Regulation of estrogen receptor-alpha expression in MCF-7 cells by taxol.

Results presented in this study demonstrate that treatment of MCF-7 cells with taxol resulted in induction of estrogen receptor-alpha (ER alpha) gene transcription with a subsequent increase in ER alpha mRNA; this effect was promoter specific since taxol did not affect total transcription in MCF-7 cells and lacked an effect on transcription of the human acidic ribosomal phosphoprotein protein PO, progesterone receptor, and pS2 genes. In contrast to the increase in transcription of the ER alpha gene, taxol inhibited translation of the ER alpha mRNA. This effect is also transcript specific since taxol did not alter total protein synthesis and did not affect the concentration of progesterone receptor protein in the cell. The overall result of taxol treatment was to decrease the concentration of ER alpha protein in the MCF-7 cells. Evidence is presented that the effects of taxol on ER alpha gene transcription may be mediated through the induction of p53.

Regulation of estrogen receptor-alpha expression by the tumor suppressor gene p53 in MCF-7 cells.

The results presented here demonstrate that p53 upregulates estrogen receptor-alpha (ER alpha) expression in the human breast cancer cell line MCF-7. Two approaches were used to alter the activity of p53 in the cells. In the first approach, stable transfectants expressing an antisense p53 were established. In the stable clones, expression of antisense p53 resulted in a decrease in the expression of ER alpha protein. In the second approach, MCF-7 cells were transiently transfected with wild-type p53. Overexpression of p53 increased the amount of ER alpha. To determine whether the effects of p53 on the expression of ER alpha were due to changes in transcription, deletion mutants of the ER alpha promoter were used. This experimental approach demonstrated that p53 up-regulates ER alpha gene expression by increasing transcription of the gene through elements located upstream of promoter A. Transfection assays using p53 mutants further demonstrated that the p53-induced increase in ER alpha gene transcription was not dependent on the ability of p53 to bind to DNA but on its ability to interact with other proteins.

Is microvascular flow rate related to ghrelin, leptin and adiponectin levels?

Ghrelin, leptin and adiponectin are three hormones which are frequently associated with metabolism, obesity and appetite. Recently, it has been shown that they may possess other physiologic roles, specially in connection with the circulation. Ghrelin infusion increases forearm blood-flow in a dose-dependent manner. Leptin has been shown to be involved not only in thermogenesis but angiogenesis as well. Adiponectin, apart from its insulin-sensitizing action, appears to modulate inflammation by inhibiting monocyte adhesion to endothelial cells. Six monkeys, which had been classified as being in the pre-diabetic state, where administered a triglyceride lowering regimen. Microvascular function was assessed using a laser Doppler flow-meter during a temperature provocation test. Percent change in flow from baseline following temperature elevation, as well as percent change in flow/degree rise in temperature were used to evaluate microvascular reserve and reactivity. Using univariate analysis, it appears that increased perfusion is significantly correlated with adiponectin, followed by leptin. Flow was also positively correlated with ghrelin, but the relationship did not attain significance. As expected, flow was also negatively and significantly correlated with fibrinogen. Trends show that flow was also negatively correlated to circulating triglyceride levels (p=0.08). The data indicate that the three hormones appear to possess microvascular actions that may impact on their other physiologic functions.

Estradiol regulates estrogen receptor mRNA stability.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

Assignment of Etfdh, Etfb, and Etfa to chromosomes 3, 7, and 13: the mouse homologs of genes responsible for glutaric acidemia type II in human.

Electron transfer flavoprotein (composed of alpha and beta subunits) is an obligatory electron acceptor for several dehydrogenases and is located in the mitochondrial matrix. Electrons accepted by electron transfer flavoprotein (ETF) are transferred to the main mitochondrial respiratory chain by way of ETF dehydrogenase (ETFDH). In humans, deficiency of ETF or ETFDH leads to glutaric acidemia type II, an inherited metabolic disorder that can be fatal in its neonatal form and is characterized by severe hypoketotic hypoglycemia and acidosis. We used cDNA probes for the Etfdh, Etfb, and Etfa genes to determine localization of these mouse genes to chromosomes 3, 7, and 13.

Assignment of FGF8 to human chromosome 10q25-q26: mutations in FGF8 may be responsible for some types of acrocephalosyndactyly linked to this region.

Emerging evidence suggests that Fgf8, a recently identified member of the fibroblast growth factor family, plays an important role in outgrowth and patterning of the face, limbs, and central nervous system of the vertebrate embryo. We report the mapping of FGF8 to human chromosome 10q25-q26, using Southern blot analyses of genomic DNAs from rodent/human somatic cell hybrid lines. Apert, Crouzon, Jackson-Weiss, and Pfeiffer syndromes are craniosynostoses genetically linked in part to 10q25-q26 and are associated with point mutations in the extracellular domain of FGFR2. Given the assignment to the same chromosomeal band(s) as FGFR2 and the probable ligand-receptor relationship of the gene products of FGF8 and FGFR2, we hypothesize that some cases of these craniosynostoses linked to 10q25-q26 that do not have mutations in FGFR2 may involve mutations in FGF8.

Cloning, structure, and chromosome localization of the mouse glutaryl-CoA dehydrogenase gene.

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped.

Assignment of the transcription factor GATA4 gene to human chromosome 8 and mouse chromosome 14: Gata4 is a candidate gene for Ds (disorganization).

We report the mapping of the human and mouse genes for transcription factor GATA-4, a newly identified member of DNA-binding proteins involved in lineage determination. The human GATA4 gene was assigned to the short arm of human chromosome 8 using genomic DNAs from human-rodent somatic cell hybrid lines. Southern blot analyses indicated the presence of a human-specific 7.6-kb fragment that was observed only in DNA from the hybrid cells containing human chromosome 8 or the proximal region of its short arm. The mouse Gata4 gene was mapped to chromosome 14, closely linked to Clu (clusterin), using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x M. spretus backcross. This mapping assignment places the Gata4 gene in the vicinity of the mouse Ds (disorganization) locus, a dominant gain-of-function mutation affecting embryonic development. We speculate that Ds is caused by a mutation in the Gata4 gene, ectopic expression of GATA-4, or a mutation in another lineage determination gene closely linked to Gata4.

Analysis of the sequences within and flanking the cyanoglobin-encoding gene, glbN, of the cyanobacterium Nostoc commune UTEX 584.

A 3.5-kb segment of DNA containing nifU glbN nifH nifD was cloned from a gene library of Nostoc 584 and sequenced. The nifU-glbN intergenic region contains short tandemly repeated repetitive sequences (5'-AATTACG). A sequence corresponding to a NifA-like upstream activator sequence (with the consensus recognition sequence for BifA in Anabaena 7120), elements of a nifH promoter and a sequence that may function as a transcription terminator, were identified downstream from glbN. GlbN, unique to certain Nostoc spp., is more homologous to protozoan myoglobins than to any other prokaryotic, vertebrate or plant globins.

Myoglobin in a cyanobacterium.

Myoglobin was found in the nitrogen-fixing cyanobacterium Nostoc commune. This cyanobacterial myoglobin, referred to as cyanoglobin, was shown to be a soluble hemoprotein of 12.5 kilodaltons with an amino acid sequence that is related to that of myoglobins from two lower eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis. Cyanoglobin is encoded by the glbN gene, which is positioned between nifU and nifH-two genes essential for nitrogen fixation-in the genome of Nostoc. Cyanoglobin was detected in Nostoc cells only when they were starved for nitrogen and incubated microaerobically.

Polysome turnover in immobilized cells of Nostoc commune (cyanobacteria) exposed to water stress.

Water stress induced changes in the polysome content of immobilized cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584. Cells maintained an intact protein synthesis complex during 2 h of drying at -99.5 MPa. Polysomes were not recovered from cells subjected to extended periods of desiccation.