Biomedical subjects

M Daheshia

Publications and source records attributed to M Daheshia.

22 records · Page 2Linked to original sources

PubMed1996

Disease in the scurfy (sf) mouse is associated with overexpression of cytokine genes.

The murine X-linked lymphoproliferative disease scurfy is similar to the Wiskott-Aldrich syndrome in humans. Disease in scurfy (sf) mice is mediated by CD4+ T cells. Based on similarities in scurfy mice and transgenic mice that overexpress specific cytokine genes, we evaluated the expression of cytokines in the lesions of sf mice by Northern blotting, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and by hybridization in situ. Overall, the phenotypic characteristics of scurfy disease correlated well with increased interleukin (IL)-4 (lymphadenopathy), IL-6 (B cell proliferation, hypergammaglobulinemia), IL-7 (dermal inflammatory cell infiltration), and high levels of tumor necrosis factor-alpha (wasting).

Animals↗

PubMed1994

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.

Aspartate Aminotransferases↗

PubMed1993

Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin.

The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.

Animals↗

PubMed1993

CCAAT/enhancer-binding protein-related proteins bind to the unusual promoter of the aspartate aminotransferase housekeeping gene.

The cytosolic aspartate aminotransferase (cAspAT) gene is ubiquitously expressed but it is regulated by hormones in a tissue-specific manner. In vitro DNase I footprinting studies of a 260-base pair fragment carrying the basal promoter activity revealed that three CCAAT sequences bind liver nuclear proteins (protected regions P2, P3, P4). Competition studies, the heat resistance of these proteins, and identical footprints obtained using a recombinant CCAAT/enhancer-binding protein (C/EBP) alpha fragment indicate that they belong to the C/EBP family of transcription factors. A fourth protected region P1, overlapping the P2 region, was observed in the liver in the presence of competing oligonucleotides containing the C/EBP site. In cotransfection experiments, the C/EBP beta protein trans-activated 10-15-fold the cAspAT gene promoter in HepG2 cells. Deletion studies revealed that regions P2 and P4 are critical for promoter activity. In gel retardation experiments, the P4 region bound different C/EBP-related proteins in different tissues: the brain protein is heat sensitive in contrast to the liver protein. The synthetic oligonucleotide OL 1-2, which covers the P1 and P2 regions, binds C/EBP-like proteins as well as NF1 and CP1 (or NFY) transcription factors, but the preferential binding of one of these protein is tissue-specific. In summary, using the cAspAT gene promoter, we have shown that ubiquitously active promoters may be recognized by different proteins in different tissues.

Animals↗