Sleep onset insomnia, daytime sleepiness and sleep duration in relationship to Toxoplasma gondii IgG seropositivity and serointensity.

Toxoplasma gondii (T. gondii) infects central nervous tissue and is kept in relative dormancy by a healthy immune system. Sleep disturbances have been found to precipitate mental illness, suicidal behavior and car accidents, which have been previously linked to T. gondii as well. We speculated that if sleep disruption, particularly insomnia, would mediate, at least partly, the link between T. gondii infection and related behavioral dysregulation, then we would be able to identify significant associations between sleep disruption and T. gondii. The mechanisms for such an association may involve dopamine (DA) production by T. gondii, or collateral effects of immune activation necessary to keep T. gondii in check. Sleep questionnaires from 2031 Old Order Amish were analyzed in relationship to T. gondii-IgG antibodies measured by enzyme-linked immunosorbent assay (ELISA). Toxoplasma gondii seropositivity and serointensity were not associated with any of the sleep latency variables or Epworth Sleepiness Scale (ESS). A secondary analysis identified, after adjustment for age group, a statistical trend toward shorter sleep duration in seropositive men (p = 0.07). In conclusion, it is unlikely that sleep disruption mediates links between T. gondii and mental illness or behavioral dysregulation. Trending gender differences in associations between T. gondii and shorter sleep need further investigation.

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains.

Knockdown of core binding factorß alters sphingolipid metabolism.

Core binding factor (CBF) is a heterodimeric transcription factor containing one of three DNA-binding proteins of the Runt-related transcription factor family (RUNX1-3) and the non-DNA-binding protein, CBFß. RUNX1 and CBFß are the most common targets of chromosomal rearrangements in leukemia. CBF has been implicated in other cancer types; for example RUNX1 and RUNX2 are implicated in cancers of epithelial origin, including prostate, breast, and ovarian cancers. In these tumors, CBF is involved in maintaining the malignant phenotype and, when highly over-expressed, contributes to metastatic growth in bone. Herein, lentiviral delivery of CBFß-specific shRNAs was used to achieve a 95% reduction of CBFß in an ovarian cancer cell line. This drastic reduction in CBFß expression resulted in growth inhibition that was not associated with a cell cycle block or an increase in apoptosis. However, CBFß silencing resulted in increased autophagy and production of reactive oxygen species (ROS). Since sphingolipid and ceramide metabolism regulates non-apoptotic cell death, autophagy, and ROS production, fumonsin B1 (FB1), an inhibitor of ceramide synthase, was used to alter ceramide production in the CBFß-silenced cells. FB1 treatment inhibited the CBFß-dependent increase in autophagy and provided a modest increase in cell survival. To document alterations to sphingolipids in the CBFß-silenced cells, ceramide, and lactosylceramide levels were directly examined by mass spectrometry. Substantial increases in ceramide species and decreases in lactosylceramides were identified. Altogether, this report provides evidence that CBF transcriptional pathways control cellular survival, at least in part, through sphingolipid metabolism.

Role of Mediator in regulating Pol II elongation and nucleosome displacement in Saccharomyces cerevisiae.

Mediator is a modular multisubunit complex that functions as a critical coregulator of RNA polymerase II (Pol II) transcription. While it is well accepted that Mediator plays important roles in the assembly and function of the preinitiation complex (PIC), less is known of its potential roles in regulating downstream steps of the transcription cycle. Here we use a combination of genetic and molecular approaches to investigate Mediator regulation of Pol II elongation in the model eukaryote, Saccharomyces cerevisiae. We find that ewe (expression without heat shock element) mutations in conserved Mediator subunits Med7, Med14, Med19, and Med21-all located within or adjacent to the middle module-severely diminish heat-shock-induced expression of the Hsf1-regulated HSP82 gene. Interestingly, these mutations do not impede Pol II recruitment to the gene's promoter but instead impair its transit through the coding region. This implies that a normal function of Mediator is to regulate a postinitiation step at HSP82. In addition, displacement of histones from promoter and coding regions, a hallmark of activated heat-shock genes, is significantly impaired in the med14 and med21 mutants. Suggestive of a more general role, ewe mutations confer hypersensitivity to the anti-elongation drug 6-azauracil (6-AU) and one of them-med21-impairs Pol II processivity on a GAL1-regulated reporter gene. Taken together, our results suggest that yeast Mediator, acting principally through its middle module, can regulate Pol II elongation at both heat-shock and non-heat-shock genes.

Association of core-binding factor ß with the malignant phenotype of prostate and ovarian cancer cells.

Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.