Manual motor speed dysfunction as a neurocognitive endophenotype in euthymic bipolar disorder patients and their healthy relatives. Evidence from a 5-year follow-up study.

BACKGROUND: Few studies have examined Manual Motor Speed (MMS) in bipolar disorder (BD). The aim of this longitudinal, family study was to explore whether dysfunctional MMS represents a neurocognitive endophenotype of BD. METHODS: A sample of 291 subjects, including 131 BD patients, 77 healthy first-degree relatives (BD-Rel), and 83 genetically-unrelated healthy controls (HC), was assessed with the Finger-Tapping Test (FTT) on three occasions over a 5-year period. Dependence of FTT on participants´ age was removed by means of a lineal model of HC samples, while correcting simultaneously the time and learning effect. Differences between groups were evaluated with an ANOVA test. RESULTS: The patients' performance was significantly worse than that of HC over time (p≤0.006), and these deficits remained when non-euthymic BD patients (n=9) were excluded from analysis. Some significant differences between BD patients and BD-Rel (p≤0.037) and between BD-Rel and HC (p≤0.033) were found, but they tended to disappear as time progressed (p≥0.057). Performance of the BD-Rel group was intermediate to that of BD and HC. Most sociodemographic and clinical variables did not affect these results in patients. (p≥0.1). However, treatment with carbamazepine and benzodiazepines may exert a iatrogenic effect on MMS performance (p≤0.006). LIMITATIONS: Only right-handed subjects were included in this study. Substantial attrition over time was detected. CONCLUSIONS: There were significant differences between the patients´ MMS performance and that of healthy relatives and controls, regardless of most clinical and sociodemographic variables. Dysfunctional MMS could be considered an endophenotype of BD. Further studies are needed to rule out possible iatrogenic effects of some psychopharmacological treatments.

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor.

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.

Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor.

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

Altered regulation of cyclin G in human breast cancer and its specific localization at replication foci in response to DNA damage in p53+/+ cells.

Cyclin G, a recent addition to the cyclin family, was initially identified in screens for new src kinase family members and soon thereafter by differential screening for transcriptional targets of the tumor suppressor gene, p53. We have identified cyclin G as being overexpressed in breast and prostate cancer cells using differential display polymerase chain reaction screening. We demonstrate here that cyclin G is overexpressed in human breast and prostate cancer cells and in cancer cells in situ from tumor specimens. Cyclin G expression was tightly regulated throughout the cell cycle in normal breast cells, peaking at the S and G2/M phases of the cell cycle with lower levels in G1. The cell cycle-dependent expression was absent in breast cancer cells. Following DNA damage in normal p53+/+ cells, cyclin G is triggered to cluster in discrete nuclear DNA replication foci that contain replication-associated proteins such as proliferating cell nuclear antigen (PCNA). While p53-/- cells displayed a faint cyclin G nuclear staining pattern, there was no increased expression and no change in distribution of the staining pattern after DNA damage. The specific subcellular localization of cyclin G at DNA replication foci provides an additional link between p53-mediated growth arrest and cell cycle regulation and suggests that cyclin G may act as an effector of p53-mediated events by functional association with replication foci protein(s).

Binding of the ubiquitous cellular transcription factors Sp1 and Sp3 to the ZI domains in the Epstein-Barr virus lytic switch BZLF1 gene promoter.

Induction of the Epstein-Barr virus lytic cycle in latently infected B cells requires the expression of the immediate-early lytic gene BZLF1. We have previously identified several cis-elements within the BZLF1 promoter that are required for induction by known inducers of the lytic cycle [E. Flemington and S. H. Speck (1990)J. Virol. 64, 1217-1226]. These include four elements termed the ZI domains (ZIA, ZIB, ZIC, and ZID) that share extensive homology and that have recently been shown to bind several cellular transcription factors [A. M. Borras, J. L. Strominger, and S. H. Speck (1996) J. Virol. 70, 3894-3901]. Here Sp1 and Sp3 are identified as the cellular factors present in crude B cell nuclear extract preparations that bind to the ZIC domain. In addition, three of the four complexes observed in electrophoretic mobility shift analyses employing probes containing either the ZIA or the ZID domains also represent Sp1 or Sp3 binding. Binding of Sp1 and Sp3 to the ZI domains was shown to be significantly weaker than binding of these factors to a consensus Sp1 site. A heterologous promoter construct containing three repeats of a consensus Sp1 site, cloned upstream of a single copy of the ZII (CREB/ AP1) element from the BZLF1 promoter linked to the beta-globin TATA box, exhibited phorbol ester inducibility. The latter observation was consistent with the functional behavior exhibited by a heterologous promoter construct containing multiple copies of the ZIC domain liked to the ZII element. However, the basal activity of the heterologous promoter construct driven by the consensus Sp1 sites was ca. 10-fold higher than that of the heterologous reporter construct containing multimerized ZIC sites. Thus, the low affinity of Sp1 binding to the ZI domains may contribute to the low-level basal activity of the BZLF1 promoter.

Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction.

Induction of the Epstein-Barr virus lytic cycle is mediated through the immediate-early BZLF1 gene and the coordinately regulated BRLF1 gene. The BZLF1 gene product, Zta, transactivates its own promoter, as well as the promoters of a number of lytic genes, thereby initiating a cascade of viral gene expression. Previous work identified four related elements (ZIA, ZIB, ZIC, and ZID) and a cyclic AMP response element binding-AP-1 element (ZII) that are involved in the induction of the BZLF1 promoter (Zp) by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (E. Flemington and S. H. Speck, J. Virol. 64:1217-1226, 1990). Here we report a detailed characterization of TPA induction mediated by the ZI domains. Mutation of individual ZI domains within the context of the intact promoter significantly diminished TPA induction. Cloning of individual ZI domains upstream of a minimal promoter demonstrated that the ZIA, ZIC, and ZID domains, but not the ZIB domain, are TPA responsive. Furthermore, cloning of the ZII domain downstream of the ZI domains significantly augmented TPA induction. The critical regions within the ZIA and ZIC elements involved in binding of cellular factors were identified by using methylation interference and electrophoretic mobility shift analyses of ZI domain mutants. Four specific complexes were observed with the ZIA and ZID domains, all of which could be specifically competed for by either the ZIA or ZID domain. Methylation interference analyses of bound complexes revealed the presence of two overlapping binding sites for cellular factors in the ZIA domain, and functional studies provided evidence that both of these sites are involved in TPA induction. Functional analyses of the ZIC domain revealed that the 5' region of this domain is largely responsible for mediating TPA induction. Binding data correlated well with functional activity and revealed that the ZIC domain binds only a subset of the cellular factors that bind to the ZIA and ZID domains. Analysis of factor binding to the ZIB domain revealed only a single shifted complex, which correlated with the most slowly migrating complex observed with the ZIA and ZID domains. These data provide a direct demonstration of TPA induction mediated by the ZIA, ZIC, and ZID domains and also provide the first evidence that the ZI domains exhibit distinct functional characteristics.

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

Characterization of the Epstein-Barr virus BZLF1 protein transactivation domain.

Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on expression of the viral transactivator Zta, which is encoded by the BZLF1 gene. Described here is an initial mapping of the regions of Zta involved in activating transcription. The data indicate that the amino-terminal 153 amino acids of Zta are important for activity, and in particular the region from residues 28 to 78 appears to be critical for Zta function. However, other features of Zta may be important for activity since a Gal4-Zta chimeric protein, generated by fusing the amino-terminal 167 residues of Zta to the DNA binding domain of the yeast transactivator Gal4, transactivated a minimal promoter containing one upstream Gal4 binding site but was unable to exhibit synergistic transactivation when assayed with a reporter containing five upstream Gal4 binding sites.