scholarly journals Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide

1977 ◽  
Vol 162 (2) ◽  
pp. 341-346 ◽  
Author(s):  
F H A Janszen ◽  
B A Cooke ◽  
H J van der Molen
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Specific Protein ◽  
Rat Testis

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as ‘protein 21’). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as “protein 33”), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.

scholarly journals Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations

1975 ◽  
Vol 150 (3) ◽  
pp. 413-418 ◽  
Author(s):  
B A Cooke ◽  
F H Janszen ◽  
W F Clotscher ◽  
H J van der Molen
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Leydig Cell ◽  
Half Life ◽  
Specific Effect ◽  
Interstitial Tissue ◽  
Protein Synthesis Inhibitors ◽  
Rat Testis ◽  
Testosterone Production

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3′:5′-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.

scholarly journals Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

1978 ◽  
Vol 170 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphodiesterase Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Rat Testis ◽  
Stimulation Of

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.

De novo protein synthesis and protein phosphorylation during anoxia and recovery in the red-eared turtle

1993 ◽  
Vol 265 (6) ◽  
pp. R1380-R1386 ◽  
Author(s):  
S. P. Brooks ◽  
K. B. Storey
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Protein Phosphorylation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Total Radioactivity ◽  
Relative Molecular Mass ◽  
De Novo Protein Synthesis

Changes in de novo protein synthesis and protein phosphorylation were monitored during anoxia and recovery in the red-eared slider Trachemys (= Pseudemys) scripta elegans. Time courses of 35S-radiolabeled methionine incorporation into acid-precipitable material showed an increase up to 5 h postinjection and remained constant after this time. Comparison of the total and acid-precipitable 35S label incorporation into tissues from 20-h control, anoxic, and recovering animals showed differences between these groups: total radioactivity in brain was 2.9-fold lower in recovering turtles, whereas protein-associated radioactivity was 2.4-fold higher in anoxic liver, 2.3-fold lower in recovering skeletal muscle, and 3.7-fold lower in recovering brain tissue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled proteins showed the existence of a newly synthesized protein band (relative molecular mass = 72 kDa) that was apparent only in 20-h recovering liver and skeletal muscle. Use of 32P labeling to monitor changes in protein phosphorylation patterns during anoxia revealed 1.6-, 1.4-, and 1.5-fold increases in 32P incorporation in anoxic brain, heart, and liver, respectively. Changes in protein phosphorylation were localized to the plasma membrane and cytosolic fractions in brain and to the cytosolic fraction in liver.

scholarly journals The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

1979 ◽  
Vol 184 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Brian A. Cooke ◽  
L. Monica Lindh ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Protein Synthesis Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Testosterone Production ◽  
Short Half Life ◽  
Regulator Protein

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32°C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61±3, 46±3, 27±4 and 18±4% lower than in the cells pretreated without cycloheximide respectively (means±s.e.m., n=4–6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36±0.5 to 36.5±1.0ng/106 cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2–4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.

scholarly journals Lutropin increases phosphorylation of a 33 000-dalton ribosomal protein in rat tumour Leydig cells

1982 ◽  
Vol 204 (3) ◽  
pp. 809-815 ◽  
Author(s):  
G H Bakker ◽  
J W Hoogerbrugge ◽  
F F G Rommerts ◽  
H J van der Molen
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Subcellular Localization ◽  
Ribosomal Protein ◽  
Molecular Mass ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Ribosomal Subunit ◽  
Ribosomal Protein S6 ◽  
Leydig Cell Tumour

Addition of lutropin (luteinizing hormone, ‘LH’) and 3-isobutyl-1-methylxanthine to tumour Leydig cells stimulated phosphorylation of five proteins, of 17 000, 22 000, 24 000, 33 000 and 57 000 Da. Phosphorylation of these proteins coincided with increased pregnenolone production. Phosphorylation of a 33 000-Da protein was lutropin-dependent in Leydig cells isolated from a Leydig-cell tumour, from immature testes or from mature testes. In tumour Leydig cells this protein was present in the small ribosomal subunit. Incubation of tumour Leydig cells with either cycloheximide or puromycin inhibited both basal and lutropin-dependent pregnenolone production, by approx. 90% and 98% respectively. In contrast, basal pregnenolone production in Leydig cells from immature and mature testes was insensitive to cycloheximide or puromycin. Cycloheximide or puromycin increased phosphorylation of the 33 000-Da phosphoprotein by approx. 130% and 80% respectively (effect of lutropin/3-isobutyl-1-methylxanthine on phosphorylation: 100%). The molecular mass, the subcellular localization and the sensitivity to phosphorylation in the presence of inhibitors of protein synthesis indicate that the 33 000-Da protein could be similar to ribosomal protein S6.

scholarly journals The effect of lutropin on specific protein synthesis in tumour Leydig cells and in Leydig cells from immature rats

1978 ◽  
Vol 172 (1) ◽  
pp. 147-153 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Leydig Cells ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Side Chain ◽  
Chain Cleavage ◽  
Immature Rats ◽  
Cleavage Enzyme ◽  
Induced Proteins ◽  
Stimulation Of

The amount of35S incorporated into the various proteins after separation by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels was used as an estimate of their synthesis in the Leydig cells. Increased synthesis of proteins with apparent mol.wts. 27000 and 29000 was observed 3h after addition of lutropin to tumour Leydig cells. Incubation of Leydig cells from immature rats with lutropin (100ng/ml) for 2h or longer resulted in increased synthesis of proteins with apparent mol.wts. 11000, 21000, 27000 and 29000. At higher concentrations (≥100ng/ml) of lutropin there was a decrease in the synthesis of a protein with apparent mol.wt. 13000. The amount of lutropin required for the stimulation of protein synthesis in both types of Leydig cells was similar to that needed for stimulation of steroidogenesis. Lutropin-stimulated specific protein synthesis was not due to increased concentrations of testosterone, however, because (1) addition of testosterone to the cells had no effect on the synthesis of the proteins, and (2) inhibition of steroidogenesis with elipten phosphate (an inhibitor of the cholesterol side-chain-cleavage enzyme complex) did not abolish the effect of lutropin. The stimulation of specific protein synthesis was also not due to contaminating follitropin in the lutropin preparation. Addition of actinomycin D to the cells at the start of the incubation prevented the effect of lutropin on specific protein synthesis, indicating that mRNA synthesis may be needed for this effect of lutropin. Incubation of the cells with cycloheximide for 30min after labelling of the proteins did not result in a detectable decrease in the amounts of the lutropin-induced proteins, indicating that their half-life is longer than 30min.

Isolation of rat Leydig cells and precursor forms after administration of ethane dimethane sulfonate

1994 ◽  
Vol 266 (6) ◽  
pp. E975-E979 ◽  
Author(s):  
G. P. Risbridger ◽  
A. Davies
Keyword(s):  
Luteinizing Hormone ◽  
Cell Population ◽  
Chorionic Gonadotropin ◽  
Leydig Cell ◽  
Human Chorionic Gonadotropin ◽  
Leydig Cells ◽  
Steroid Production ◽  
Microscopic Appearance ◽  
Rat Testis ◽  
Adult Rat

The cytotoxic drug ethane dimethane sulfonate (EDS) has been extensively used as a means of studying the regeneration of Leydig cells in the adult rat testis. This study used the EDS-treated rat testis as a source of material for the isolation of regenerating Leydig cells and their precursors and describes the procedures required for the isolation of these cell preparations. As early as 13-15 days after EDS, cells in the precursor fraction can bind low, but detectable, levels of iodinated purified human chorionic gonadotropin. However, no luteinizing hormone (LH) response was detected in terms of steroid production. The precursor fraction of cells isolated from the EDS-treated rat testis 17-19 days after the administration of EDS was heterogeneous in light-microscopic appearance, but identifiable Leydig-like cells were present. The cells in this fraction were the first to exhibit the ability to respond to LH with the production of detectable levels of the reduced androgen, 5 alpha-androstane-3 alpha,17 beta-diol. The amount of androgen produced by both the Leydig cell and precursor fractions had increased by 21 days after EDS and reached the levels produced by immature adultlike Leydig cells, which can be isolated from the 20-day-old rat testes. These studies demonstrate that steroidogenically responsive precursor forms of Leydig cells can be isolated from the EDS-treated testes 17-19 days after depletion of the adult Leydig cell population.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

Effects of prolactin on testosterone-induced growth and protein synthesis in rat accessory sex glands

1983 ◽  
Vol 96 (3) ◽  
pp. 407-416 ◽  
Author(s):  
R. Jones ◽  
P. R. Riding ◽  
M. G. Parker
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Sodium Dodecyl ◽  
Chronic Administration ◽  
Specific Protein ◽  
Ventral Prostate ◽  
Plasma Prolactin ◽  
Accessory Sex Glands ◽  
Caput Epididymidis

The relative importance of testosterone and prolactin in regulating growth and protein synthesis in rat accessory sex glands has been investigated. Protein synthesis was measured by incubating tissue minces in vitro with [35S]methionine and analysing labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Plasma prolactin was assayed by radioimmunoassay. Results showed that castration for 8 days significantly reduced wet weights and total protein synthesis in the ventral prostate, dorsolateral prostate and caput epididymidis, but that these effects could be reversed by exogenous testosterone. Similarly, the specific incorporation of [35S]methionine into four polypeptides in the ventral prostate, two polypeptides in the dorsolateral prostate and two polypeptides in the caput epididymidis was lowered by castration but markedly stimulated by testosterone. Acute or chronic administration of 2-bromo-α-ergocryptine to animals in combination with testosterone had no significant effect on any of the parameters measured, although the drug reduced circulating prolactin to undetectable levels. In addition, exogenous prolactin given alone, or in combination with testosterone, to hypophysectomized rats had no effect on general or specific protein synthesis. The induction of hyperprolactinaemia in immature or mature rats with pituitary homographs had no effect on testosterone-stimulated growth of any accessory gland, although it caused a significant stimulation of total protein synthesis in the dorsolateral prostate and coagulating glands. However, this was a generalized effect as it did not increase the specific incorporation of [35S]methionine into androgen-dependent proteins. The results do not indicate a major role for prolactin in regulating androgen responsiveness of male accessory sex glands in the rat.

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