Covalent and noncovalent receptor-glucocorticoid complexes preferentially bind to the same regions of the long terminal repeat of murine mammary tumor virus proviral DNA

Dexamethasone 21-mesylate, an irreversible antiglucocorticoid in HTC cells, forms a covalent receptor-steroid complex which can be activated in cell-free systems. The molecular basis of its antiglucocorticoid activity is unknown; it might result from altered DNA sequence preferences and/or affinities of the covalent receptor-steroid complex. To test this hypothesis, the affinities of both covalent receptor-antagonist and noncovalent receptor-agonist complexes for defined DNA sequences were measured in a DNA binding competition assay. This assay requires neither purified complexes nor large quantities of DNA, yet it provides quantitative comparisons of the affinities of different double-stranded DNAs for binding receptor-steroid complexes. In this assay, activated covalent receptor-dexamethasone 21-mesylate complexes in crude cytosol bound to calf thymus DNA and cloned subregions of the long terminal repeat (LTR) of murine mammary tumor virus (MMTV) proviral DNA with approximately the same relative affinities as did noncovalent receptor-dexamethasone complexes. Both types of complex exhibited similar orders of preferential binding to DNA sequences. LTR subregions, as well as the entire LTR, were 2-20 times more potent competitors than calf thymus DNA. Cloned sequences from the 3' terminus of the LTR were more effective competitors than either the entire LTR or comparably sized DNAs from the 5' terminus. The DNA sequences with the greatest affinities for both covalent and noncovalent complexes are located within the region of -221 to -67. These studies support the theory that recognition by regulatory elements of specific DNA sequences upstream of responsive genes is an integral step of hormone action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoprotein phosphatase activity of human prostate acid phosphatase

Human prostate acid phosphatase (EC 3.1.3.2) has been shown to dephosphorylate different phosphoproteins with the maximum rate at pH 4.0-4.5. The activity with phosvitin is distinctly higher than with beta-casein, casein and most of all than with riboflavin-binding protein. The native phosvitin is homogeneous on isoelectric focusing with pI value of 2.1, whereas phosvitin partially dephosphorylated (in about 15%) by the prostate acid phosphatase shows multiple bands with pI values of 3.5 - 6.8 or higher. The phosphate groups bound to serine residues are removed enzymatically twice as fast as phosphothreonine residues. The apparent Km value for phosvitin was 2.4 X 10(-7) M, and is by three orders of magnitude lower than Km of p-nitrophenyl phosphate (2.9 X 10(-4) M). The competitive inhibitors of prostate acid phosphatase, fluoride and L(+)-tartrate, show the same Ki values for phosvitin and p-nitrophenyl phosphate.     

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.     

Life-history studies of heterophyid trematodes in the Neotropical Region: Ascocotyle (Phagicola) diminuta (Stunkard & Haviland, 1924) and A. (P.) angrense Travassos, 1916

The life-cycle of Ascocotyle (Phagicola) diminuta (Stunkard & Haviland, 1924) was reproduced experimentally, starting from cercariae from naturally infected Littoridina castellanosae and L. parchappei (Hydrobiidae) collected from artificial ponds in the Zoological Garden in Buenos Aires and from Los Ranchos stream, Buenos Aires Province, respectively. Metacercariae were found encysted in the gills of experimentally exposed Cnesterodon decemmaculatus (Poecilidae) and of other naturally infected freshwater fishes. Adults were obtained experimentally in chicks and mice and from a naturally infected egret, Egretta thula. A. (P.) angrense Travassos, 1916 was found parasitising the egret Ixobrychus involucris; it is considered a valid species and the morphological differences between it and A. (P.) diminuta were established. The “Phagicola-form” of the cercaria in the present life-cycle is also known in the genus Pygidiopsis.     

Protein kinase C gamma expression mimics phorbol ester-induced transcriptional activation of a murine VL30 enhancer element

Protein kinase C (PKC), a critical component in the regulation of cell growth, is thought to participate in transmitting the signals of certain cell surface receptor activation events to the nucleus. We have previously shown that stable expression of the PKC gamma isoenzyme in NIH 3T3 cells causes altered growth and enhanced tumorigenicity. In this report, we show that transient expression of the PKC gamma isoenzyme can trans-activate a murine VL30 enhancer element in a pattern similar to that of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In contrast, ras activation of this element is distinct both quantitatively and qualitatively from PKC gamma and 12-O-tetradecanoylphorbol-13-acetate activation. These results provide direct evidence that PKC is the cellular mediator in the activation of phorbol ester-responsive genes and suggest a mechanism by which abnormal PKC expression might lead to altered growth control by changing the pattern of cellular gene expression.     

Generation of phosphatidic acid and diacylglycerols following ligation of surface immunoglobulin in human B lymphocytes: potential role in PKC activation.

We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.     

Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism.

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.