Biomedical subjects

E Wieben

Publications and source records attributed to E Wieben.

6 recordsLinked to original sources

PubMed1992

The expression of milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system.

We expressed milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system in which the cloned cDNA for the hVDR was expressed under the control of bacterial T7 polymerase. The hVDR protein comprised approximately 60% of total bacterial protein. It migrated on polyacrylamide-sodium dodecyl sulfate gels with an M(r) of 48,000. It had the predicted amino acid composition and amino acid sequence analysis. The expressed protein was bound by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a Kd in the nanomolar range. It sedimented on sucrose density gradients at 3.5S. Furthermore, the expressed protein bound to the osteocalcin vitamin D response element (VDRE) as assessed by a gel mobility shift assay. The expression of large amounts of hVDR protein should allow for the use of this protein in structure-function and x-ray crystallography studies.

Amino Acid Sequence↗

PubMed1992

Androgens are necessary for the establishment of secretory protein expression in the guinea pig seminal vesicle epithelium.

The guinea pig seminal vesicle epithelium (GPSVE) synthesizes and secretes milligram quantities of four related secretory proteins in an androgen-dependent manner. To investigate the role of androgens in the establishment of secretory protein synthesis during the development of the GPSVE, animals were castrated at Day 5, approximately 10 days before secretory protein accumulation begins in intact animals. Castration did not eliminate secretory protein mRNA from the SVE, but it did indefinitely postpone the developmentally programmed increase in secretory protein mRNA. Injection of neonatally castrated guinea pigs with either estradiol or dexamethasone did not alter levels of secretory protein mRNAs. However, treatment of castrated neonates with either testosterone propionate or dihydrotestosterone (DHT) led to specific increases in secretory protein mRNAs within 4 days. Although neonatally castrated animals accumulated and translated significant amounts of secretory protein mRNA, the newly synthesized secretory proteins failed to accumulate until exogenous androgens were provided. This observation suggests that androgens regulate both the accumulation of secretory protein mRNA and the accumulation of secretory proteins in the GPSVE.

Androgens↗

PubMed1991

Post-transcriptional regulation of secretory protein production during the development of the guinea pig seminal vesicle.

To investigate the influence of androgens on secretory protein expression during the development of the guinea pig seminal vesicle epithelium, we examined the patterns of mRNA and protein accumulation during the first 2 wk after birth. Hybridization of total seminal vesicle RNA to cDNA probes revealed that the secretory protein genes were active as early as 5 days after birth. However, the accumulation of secretory proteins was barely detectable between Days 5 and 10, and could not be enhanced by treatment of neonatal animals with exogenous androgens. Secretory protein mRNA and protein levels both increased rapidly between Days 10 and 15. However, the 800-fold rise in protein levels between Days 5 and 15 greatly exceeded the magnitude of the increase in secretory protein mRNA that occurred during this interval. These data indicate that the rate of secretory protein accumulation in the guinea pig seminal vesicle is not determined strictly by the availability of secretory protein mRNA, and suggest that post-transcriptional mechanisms may contribute to the regulation of secretory protein accumulation in neonatal guinea pigs.

Aging↗

PubMed1989

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins.

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.

Amino Acid Sequence↗

PubMed1986

Solid-phase processing of U2 snRNA precursors.

HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.

Antibodies↗

PubMed1979

Molecular signals for initiating protein synthesis in organ hypertrophy.

When chronically provoked to increased physiologic activity, organs increase in mass through augmented protein protein synthesis. This process of compensatory hypertrophy can involve cell division as well as cell growth. To test for molecules that might regulate organ size, by inducing hypertrophy, we performed a series of experiments using isolated, perfused, canine hearts in which the left ventricle was beating but performed no work. Hypertrophying hearts and kidneys as well as normal control organs were extracted and the extracts were perfused through isolated heart preparations. Before and after perfusion, RNA was extracted from fragments of the isolated hearts and translated in cell-free media containing [35S]methionine. Incorporation of methionine into protein was measured by liquid scintillation spectrometry. When perfused through normal hearts, extracts from hypertrophying heart and kidney were able to increase greatly the translational ability of RNA extracted from the normal hearts; corresponding perfusates from nonhypertrophying hearts and kidneys had no effect. Our results indicate that molecules that initiate hypertrophic organ growth are extractable, are generated by the cells of the organ under stress, and are probably similar in heart and kidney and perhaps in many other organs as well.

Animals↗