of proteins, e.g., CaM-dependent protein kinase II and the CaM-dependent protein phosphatase calcineurin (11,51,54). The role of the specific complexes of CaM with these CaM-binding proteins in the nuclear functions still remains obscure, although some possible pathways have recently emerged. It has recently been reported that the addition of antiactin antibodies blocks the transport of macromolecules into purified nuclei in in vitro experiments, suggesting that the nuclear actin-myosin contractile system can play a role in nucleocytoplasmic transport (55). The transcription factors which are able to bind CaM and belong to the basic helix-loop-helix grou...
The effects of very brief and recurrent coronary occlusions on myocardial regional shortening and its ultrastructure have been analysed. Ultrasonic crystals were implanted in the left ventricular subendocardium of 23 anaesthetized dogs with the thorax open, to measure the shortening fractions of an ischaemic and a control segment. Twenty 2 min total occlusions were provoked in the left anterior descending coronary artery, with 3 min recovery intervals (reperfusion) between occlusions. The shortening fraction decreased progressively with each occlusion, reaching a value 18.9% lower than the basal after the last ischaemic episode (P < 0.05); 32.3% after 4 h of reperfusion (P < 0.01), and 28.6% after 24 h (P < 0.01). Qualitative and quantitative ultrastructural analysis showed an increase in the mitochondrial volume of the ischaemic tissue (158% vs control, P < 0.001) with significant damage to the cell components (7.7-fold increases vs control mitochondria). These results show that when the myocardium is subjected to very brief and repeated coronary occlusions, there is progressive deterioration of systolic function with structural alterations, mainly at the mitochondrial level. These modifications are still observable 24 h after the end of ischaemic stimulation and could be the cause of transitory and/or chronic systolic dysfunctions in the absence of previous heart attack.
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.
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