5α-DIHYDROTESTOSTERONE AND THE SYNTHESIS OF RNA IN THE RAT VENTRAL PROSTATE IN VITRO. II

1973 ◽  
Vol 73 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Ingrid Alfheim ◽  
Bjørg Fjell
Keyword(s):  
Gel Electrophoresis ◽  
Rna Synthesis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extraction Procedure ◽  
Ventral Prostate ◽  
Double Labelling ◽  
Phenol Extraction ◽  
Rat Ventral Prostate

ABSTRACT A double labelling technique has been used to study RNA synthesis in explanted tissues obtained from ventral prostates of castrated rats, incubated with or without 5α-dihydrotestosterone in the medium. RNA was isolated by a phenol extraction procedure, and analysed by sucrose gradient centrifugation and polyacrylamide gel electrophoresis. No specific hormonal response could be detected.

scholarly journals Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate)

1976 ◽  
Vol 156 (3) ◽  
pp. 619-626 ◽  
Author(s):  
S M Tilghman ◽  
L M Fisher ◽  
L Reshef ◽  
F J Ballard ◽  
R W Hanson
Keyword(s):  
Gel Electrophoresis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Messenger Ribonucleic Acid ◽  
Partial Isolation ◽  
Heterologous Cell ◽  
Rabbit Reticulocytes

1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.

5α-DIHYDROTESTOSTERONE AND THE SYNTHESIS OF RNA IN THE RAT VENTRAL PROSTATE IN VITRO

1971 ◽  
Vol 68 (3) ◽  
pp. 567-575 ◽  
Author(s):  
Ingrid Alfheim ◽  
Olav Unhjem
Keyword(s):  
Incubation Medium ◽  
Rna Synthesis ◽  
Ventral Prostate ◽  
Isolated Nuclei ◽  
Quantitative Effect ◽  
Rat Ventral Prostate

ABSTRACT The effect of 5α-dihydrotestosterone on RNA synthesis in the rat ventral prostate has been studied in vitro. The steroid was added to the incubation medium in a concentration of 10−6 m. Experiments have been performed with slices, homogenates, crude nuclear preparations and isolated nuclei from the rat ventral prostate. The results appear to indicate that there is no immediate quantitative effect of the steroid on RNA synthesis when administered in vitro.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

FURTHER STUDIES ON A SOLUBLE ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

1970 ◽  
Vol 65 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Olav Unhjem
Keyword(s):  
Hydrogen Atom ◽  
Keto Group ◽  
Ventral Prostate ◽  
Metabolic Inhibitors ◽  
Macromolecular Complex ◽  
Structural Requirements ◽  
Macromolecular Complexes ◽  
Rat Ventral Prostate

ABSTRACT The ability of various steroids and metabolic inhibitors to influence the binding of androgen to soluble macromolecules in the rat ventral prostate was evaluated in vitro. The results obtained revealed some structural requirements of steroids for binding to the macromolecules. An androstane skeleton with the α-configuration of the hydrogen atom at position 5 seemed to be essential for binding as well as a keto group at position 3. N-ethylmaleimide, Na-iodoacetate and p-hydroxymercuribenzoate inhibited the binding of androgen to macromolecules. The androgen-macromolecular complexes appeared to be rather stable at temperatures below 5°C.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Incorporation of Testosterone and 5α-Dihydrotestosterone into Rat Ventral Prostate in vitro

1970 ◽  
Vol 17 (6) ◽  
pp. 453-458 ◽  
Author(s):  
JUN SHIMAZAKI ◽  
JIN SATO ◽  
HISAKO NAGAI ◽  
KEIZO SHIDA
Keyword(s):  
Ventral Prostate ◽  
Rat Ventral Prostate

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

scholarly journals Initiation and elongation of polyribonucleotide chains on rat ventral-prostate chromatin transcribed by homologous ribonucleic acid polymerase B.

1977 ◽  
Vol 166 (2) ◽  
pp. 189-198 ◽  
Author(s):  
P Thomas ◽  
P Davies ◽  
K Griffiths
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Control Of Gene Expression ◽  
Rat Ventral Prostate ◽  
Associated Proteins ◽  
Ribonucleoside Triphosphate ◽  
Androgenic Steroids ◽  
Transcriptional Process

The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression.

Organophosphorus insecticide resistance in a new strain of Culex quinquefasciatus (Diptera: Culicidae) from Tanga, Tanzania

1993 ◽  
Vol 83 (1) ◽  
pp. 67-74 ◽  
Author(s):  
A. Khayrandish ◽  
R.J. Wood
Keyword(s):  
Insecticide Resistance ◽  
Culex Quinquefasciatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Laboratory Culture ◽  
Fourth Instar ◽  
Resistance Allele ◽  
Susceptible Control ◽  
The Mean

AbstractFourth instar larvae of a new strain of Culex quinquefasciatus Say from Tanzania (TANGA) were tested for insecticide resistance. Initially, the resistance ratio (RR) to chlorpyrifos was 41.8, to temephos 30.8, to propoxur 3.7. After 2–3 years of laboratory culture, resistance to chlorpyrifos and propoxur had declined (chlorpyrifos 5.7, 3.8; propoxur 1.9, permethrin 1.9). Significant synergism was found between s, s, s-tributyl trithiophosphate (DEF) and chlorphyrifos, reducing the RR from 8.0 to 2.5. Synergism between piperonyl butoxide and permethrin was less than in a susceptible control strain. Twelve esterase isozymes of different relative mobilities (Rm) on polyacrylamide gel electrophoresis were identified, ten of which remained when the strain was reinvestigated two years (approximately 32 generations) later. Null activity for all but one of these bands was observed in some larvae. Four esterase bands (Rm 0.25, 0.27, 0.31, 0.34, designated A2, A3, B2, B3) showed polymorphism in activity, with very intense bands in some larvae. The mean frequency of bands with activity greater than standard, declined as organophosphorus (OP) resistance declined, but resistance was unconnected with the frequency of nulls at these positions. In mass larval assays of in vitro sensitivity of acetylcholinesterase (AChE) to propoxur, the I50 exceeded 10x10−4M, compared with 0.1x10−4M in a reverted resistant strain (RANGOON). Single larvel assays revealed heterogeneity, which was interpreted on the basis of an AChE resistance allele (AceR) with a frequency of 0.23.

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