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Cardiac memory (CM) has short- (STCM) and long-term (LTCM) components modulated by calcium and angiotensin II. LTCM is associated with reducedItoand Kv4.3 mRNA levels. Because the cAMP response element binding protein, CREB, contributes to CNS memory transcription, we hypothesized that it might be a transcriptional factor in CM, influenced by calcium and angiotensin II. We studied STCM in dogs that were AV sequentially paced (AVP) for 2 hours or sham-operated. STCM was evaluated with ECG and vectorcardiogram (VCG), and subepicardial biopsies were taken at 5 to 120 minutes and investigated for CREB. LTCM was studied in dogs paced for 3 weeks and in sham controls. At 3 weeks the heart was excised, biopsies obtained, and CRE binding tested. STCM induction occurred in AVP dogs but not in sham or AVP dogs treated with saralasin or nifedipine. Nuclear CREB was significantly decreased at 2 hours in the AVP no-drug group only. LTCM dogs manifested reduced binding of nuclear proteins to CRE, and CRE binding activity in the promoter region of Kv4.3. In conclusion, there is an association between STCM induction and decreased nuclear CREB that is angiotensin-modulated and calcium-dependent. Moreover, the decreased CRE binding after 3 weeks of AVP combined with CRE binding activity in the Kv4.3 promoter can explain the Kv4.3 mRNA andItodownregulation that characterize LTCM.

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.

Prenatal exposure to cocaine has been shown to induce an increase in the myocardial expression and activation of the cAMP response binding protein (CREB), a transcriptional factor that has been shown to regulate gene expression. Several different kinases, including protein kinase A, calcium calmodulin kinase II, and mitogen-activated protein kinase can induce phosphorylation of CREB at serine 133, a necessary step for CREB activation. We examined whether the mitogen-activated protein kinase-extracellular receptor kinase (ERK) pathway may be involved in mediating the serine 133 CREB phosphorylation in cardiac nuclei after perinatal cocaine exposure. Pregnant rats were treated daily with saline or cocaine at 60 mg/kg (C60) by intragastric administration during the entire gestational period, and treatment was continued in the nursing dams after delivery until the time of the study. Nuclear extracts were isolated from hearts of 1-d- and 7-d-old neonatal rats. We performed immunoblotting experiments using an antibody that recognized CREB with phosphorylation specifically at the serine 133 site and an antibody that recognized both the phosphorylated and the unphosphorylated forms of CREB, as well as antibodies for total ERK, phospho-ERK, total ribosomal S6 kinase 1 (RSK1), RSK2, and phospho-RSK. We assessed the interaction of RSK with CREB or CREB-binding protein by performing co-immunoprecipitation experiments. We found that perinatal cocaine exposure increased both phospho-ERK and phospho-RSK expression, indicative of an increased activity of these two enzymes. Furthermore, we demonstrated that phospho-RSK was immunoprecipitated with CREB in all neonatal cardiac nuclei and that the greatest interaction was found in day 7 hearts after perinatal cocaine exposure. Our results thus illustrate that the ERK-RSK pathway was active in the postnatal rat heart at 1 and 7 d of age and that this pathway may mediate the increase in myocardial CREB activation after perinatal cocaine exposure in the day 7 hearts.

cAMP response binding protein (CREB) is a transcriptional factor known to regulate gene expression. Phosphorylation of CREB at serine 133 is necessary for CREB activation, and quantification of phospho-CREB (p-CREB) expression is an index of CREB activation. Because CREB expression and activation in specific brain regions are modified after chronic cocaine administration, we sought to determine whether chronic perinatal cocaine exposure affects the expression of CREB and p-CREB in the postnatal rat heart. Pregnant rats were treated daily with saline (control) or cocaine at 20 mg/kg (C20) or 60 mg/kg (C60) by intragastric administration throughout gestation. The expression of total CREB and p-CREB was quantified in nuclear extracts isolated from 1- and 7-d-old neonatal rat hearts. Cardiac nuclear p-CREB was increased in the C20 and C60 groups on d 1 and 7 of age compared with their respective age-matched control groups. The increase in p-CREB expression corresponded to an increase in cAMP response element binding activity. We also assayed nuclear protein kinase A activity, which was up-regulated in d 1 animals with prenatal cocaine exposure, but was comparable in all groups at d 7. Our results suggest that perinatal cocaine exposure stimulates CREB activation in the neonatal heart, and it may be mediated by different mechanisms at d 1 and d 7. The changes in myocardial CREB activation induced by perinatal cocaine exposure are likely to result in modified gene expression in the neonatal heart that may account for the cardiac dysfunction reported in human neonates born to cocaine-abusing mothers.

Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase delta (pol delta), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol delta.PCNA.template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol delta.template-primer complex.