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D J Shapiro

Publications and source records attributed to D J Shapiro.

At least 73 records · Page 4Linked to original sources
PubMed1983

Nuclease sensitivity and DNA methylation in estrogen regulation of Xenopus laevis vitellogenin gene expression.

Estrogen activates transcription of the vitellogenin genes in livers of male Xenopus laevis. We have examined the conformation of the vitellogenin genes in chromatin and the methylation state of one vitellogenin gene during the process of estrogen stimulation and withdrawal. Sensitivity of the vitellogenin genes to DNase I digestion parallels transcription. The vitellogenin genes are insensitive to DNase I digestion in unstimulated liver cells, become more sensitive to DNase I digestion following estrogen activation of vitellogenin gene transcription, and are insensitive to DNase I digestion in liver cells withdrawn from estrogen. In contrast, the methylation state of nine potential methylation sites within the vitellogenin A1 gene is identical in red blood cells, unstimulated and withdrawn liver, estrogen-stimulated liver, and hepatocytes purified from estrogen-stimulated liver. Rapid transcription of the vitellogenin genes in estrogen-stimulated liver cells occurs with six of the nine methylation sites examined fully methylated.

Animals↗
PubMed1983

Estrogen regulates the absolute rate of transcription of the Xenopus laevis vitellogenin genes.

Estrogen regulates the synthesis of the egg yolk precursor protein, vitellogenin, by causing both a 20-60-fold increase in the absolute rate of total nuclear RNA synthesis and a selective increase of at least several thousand fold in the absolute rate of vitellogenin gene transcription. Vitellogenin gene transcription is undetectable in unstimulated and withdrawn Xenopus laevis liver cells and in cultured Xenopus kidney cells allowing us to set a very low upper limit (less than 1 transcript/vitellogenin gene/day) on potential basal rates of vitellogenin gene transcription. The elevated rates of vitellogenin mRNA accumulation previously observed during restimulation of withdrawn liver cells (secondary estrogen stimulation) appear to be due to an increased rate of vitellogenin gene transcription. Both the maximum transcription rate and the rapidity of the early response increase on secondary estrogen stimulation. Relative transcription rates were determined by hybridization of pulse-labeled nuclear RNA to vitellogenin cDNA clones immobilized on nitrocellulose filters. The conversion of relative transcription rates to absolute transcription rates, was facilitated by development of a sensitive high performance liquid chromatography method for quantitation of the specific radioactivity of the cellular UTP pool.

Animals↗
PubMed1983

Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation.

We have demonstrated, by DNA excess filter hybridizations to pulse-labeled cell RNA, that estrogen selectively stabilizes Xenopus liver vitellogenin mRNA against cytoplasmic degradation. The half-life of vitellogenin mRNA is approximately 3 weeks in the presence of estrogen and 16 hr after estrogen is withdrawn from the culture medium. Total poly(A) mRNA exhibits the same half-life (16 hr) in the presence or absence of estrogen. The rapid cytoplasmic degradation of vitellogenin mRNA in the absence of estrogen is fully reversible upon restimulation with estrogen, indicating that nuclear modification of vitellogenin RNA transcripts is not responsible for their stability. Intermediate levels of vitellogenin mRNA stability and changes in the relative rate of vitellogenin gene transcription are not observed late in estrogen induction, when vitellogenin mRNA levels plateau. Instead, Xenopus liver cells achieve fine control over the level of vitellogenin mRNA through down-regulation of the overall rate of total nuclear RNA synthesis.

Animals↗
PubMed1982

Activation of vitellogenin gene transcription is a direct response to estrogen in Xenopus laevis liver.

Estrogen induces the synthesis of vitellogenin mRNA by activating transcription of the vitellogenin genes. Quantitative inhibition of liver protein synthesis by cycloheximide does not prevent activation of vitellogenin gene transcription. The relative transcription rate of the vitellogenin genes in estrogen stimulated liver is similar in control and cycloheximide treated animals (800-1000 ppm). Selective estrogen activation of vitellogenin gene transcription therefore represents a direct effect of estrogen on vitellogenin gene transcription which can occur without any change in the cells' protein complement. Two other cellular responses to estrogen, the induction of nuclear estrogen receptor, and an increased rate of total nuclear RNA synthesis, are blocked by cycloheximide administration. Since the overall rate of vitellogenin mRNA synthesis is a function of both the selective estrogen activation of vitellogenin gene transcription which is not blocked by cycloheximide and the increased rate of total nuclear RNA synthesis which is blocked by cycloheximide, the total rate of vitellogenin mRNA synthesis is markedly reduced following cycloheximide administration.

Animals↗
PubMed1982

Quantitation of estrogen effect on Xenopus laevis albumin mRNA levels by hybridization to cloned albumin cDNA.

The isolation and characterization of a Xenopus laevis albumin cDNA clone, pUN18, is described. This clone was identified as a X. laevis albumin cDNA clone by hybridization-mRNA selection-translation and by other techniques. The clone was used in hybridizations to determine the effect of estrogen on albumin synthesis in livers of male X. laevis. We find that albumin mRNA levels remain relatively constant during the massive induction of vitellogenin mRNA synthesis and vitellogenin secretion which follow administration of estrogen to male X. laevis.

Albumins↗
PubMed1982

Production and characterization of monoclonal antibodies to rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the key regulatory enzyme in cholesterol biosynthesis, has been purified to apparent homogeneity. Purified HMG-CoA reductase yields a single diffuse band when NaDodSO4/polyacrylamide gels are stained with Coomassie blue and yields two adjacent bands when gels are stained with silver. Purified reductase was used to elicit the production of monoclonal antibodies. Spleen cells from BALB/c mice immunized with purified HMG-CoA reductase were fused with Sp-2/0 myeloma cells. Clones producing monoclonal antibodies to HMG-CoA reductase were identified by using a solid-phase radioimmunoassay and were subcloned in soft agar. The three relatively stable hybridoma lines isolated secrete different Igs as judged by their antibody subclasses and differing abilities to inhibit HMG-CoA reductase in solution. Efficient precipitation of solubilized HMG-CoA reductase was achieved with the two IgG antibodies but not with the IgM. A mixture of all three monoclonal antibodies immunoprecipitates more than 90% of the HMG-CoA reductase activity in solubilized rat liver extracts. These monoclonal antibodies should be useful probes for investigation of the regulation of HMG-CoA reductase and cholesterol synthesis.

Animals↗
PubMed1982

The role of estrogen receptor in Xenopus laevis vitellogenin gene expression.

Administration of estradiol 17 beta [estra-1,3,5(10)-triene-3,17-beta-diol] to male Xenopus laevis induces the massive synthesis by the liver of the egg yolk precursor phospholipoglycoprotein, vitellogenin, and its cognate mRNAs. Restimulation of male X. laevis that have been previously induced to synthesize vitellogenin mRNA but are inactive in vitellogenin mRNA synthesis at the time of restimulation with estrogen results in more rapid accumulation of vitellogenin mRNA and more efficient transcription of the vitellogenin genes than occurs following primary estrogen stimulation. The estrogen receptor system that mediates estrogen action in this organism exhibits several unusual properties. The cytoplasm of unstimulated liver cells contains high levels of a middle-affinity estrogen-specific binding protein and little if any estrogen receptor. The properties of the estrogen binding protein are consistent with a role in protecting estradiol 17 beta against metabolism, as a fraction of cytoplasmic estradiol 17 beta is not subject to rapid metabolism. In addition, similar binding activities are found in all Xenopus tissues surveyed that respond to steroid hormones. The induction of nuclear estrogen receptor is coincident with the onset of vitellogenin mRNA accumulation. However, an increased level of estrogen receptor is not responsible for the elevated rate of vitellogenin gene transcription observed following restimulation with estrogen.

Animals↗
PubMed1981

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis.

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.

Acetyl-CoA Carboxylase↗
PubMed1980

Induction of estrogen receptor and reversal of the nuclear/cytoplasmic receptor ratio during vitellogenin synthesis and withdrawal in Xenopus laevis.

The levels of cytoplasmic and nuclear estrogen receptor have been determined in livers of male Xenopus laevis stimulated by estradiol-17 beta to synthesize vitellogenin mRNA. Estrogen receptor levels were also determined in unstimulated liver and following long term withdrawal of estrogen. In unstimulated liver cells, which do not contain detectable vitellogenin mRNA, more than 80% of the estrogen receptor is located in the nucleus (550 high affinity estrogen binding sites/nucleus), while the cytoplasm contains only 100 high affinity estrogen binding sites/cell. Administration of estradiol-17 beta, which induces massive synthesis and accumulation of vitellogenin mRNA, induces the estrogen receptor as well. The nuclear receptor level rises to approximately 2,000 estrogen binding sites/cell, while the cytosol receptor increases to only 150 sites/cel. Liver cells of male X. laevis which have been withdrawn from estrogen for 70 days exhibit a striking change in receptor levels. The nuclear receptor returns to the level prevailing in unstimulated cells (approximately 500 sites/cell) while the cytosol receptor level rises to more than 1,200 sites/cell (equivalent to 260 fmol/g of tissue). The existence of a pool of cytosol receptor, which is rapidly available for induction of vitellogenin mRNA, may in part explain the shorter lag period and more rapid induction of vitellogenin mRNA observed during secondary estrogen stimulation of withdrawn Xenopus liver cells.

Animals↗
PubMed1979

Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]-mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.

Acid Phosphatase↗
PubMed1978

Rapid accumulation of vitellogenin messenger RNA during secondary estrogen stimulation of Xenopus laevis.

Accurate quantitation of low concentrations of vitellogenin mRNA by hybridization to vitellogenin cDNA allows analysis of the accumulation of new vitellogenin mRNA sequences throughout secondary estrogen stimulation. Administration of a secondary injection of estradiol-17 beta to male Xenopus laevis which have been withdrawn from estrogen for 60 days results in synthesis of complete vitellogenin mRNA molecules in as little as 1 h after restimulation. Vitellogenin mRNA accumulates at a rate of 13 molecules/cell/min--at least four times the rate observed in primary estrogen stimulation and peaks at a level twice that observed in primary stimulation. Administration of estrogen to male Xenopus laevis evokes stable long lived changes in the pattern of vitellogenin gene expression and constitutes a type of cellular "memory effect."

Animals↗
PubMed1976

Strongyloides stercoralis hyperinfection in a renal allograft recipient.

A patient who had received a renal allograft required intensive immunosuppression for a severe rejection episode. Four months after the graft he died of septicaemia and respiratory failure caused indirectly by a Strongyloides stercoralis hyperinfection. Patients from endemic areas who are to undergo a renal transplant should be screened for the parasite before receiving immunosuppressive therapy. Should this infection occur after transplantation, early diagnosis and treatment with thiabendazole is essential to prevent the high mortality rate associated with Strongyloides hyperinfection.

Adult↗
PubMed1976

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.

Animals↗