Biomedical subjects

Loren J Miraglia

Publications and source records attributed to Loren J Miraglia.

3 recordsLinked to original sources

PubMed2006

Feedback repression is required for mammalian circadian clock function.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

3T3 Cells↗

PubMed2004

A functional genomics strategy reveals Rora as a component of the mammalian circadian clock.

The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes. The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input. Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function. Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN. Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.

ARNTL Transcription Factors↗

PubMed2003

Protein kinase C delta is a prosurvival factor in human breast tumor cell lines.

Protein kinase C (PKC) promotes cell survival in response to ionizing radiation in a variety of experimental models including human carcinoma, human glioblastoma, and transformed mouse embryo fibroblast cell lines. We have introduced specific antisense oligonucleotides into human mammary tumor cell lines in vitro to analyze the role of individual PKC isoforms in radiation-induced cell death in breast cancer. MDA-MB-231 and MCF-7 cells treated with oligonucleotide directed against the PKC delta isoform exhibited impaired survival in response to 5.6 Gy gamma-radiation as measured by mitochondrial metabolism of tetrazolium dye. The role of PKC delta in the breast tumor cell lines was of particular interest, because contradictory reports exist in the literature regarding the role of PKC delta in cell survival and apoptosis. A comparison of the effects of the PKC delta antisense oligonucleotide and a nucleotide scrambled version of this nucleotide revealed only the antisense oligonucleotide decreased cell survival. The PKC delta antisense oligonucleotide decreased cell survival after exposure to low (1.5 Gy) radiation doses and in the absence of radiation insult. We found 3 micro M rottlerin, a selective PKC delta inhibitor, to reduce MCF-7 and MDA-MB-231 cell survival. Furthermore, MCF-7 cells transformed to express a dominant-negative mutant of PKC delta exhibited reduced survival. Comet analysis showed that PKC delta oligonucleotide treatment caused an accumulation of cells containing damaged DNA similar to that seen in 1.5 Gy radiation-treated cells. We conclude that PKC delta acts as a prosurvival factor in human breast tumor cells in vitro.

Acetophenones↗