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Biomedical subjects

E M Wilson

Publications and source records attributed to E M Wilson.

At least 145 records · Page 8Linked to original sources
PubMed1987

Transformation of the 10S androgen receptor in a hamster ductus deferens tumor cell line.

Androgen receptor transformation was studied in a hamster ductus deferens tumor cell line using whole cell and cell-free labeling conditions. After cell incubations with [3H] dihydrotestosterone at 0 or 23 C, receptor was recovered predominantly in cytosol as 406,000 mol wt (10.1 +/- 0.4S; 89 +/- 1 A) and 237,000 mol wt (7.1 +/- 0.4S; 74 +/- 1 A). At 37 C, increasing amounts of receptor were retained with time by nuclei as receptor size decreased to 127,000 mol wt (5.5 +/- 0.3S; 51 +/- 2 A). The half-time of nuclear association was approximately 3 min at 37 C. DNA-binding activity was acquired upon conversion of the 10S receptor to 7S and 5.5S forms. The use of serum-free culture conditions ruled out the possible involvement of serum proteins in the formation of multiple receptor forms. In cell-free preparations, dihydrotestosterone binding was required for receptor dissociation from 10S to 7S, which at short time intervals was enhanced by mercaptoethanol. It is concluded that the 7S receptor is a transformation intermediate that displays DNA-binding activity but is not well retained in nuclei. Complete androgen receptor transformation is a steroid- and temperature-dependent reaction involving a decrease in receptor size from 10S to 5.5S.

Animals↗
PubMed1987

Androgen regulation of c-myc messenger ribonucleic acid levels in rat ventral prostate.

Androgens regulate growth of the rat ventral prostate gland. In a search for possible mediators of androgen stimulated growth we have studied c-myc proto-oncogene expression in ventral prostate after androgen withdrawal and replacement. Steady state levels of c-myc mRNA were determined by Northern hybridization and compared with mRNA levels for prostatein, the major androgen dependent protein of ventral prostate. C-myc mRNA in ventral prostate increased nearly 4-fold within 1 day and 6- to 7-fold within 2 days after castration. Administration of androgen at the time of castration prevented this increase in c-myc mRNA levels. Androgen treatment of 4-day castrate rats caused c-myc mRNA levels to decrease within 4 h. Cycloheximide increased c-myc mRNA severalfold within 2 h. The net increase in c-myc mRNA after cycloheximide treatment was greater in the castrate than in the noncastrate or in androgen-treated castrate rats. These results suggest that androgen may influence both transcription and turnover of c-myc mRNA. Prostatein C3 mRNA decreased rapidly after castration and increased after androgen treatment of the castrate but was only slightly influenced by cycloheximide. Steady state levels of c-myc mRNA were higher in the 10-day-old rat and decreased with age while prostatein C3 mRNA increased with age. In situ hybridization demonstrated that both c-myc and prostatein mRNAs are expressed in the epithelial cells of ventral prostate acinar glands. These data indicate that androgens regulate the expression of c-myc in the ventral prostate.

Aging↗
PubMed1987

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures.

The capacity of individual muscle cells and neurons to establish synaptic specializations along the entire extent of their neurite-muscle contacts was assessed in culture. Spinal cord neurons derived from embryos of Xenopus laevis were plated at low density in cultures of Xenopus myotomal muscle cells in order to obtain isolated motor units whose neuron and muscle cells were not contacted by any other neuron. These isolated motor units were examined for localization of acetylcholine receptors (AChRs) after staining with fluorescent alpha-bungarotoxin and, in some cases, for localization of a synaptic vesicle antigen by immunofluorescence. The neurite-muscle contacts formed by competent neurons exhibited discontinuous sites of AChR localization occupying about 25% of the contact length as compared with 2% for incompetent neurons. Competent neurons, unlike incompetent ones, established these neurite-associated receptor patches (NARPs) on virtually all the muscle cells they contacted and functionally innervated them. These and other observations on the distribution of NARPs throughout the isolated motor units indicate that the capacity of competent neurons to establish NARPs extends to the limits of growth of most if not all of their neurites, that this capacity is least in the most proximal portions of initial neuritic segments, and that the overall capacity of muscle cells to generate NARPs can be saturated by long lengths of neurite-muscle contact. The results also suggest that even in the absence of competitive interactions between neurons there are spatial discontinuities in neuritic action and/or muscle response during the establishment of NARPs. Synaptic vesicle antigen patches (SVAPs) occurred along the neuritic arbor of all neurons, but their distribution in competent neurons (those which established NARPs) and in incompetent ones differed. For competent neurons the percentage of neurite length occupied by SVAPs was 4.8-fold greater on muscle cells than off, whereas the corresponding value for incompetent neurons was only 1.5-fold. This large preferential localization of SVAPs along neurite-muscle contacts of competent neurons was further associated with a colocalization of SVAPs and NARPs that was greater than predicted by chance. These results suggest that muscle cells are much more effective in influencing the distribution of synaptic vesicles along the neuritic arbor of competent neurons than along the arbor of incompetent neurons and that this influence is greatest at sites of postsynaptic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

The possible role of disulfide bond reduction in transformation of the 10 S androgen receptor.

Dissociation of the 10 S androgen receptor to 8, 6, and 4.5 S forms was dependent on temperature, the reducing and ionic environment, and the binding of androgen. The [3H]dihydrotestosterone-labeled 10 S receptor was observed at low ionic strength using rat Dunning prostate tumor cytosol freshly prepared in the absence of an exogenous sulfhydryl reducing agent. Addition of mercaptoethanol caused 10 S receptor dissociation to 8 S following incubation at 0 degrees C for 30 min, to 6 S after a 30-min incubation at 23 degrees C at low ionic strength, and to 4.5 S at high ionic strength. Mercaptoethanol-induced dissociation required binding of [3H]dihydrotestosterone. Treatment with cupric phenanthroline, a disulfide-forming reagent, stabilized the 10 S receptor in 0.4 M KCl, but the receptor remained sensitive to dissociation by mercaptoethanol. Zn2+ (25 microM) and sodium molybdate (10 mM) also stabilized the 10 S receptor. A Stokes radius of 96 +/- 5 A was determined for the 10 S receptor by Sepharose-6B chromatography, with a calculated Mr of 396,000. The 10 S receptor was not retained by DNA-Sepharose, while dissociated forms displayed binding affinity for DNA. It is proposed that the 10 S receptor represents the nontransformed androgen receptor, composed of the 4.5 S steroid binding units plus a nonsteroid binding protein, perhaps in a tetrameric configuration. Binding of dihydrotestosterone appears to sensitize the 10 S receptor to disulfide bond reduction, resulting in transformation by subunit dissociation.

Animals↗
PubMed1986

Construction of recombinant vaccinia virus strains using single-stranded DNA insertion vectors.

The ability of single-stranded (ss) DNA, isolated from recombinant M13 bacteriophage, to direct the insertion of foreign genetic elements into the vaccinia virus (VV) genome was examined. An identical chimeric transcriptional unit [VV promoter/chloramphenicol acetyl transferase (CAT) gene embedded in DNA sequences encoding vaccinia virus thymidine kinase (TK)] was inserted into either the previously characterized plasmid insertion vector, pGS20, or into M13mp18. It was found that the ss vector (M13mp18:TK/CAT) was four times more efficient than the plasmid vector (pGS20:CAT) in catalyzing homologous recombination of the cat gene by marker transfer into the VV genome. Furthermore, Southern blot analyses and CAT enzymatic activity assays confirmed that the structure of the M13-derived recombinant genomes were as expected and that the chimeric genes were fully active. Although the precise mechanism responsible for the ss DNA-catalyzed insertion event is not known, these results are discussed with respect to the advantages of using M13-based vectors with which to manipulate and insert genetic information into infectious VV recombinants.

Cloning, Molecular↗
PubMed1985

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+.

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.

Ammonium Sulfate↗
PubMed1984

Zinc potentiation of androgen receptor binding to nuclei in vitro.

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Androgen receptor binding to nuclear matrix in vitro and its inhibition by 8S androgen receptor promoting factor.

The partially purified 4.5S [3H]dihydrotestosterone receptor binds to nuclear matrix isolated from rat Dunning prostate tumor with properties similar to those reported for androgen receptor binding in intact nuclei [Colvard, D.S., & Wilson, E.M. (1984) Biochemistry (preceding paper in this issue)] in that it requires Zn2+ and mercaptoethanol, is saturable, and is temperature dependent and of high affinity (Ka approximately 10(13) M-1). On a milligrams of DNA equivalent basis, the extent of matrix binding of androgen receptor (700 fmol of receptor bound/mg of matrix protein) is similar to that of intact nuclei, corresponding to approximately 1400 sites/nucleus. Association rate constants (ka) for 4.5S androgen receptor binding to matrix at 0, 15, and 25 degrees C are 2.7 X 10(5), 1.2 X 10(6), and 2.4 X 10(6) M-1 min-1, respectively, indicating an energy of activation of 15 kcal/mol. Up to 50% of matrix-bound receptor is extractable in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 5 mM pyridoxal 5'-phosphate. A protein fraction designated 8S androgen receptor promoting factor that promotes conversion of the 4.5S androgen receptor to 8 S [Colvard, D. S., & Wilson, E. M. (1981) Endocrinology (Baltimore) 109, 496-504] has been further purified and found to inhibit the binding of the 4.5S androgen receptor to isolated nuclei and nuclear matrix in a concentration-dependent manner. The results support the hypothesis that the 8S steroid receptor is a complex of the activated 4.5S androgen receptor with a non-steroid binding protein that renders the receptor incapable of binding in nuclei.

Animals↗
PubMed1984

In vitro synthesis and glycosylation of androgen-dependent secretory proteins of rat dorsal prostate and coagulating gland.

The two major androgen-dependent secretory proteins of rat dorsal prostate and coagulating gland, DP I and DP II, were compared by in vitro translation of total poly(A)RNA and by pulse-chase techniques by means of [35S]methionine and tissue minces of coagulating gland. DP I is a major in vitro translation product of isolated poly(A)RNA, whereas DP II is undetectable in a mouse embryo fibroblast cell-free system where glycosylation does not occur. DP I is synthesized within 20 min in minces of coagulating gland incubated in the presence of [35S]methionine and is secreted in 40 min. DP II is detectable in the medium only after 8 hr of labeling. Inhibition of asparagine-linked protein glycosylation with tunicamycin (10 micrograms/ml) blocked the synthesis and secretion of DP II with an apparent increase in DP I secretion. Inhibition of DP II synthesis by monensin implicates the Golgi in the processing of DP II oligosaccharides. The data are consistent with the proposal that DP I enters a pathway of rapid secretion that is enhanced by the absence of core glycosylation, whereas DP II follows a slow pathway through the Golgi that involves extensive glycosylation.

Androgen-Binding Protein↗
PubMed1984

Relevance of sensorimotor task components to the interpretation of task related blood flow changes.

This study demonstrated the importance of documenting rCBF changes produced by the sensorimotor components of a cognitive task when making inferences regarding brain-behavior relations. Subjects were ten young, non-smoking adult, right-handed, normal male volunteers. They were administered two tasks having identical cognitive and similar sensory components but different response modalities (oral vs manual). The two tasks produced highly divergent rCBF landscapes. In conjunction with the results from a previous rCBF activation study, these data were used to illustrate the necessity of including sensorimotor control tasks in cognitive activation studies designed to elucidate brain-behavior relations.

Adult↗
PubMed1983

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein.

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.

Amino Acid Sequence↗
PubMed1983

Effect of dietary compliance on metabolic control in insulin-dependent diabetics.

One hundred and seventy eight insulin dependent diabetics (IDDM) have been studied regarding their understanding of and compliance with a controlled carbohydrate diet. Forty per cent could not remember their dietary prescription, 35 per cent did not estimate carbohydrate and many scored badly in a 'quiz' on the carbohydrate content of common foods. Quiz scores were significantly associated with HbA1 levels (P less than 0.001). One hundred and thirty six patients completed a seven-day food record from which the pattern of eating was established. Of these 75 were injecting twice daily short and intermediate acting insulin; in this group there were significant relationships between HbA1 and both care with carbohydrate estimation and eating pattern (P less than 0.05). Thus, in IDDM, the understanding of a controlled carbohydrate diet and adherence to it are associated with better glycaemic control but adequate compliance is rare. Future dietary recommendations for insulin taking diabetics should be simple, easy to understand, acceptable in practice and of proven effectiveness if they are to appreciably influence the quality of diabetic control.

Adolescent↗
PubMed1982

Focal changes in cerebral blood flow produced by a test of right-left discrimination.

A sensorimotor control activation task was used to isolate the focal cerebral blood flow changes resulting from the visual and cognitive processing of a right-left discrimination task. Eleven normal right-handed males participated. The sensorimotor control task produced significant bilateral increases in flow in most cortical channels. Significant bilateral parieto-occipital activation was found for the right-left discrimination task over and above the flow changes produced by the sensorimotor control task. The left occipital flow increase resulting from the right-left discrimination task was found to be negatively related to task performance. An inverse relationship was also found between WAIS Performance IQ and the blood flow change in the left parietal channel. These results suggest areas for further testing concerning potential individual differences in cognitive processing during the performance of a right-left discrimination task.

Adult↗