Regions outside of the Bcl-2 homology domains, BH1 and BH2 protect a dopaminergic neuronal cell line from staurosporine-induced cell death.

Recent evidence demonstrates that the proto-oncogene product, Bcl-2 can protect cells from a variety of cell death-inducing stimuli. Because previous studies have demonstrated that protein kinase (PK) pathways may be involved in the regulation of cell death, we tested various PK inhibitors for their effects on cell death in a dopaminergic neuronal cell line, MN9D, as well as the potential of Bcl-2 family members and structural mutants to block this process. Cells expressing either human Bcl-2 (MN9D/Bcl-2), or neomycin (MN9D/Neo; control cells) were treated with either staurosporine (0.

An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein.

In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated.

Inhibition of dopamine synthesis by dopamine D2 and D3 but not D4 receptors.

The goal of the current study was to determine which of the D2-like receptors (D2, D3 or D4) are involved in autoreceptor regulation of dopamine synthesis. We have derived a model system utilizing a mouse mesencephalic cell line, MN9D, which both synthesizes and releases dopamine, to characterize the modulation of tyrosine hydroxylase activity, the rate limiting enzyme in the conversion of tyrosine to dopamine, by the D2-like receptors. Previously, we have shown that stimulation of D2 and D3, but not D4, dopamine receptors transfected into MN9D cells inhibited the release of dopamine.

The human aromatic L-amino acid decarboxylase gene can be alternatively spliced to generate unique protein isoforms.

Aromatic L-amino acid decarboxylase (AADC) is expressed in a wide variety of tissues, including those where it is known to convert L-DOPA and 5-hydroxytryptophan to dopamine and serotonin, respectively. AADC has been cloned from many species and shown to undergo alternative splicing within its 5' untranslated region. Here, we report that the human AADC gene can undergo additional alternative splicing of exon 3, generating two different protein isoforms (termed AADC480 and AADC442).

The role of vitamin D in chorioallantoic membrane calcium transport.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (CAM). CAM contains the vitamin D receptor that increases following 1,25-(OH)2D3 injection into embryos at day 10 of incubation. Further, a single injection of 100 ng of 1,25-(OH)2D3 into vitamin D-deficient quail eggs at day 10 of incubation resulted in a significant increase in both body and yolk calcium.

1,25-dihydroxycholecalciferol analogs cannot replace vitamin D in normocalcemic male rats.

A possible role of vitamin D in the growth and development of rats was investigated. Impaired development was observed in normocalcemic, vitamin D-deficient male and female rats, as revealed by low intestinal calcium transport, low renal vitamin D receptor levels and poor bone mineralization. Analogs of 1,25-dihydroxycholecalciferol, possessing reduced calcium-mobilizing activity in intestine and bone but retaining differentiation activity in cultured cells, were unable to support normal development of normocalcemic, vitamin D-deficient male rats.

The necessity for calcium for increased renal vitamin D receptor in response to 1,25-dihydroxyvitamin D.

To further investigate the regulation of the vitamin D receptor in the kidney of the rat, we analyzed the response of the receptor to 1,25-dihydroxyvitamin D-3 under conditions of calcium supplementation and calcium restriction. Vitamin D-deficient, male weanling rats, fed a calcium-restricted or calcium-supplemented, vitamin D-deficient diet, were treated for 4 weeks with vitamin D (orally) or 1,25-dihydroxyvitamin D-3 (60 pmol/d by mini-osmotic pump). We also extended the treatment to 8 weeks for one group of animals fed the calcium-supplemented diet.

Short-term regulation of the renal vitamin D receptor in rats by 1,25-dihydroxycholecalciferol is calcium insensitive.

Acute regulation of the vitamin D receptor in kidney by 1,25-dihydroxycholecalciferol and dietary calcium was investigated using vitamin D-deficient rats. 1,25-Dihydroxycholecalciferol administered to normocalcemic, vitamin D-deficient rats increased the renal receptor level, whereas serum calcium and phosphorus concentrations remained nearly constant. In hypocalcemic, vitamin D-deficient rats, 1,25-dihydroxycholecalciferol caused a sharp response at 4 h, which remained elevated for the remaining 20 h.

Up-regulation of the vitamin D receptor in response to 1,25-dihydroxyvitamin D3 results from ligand-induced stabilization.

Several studies have shown that the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor protein levels increase in response to 1,25-(OH)2D3. We have studied the mechanism of this regulation in both mouse fibroblasts and rat intestinal epithelial cells. Cell extracts and total RNA were prepared at varying times after addition of 1,25-(OH)2D3.