Calcium dynamics in the ventricular myocytes of SERCA2 knockout mice: A modeling study.

We describe a simulation study of Ca²(+) dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²(+) flux through the L-type Ca²(+) channels and the Na(+)/Ca²(+) exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2(flox/flox) mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²(+) handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na(+)/Ca²(+) exchanger. The FF and KO models were then validated against experimentally measured [Ca²(+)](i) transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²(+)](i) transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²(+)](i) rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²(+)](i). Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na(+)/Ca²(+) exchanger and increased L-type Ca²(+) current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function.

Regulation of neuronal type genes in congestive heart failure rats.

AIM: After myocardial infarction (MI), complex changes in the heart occur during progression into congestive heart failure (CHF). This study sought to identify regulated genes that could have a functional role in some of the changes seen in CHF. METHODS: Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD) in Wistar rats. Gene expression changes in 1- and 7-day MI left ventricular myocardium was analysed using complementary DNA (cDNA) filter arrays. Regulated genes were identified by repeated measurements and a ranked ratio analysis method. RESULTS: A total of 135 genes were identified as differentially expressed. A few genes were robustly regulated at 1-day MI. In 7-day CHF hearts, changes in the expression of neuronal type genes was prominent (32%, n = 28). Eleven of these genes with no described association with CHF were selected for validation. One gene failed the validation. In CHF hearts, the expression of the muscarinic m4 (Chrm4) and nicotinic alpha4 (Chrna4) acetylcholin receptors, the ATP receptor P2rx4, nerve growth factor receptor (Ngfr), discoidin domain receptor 1 (Ddr1), neuronal pentraxin receptor (Nptxr), peripheral myelin protein Pmp-22, leukocyte type 12-lipoxygenase (Alox15), cytochrome P450 4F5 (Cyp4F5) and cardiac Kcne1 were all increased (range 1.6-6.0-fold, P < 0.01 for all genes). The lack of significant regulation of these genes at 1-day post-MI, suggests that the induction of these genes at 7-day post-MI is not a short-term response induced by the infarct itself. CONCLUSION: These neuronal type genes may participate in underlying processes that affect contractility, intracardiac nerve function and development of arrhythmias in CHF hearts.

Leukaemia inhibitory factor alters expression of genes involved in rat cardiomyocyte energy metabolism.

AIM: Cardiac remodelling is associated with changes in contractile proteins and their performance, alterations in energy production and intracellular calcium homeostasis, as well as changes in extracellular matrix proteins. Some of these processes may be mediated through the gp130 receptor complex. Patients with heart failure have increased cardiac gene expression of leukaemia inhibitory factor (LIF), a cytokine that signals through the gp130 receptor. The aim of this study was to identify alterations in gene expression in LIF-stimulated neonatal cardiomyocytes. METHODS: Cardiomyocytes were isolated from 1- to 3-day-old Wistar rats and stimulated for 48 h with LIF. Gene expression was examined by repeated cDNA filter array analysis (n = 5) and key results verified by complementary methods. RESULTS: In LIF-stimulated cultures we observed increased cell area and changes in gene expression. The intracellular signal regulators signal transducer and activator of transcription 3, calcium/calmodulin-dependent protein kinase IV, protein kinase Cdelta and the transcription factor ID1 were upregulated. Adenylyl cyclase V was downregulated. LIF also induced altered expression of tissue inhibitor of metalloproteinase-1. Receptor genes for tumour necrosis factor, interleukin-4, neurotensin and somatostatin were upregulated. Finally, LIF reduced the expression of components in the adenosine triphosphate synthase complex, epidermal fatty acid-binding protein and insulin-like growth factor-binding proteins 1 and 6. CONCLUSIONS: Array analysis revealed changes in mRNA levels of several genes not previously associated with activation of the gp130/LIF receptor complex. Our findings indicate a role for LIF in regulation of cardiomyocyte energy metabolism.

A novel yeast system for in vivo selection of recognition sequences: defining an optimal c-Myb-responsive element.

Yeast (Saccharomyces cerevisiae) has proved to be a highly valuable tool in a range of screening methods. We present in this work the design and use of a novel yeast effector-reporter system for selection of sequences recognised by DNA-binding proteins in vivo. A dual HIS3-lacZ reporter under the control of a single randomised response element facilitates both positive growth selection of binding sequences and subsequent quantification of the strength of the selected sequence. A galactose-inducible effector allows discrimination between reporter activation caused by the protein under study and activation due to endogenous factors. The system mimics the physiological gene dosage relationship between transcription factor and target genes in vivo by using a low copy effector plasmid and a high copy reporter plasmid, favouring sequence selectivity. The utility of the novel yeast screening system was demonstrated by using it to refine the definition of an optimal recognition element for the c-Myb transcription factor (MRE). We present screening data supporting an extended MRE consensus closely mimicking known strong response elements and where a sequence of 11 nt influences activity. Novel features include a more strict sequence requirement in the second half-site of the MRE where a T-rich sequence is preferred in vivo.

The highly conserved DNA-binding domains of A-, B- and c-Myb differ with respect to DNA-binding, phosphorylation and redox properties.

In the Myb family, as in other families of transcription factors sharing similar DNA-binding domains (DBDs), diversity of function is believed to rely mainly on the less conserved parts of the proteins and on their distinct patterns of expression. However, small conserved differences between DBDs of individual members could play a role in fine-tuning their function. We have compared the highly conserved DBDs of the three vertebrate Myb proteins (A-, B- and c-Myb) and found distinct functional differences. While A- and c-Myb behaved virtually identically in a variety of DNA-binding assays, B-Myb formed complexes of comparatively lower stability, rapidly dissociating under competitive conditions and showing less tolerance to binding site variations. The three protein domains also differed as substrates for protein kinases. Whereas PKA in theory should target the DBDs of A- and c-Myb, but not B-Myb, only c-Myb was phosphorylated by PKA. CK2 phosphorylated all three proteins, although on different sites in the N-terminal region. Finally, B-Myb was remarkably sensitive to cysteine-directed oxidation compared to the other Myb proteins. Our data suggest that the small differences that have evolved between individual Myb family members lead to clear differences in DBD properties even if their sequence recognition remains the same.

Sequence selectivity of c-Myb in vivo. Resolution of a DNA target specificity paradox.

We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R(2)R(3) in vitro and the more restricted preference of a R(2)R(3)VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1-360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1-360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R(2)R(3) domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell.

[Genetic modification of the mouse]. / Genetisk skreddersøm av mus.

Genetic modification of mice by homologous recombination has rapidly become a central tool within molecular medicine and molecular biology. The use of such techniques allows precise mutation of single genes and study of the direct consequences in intact animals. A decade has passed since the a series of landmark studies demonstrated the feasibility of genetically modifying embryonic stem cells in mice by homologous recombination. This initiated the whole field of gene targeting, or "knockout" technology in mice. This article reviews briefly the historical and technical development of knockout technology, and the application to selected mouse models within cancer biology, immunology and neurobiology. Refinements of this type of genetic modification, with tissue- or time-specific genetic ablation or mutations, represent the next step in technology development. The combined use of Cre/loxP and homologous recombination has opened for a wide spectrum of possibilities, reaching far beyond null mutations. The rapid evolvement of the whole field has opened for genetic engineering--in the presise sense of the word--in whole animals.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIalpha) was present in all the cells investigated, in contrast to the other R subunits (RIbeta, RIIalpha, and RIIbeta). Furthermore, three T cell lines and one B cell line expressed only RIalpha and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIalpha protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes.

Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c-Myb in vitro.

In an attempt to elucidate signal transduction pathways which may modulate DNA binding of the transcription factor c-Myb, we investigated whether c-Myb could be a target for the signaling molecule nitric oxide (NO) in vitro. NO-generating agents severely inhibited specific DNA binding of the c-Myb minimal DNA-binding domain R2R3. This inhibition was readily reversible upon treatment with excess DTT. A redox-sensitive cysteine (C130) was required for this NO sensitivity. Moreover, a DNA-binding domain carrying two of the avian myeloblastosis virus (AMV)-specific mutations (L106H, V117D) appeared to be less sensitive to S-nitrosylation than the wild-type c-Myb. This difference in NO sensitivity may influence the regulation of wild type versus AMV v-Myb protein function.

Characterization of the expression and gene promoter of CD22 in murine B cells.

CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo.

CD22 regulates thymus-independent responses and the lifespan of B cells.

The B-lymphocyte-restricted glycoprotein CD22 is expressed on mature IgM+IgD+ B cells, and is capable of binding to ligands on T and B cells. CD22 can interact with both the B-cell antigen receptor (BCR) complex and signalling molecules, including the protein tyrosine phosphatase SHP1 (PTP1C, SHP), a putative negative regulator of BCR signalling. Thus CD22 may facilitate interactions with lymphocytes and regulate the threshold of BCR signalling. To define the in vivo function of CD22, we generated CD22-deficient mice. Here we show that CD22 is required for normal antibody responses to thymus-independent antigens and regulates the lifespan of mature B cells.

Regulation of growth in a neoplastic B cell line by transfected subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

cAMP inhibits the proliferation of both normal peripheral blood B and T lymphocytes as well as the proliferation of a human neoplastic B precursor cell line (Reh). To positively show that this is mediated via the the catalytic subunit, C alpha, of cAMP-dependent protein kinase, stably transfected Reh cell lines overexpressing C alpha were established. This was achieved by transfection with a construct confering hygromycin resistance together with a zinc-inducible expression of C alpha from the human metallothionine promoter. C alpha transfected clones were shown to confer a 2- to 2.5-fold zinc-dependent increase in C alpha messenger RNA, immunoreactive C, and phosphotransferase activity. The growth rate of clones transfected with C alpha was retarded, and a zinc-dependent inhibition of cell proliferation was demonstrated in the presence of a small trigger dose of forskolin. In contrast, overexpression of the regulatory subunit I alpha had no effect on cAMP-dependent inhibition of cell proliferation. Furthermore, expression of mutant regulatory subunit I alpha AB, which renders cAMP-dependent protein kinase unresponsive to cAMP, clearly protected against that inhibitory effect of cAMP. These data provides evidence that activation of the C subunit (C alpha) of cAMP-dependent protein kinase mediates the inhibitory action of cAMP on cell proliferation in Reh cells.

Retinoblastoma protein is rapidly dephosphorylated by elevated cyclic adenosine monophosphate levels in human B-lymphoid cells.

Elevated cyclic AMP levels induce a rapid block in the mid-G1 phase of the cell cycle in B-lymphoid Reh cells, accompanied by a transient block in G2. The retinoblastoma (Rb) gene product has been implicated as a key regulator of eukaryotic cell growth. The Rb protein enforces its growth-suppressive effect in early G1, where it is underphosphorylated and firmly bound in the nucleus. A possible link between the cyclic AMP-mediated growth arrest and regulation of Rb protein phosphorylation was explored by Western blot analysis. We found that both forskolin and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate induced a rapid (within 3 h) dephosphorylation of Rb protein. These data were confirmed by flow-cytometric analysis of isolated nuclei costained with anti-Rb antibodies and propidium iodide. The percentage of cells containing underphosphorylated Rb protein (i.e., G1 nuclei with bound Rb protein) increased from 9 to 87% after 4 h of forskolin treatment. During the first 4 h of forskolin treatment, the cells were transiently blocked in the G2 phase of the cell cycle, and virtually no cells had passed through mitosis. The increased level of dephosphorylated Rb protein at 4 h was therefore not due to an accumulation in early G1 of cells containing underphosphorylated Rb protein. Instead, our data indicated that dephosphorylation of Rb protein occurred in cells that had already passed the point in G1 of Rb protein phosphorylation. Dephosphorylation of Rb protein was prevented by high concentrations of the protein phosphatase inhibitor okadaic acid, indicating that activation of a phosphatase is involved in the cyclic AMP-mediated dephosphorylation of Rb protein. We suggest that the dephosphorylation of Rb protein is required for the forskolin-mediated arrest of the Reh cells in mid-G1.

Cyclic AMP downregulates c-myc expression by inhibition of transcript initiation in human B-precursor Reh cells.

In the human pre-B cell line Reh, activation of the cyclic AMP signal transduction pathway induces a rapid, transient 10-fold down-regulation of steady-state c-myc mRNA. We have investigated the mechanisms involved in this cAMP-mediated regulation of c-myc expression. Forskolin did not alter c-myc mRNA stability. Initiation of c-myc transcripts was strongly inhibited after 1 h of forskolin treatment, as measured by nuclear run-on assays. Reinitiation of c-myc transcription was apparent after 3-4 h, and full transcriptional elongation was detected after 8 h of forskolin treatment. These data suggest that cyclic AMP downregulates c-myc expression by inhibition of transcriptional initiation.

Reciprocal regulation of mRNA and protein for subunits of cAMP-dependent protein kinase (RI alpha and C alpha) by cAMP in a neoplastic B cell line (Reh).

The present study examines the activity, levels of expression and regulation of cAMP-dependent protein kinase subunits during cAMP-mediated inhibition of Reh cell proliferation. Human Reh cells express mRNAs for the RI alpha and C alpha subunits of cAK at high levels and are practically devoid of cAMP-dependent protein kinase type II. Treatment with isoproterenol, forskolin, or a cAMP analog increased RI alpha mRNA in a time- and concentration-dependent manner (maximal, 4-fold, at 4-8 h). Messenger RNA for C alpha was also stimulated by cAMP, although with slower kinetics (maximal, 2-fold, at 16-24 h). Nuclear run-on assays showed a 2-fold increase in RI alpha gene transcription, whereas that of C alpha was unchanged. In spite of the stimulatory effects of cAMP on mRNAs for both RI alpha and C alpha, phosphotransferase activity and specific [3H]cAMP binding decreased rapidly after treatment with either cAMP or forskolin. Interestingly, the decrease in R and C activity preceded the increase in RI alpha and C alpha mRNA levels, raising the question whether increased mRNA levels may be secondary to the decrease in RI alpha or C alpha protein. The finding that the protein synthesis inhibitor cycloheximide gave changes in RI alpha and C alpha mRNA similar to cAMP and that co-treatment with cycloheximide and cAMP resulted in additive effects tend to support this notion.

Constitutive expression of c-myc does not relieve cAMP-mediated growth arrest in human lymphoid Reh cells.

The Reh cell system is suitable for evaluating events important for control of proliferation independently of mechanisms involved in differentiation, as Reh cells are unable to differentiate. In the human pre-B cell line Reh, activation of adenylate cyclase by forskolin induces a five to tenfold rapid, transient down-regulation of steady-state c-myc RNA within 4 hours. Concurrently, the cells are strongly growth arrested in the G1 phase of the cell cycle. To clarify if the observed growth arrest could be relieved by constitutive expression of c-myc, an exogenous c-myc gene under constitutive promoter control was introduced into Reh cells by electroporation. The c-myc-expressing construct pDMmycHyg contained human c-myc exons 2 and 3 driven by the Mo-MLV LTR and conferred hygromycin resistance. Exogenous c-myc RNA transcripts and protein were constitutively expressed in the transfected clones at levels roughly twice as high as the level in nontransfected cells. Total c-myc protein levels were unchanged upon treatment of transfected clones with forskolin. Yet, the transfected cells were not released from growth arrest. Furthermore, the transfected Reh cells did not differentiate upon forskolin treatment. Constitutive overexpression of c-myc is therefore not sufficient for relieving forskolin-mediated effects on growth arrest in Reh cells.

Novel isozymes of cAMP-dependent protein kinase exist in human cells due to formation of RI alpha-RI beta heterodimeric complexes.

We report that a human neoplastic B cell line (Reh) contains cAMP-dependent protein kinase (cAK) type I (cAKI), but is practically devoid of cAK type II (cAKII). However, these cells contain a novel cAKI isozyme consisting of an RI alpha-RI beta heterodimer in association with phosphotransferase activity (RI alpha RI beta C2) eluting from DEAE-cellulose columns at a salt concentration characteristic of a cAKII. Immunoprecipitation of 8-azido-[32P]cAMP-labeled extracts and DEAE fractions employing specific antibodies directed against RI alpha and RI beta clearly demonstrated the presence of RI alpha-RI beta heterodimers. RI alpha was precipitated with RI beta antiserum and vice versa. Furthermore, disruption of disulfide bridges by reduction-alkylation abolished this coimmunoprecipitation. In addition, formation of heterodimeric complexes of RI alpha and RI beta could be demonstrated in vitro using recombinant RI proteins. Finally, the presence of low levels of RI alpha-RI beta heterodimers could also be demonstrated in human peripheral blood T lymphocytes. RI alpha-RI beta heterodimers complexed with the catalytic subunit represent a novel isozyme of cAKI (RI alpha RI beta C2), which enhances the possibilities for diversification of cAMP-mediated effects.

Expression of CD18 (integrin beta 2 chain) correlates with prognosis in malignant B cell lymphomas.

Several studies have shown that cell cycle related parameters including DNA synthesis and activation antigen expression can predict patient survival in lymphoma patients. In this study of 69 malignant B cell lymphomas we have examined the cell surface expression of several cell interaction and activation molecules by flow cytometry. Expression of CD18 (integrin beta 2 chain) was found to correlate strongly with patient survival (median follow up 50 months) even when adjusting for other important prognostic factors (P = 0.0001). The percentage of cells positive for CDw75 proved important both as a single parameter and in the multivariate analysis. Histology, classified as low versus high grade malignancy, bulky versus not bulky disease and high versus low thymidine incorporation, was also found to correlate with prognosis in this study.

The leucocyte L1 protein: identity with the cystic fibrosis antigen and the calcium-binding MRP-8 and MRP-14 macrophage components.

The partial amino acid sequence of L1 protein light and heavy chains reveals an overall structure identical to the two macrophage proteins, MRP-8 and MRP-14, deduced from the sequence of the cDNA encoding the polypeptides. The light chain of L1 protein (L1-L) was shown to contain two modified amino acid residues.