scholarly journals Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

1977 ◽  
Vol 166 (2) ◽  
pp. 167-173 ◽  
Author(s):  
Carlo M. Veneziale ◽  
John M. Burns ◽  
Jon C. Lewis ◽  
Kaspar A. Büchi
Keyword(s):  
Protein Synthesis ◽  
Guinea Pig ◽  
Protein Content ◽  
Seminal Vesicle ◽  
Specific Protein ◽  
Absolute Amount ◽  
Cytoplasmic Protein ◽  
Specific Proteins ◽  
Wet Weight

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [14C]glycine and [14C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. ‘non-specific’. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.

Development of Haemoglobin by De-embryonated Chick Blastoderms Cultured in vitro and the Effect of Abnormal RNA upon its Synthesis

Development ◽  
1961 ◽  
Vol 9 (1) ◽  
pp. 202-221
Author(s):  
B. R. A. O'Brien
Keyword(s):  
Protein Synthesis ◽  
Developmental Stages ◽  
Specific Protein ◽  
Cytoplasmic Protein ◽  
Whole Cells ◽  
Restricted Group ◽  
Dividing Cells ◽  
First Time ◽  
Structural Code

The embryo provides a sequence of developmental stages in which proteins both structural and enzymatic appear or become detectable for the first time in a restricted group of dividing cells. The cells or tissues can be maintained in vitro for a period that may precede and include the synthesis of a specific ‘cytoplasmic’ protein. In this way systems of protein synthesis within the cells of higher organisms can be studied during those stages in which current hypotheses suggest that some structural code is passed on from the DNA of the nucleus to the cytoplasm where the synthesis of the protein becomes maximal. Acellular preparations have contributed much to the elucidation of protein synthesis, but it is doubtful whether actual net synthesis has been obtained in systems less complex than the ‘protoplast’ developed by Spiegelman (1957). In order to study the synthesis of a specific protein it seems necessary at this stage to use whole cells.

scholarly journals Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features

1977 ◽  
Vol 166 (2) ◽  
pp. 155-166 ◽  
Author(s):  
Carlo M. Veneziale
Keyword(s):  
Guinea Pig ◽  
Seminal Vesicle ◽  
Deae Cellulose ◽  
Specific Protein ◽  
Total Soluble Protein ◽  
High Concentration ◽  
Γ Globulins ◽  
Specific Proteins ◽  
Secondary Antibodies ◽  
Sexually Mature

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat γ-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from14C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [14C]lysine plus [14C]glycine, [14C]histidine plus [14C]glutamate, [14C]glutamine alone and [14C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10–15min of incubation. Only 4–9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.

Protein Synthesis In Megakaryocytes Stimulated By Thrombo- Poietin In Vitro

1981 ◽  
Author(s):  
K L Kellar ◽  
B L Evatt ◽  
C R McGrath ◽  
R B Ramsey
Keyword(s):  
Protein Synthesis ◽  
Guinea Pig ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leucine Incorporation ◽  
Ploidy Levels ◽  
Percoll Gradients ◽  
Specific Proteins ◽  
Phosphate Buffered Saline

Studies in our laboratories have been concerned with the responses of megakaryocytes to thrombopoietin in vitro. We have shown that preparations of thrombopoietin stimulate DNA synthesis in guinea pig megakaryocytes. The increase in 3H-thymidihe incorporation correlates with an increase in the labeling index of the megakaryocytes. After 2 and 3 days of incubation an increase in the ploidy levels of the megakaryocytes has been observed in thrombo- poietin-supplemented cultures compared to controls.Recent studies have examined the incorporation of 3H-leucine in megakaryocyte cultures. Megakaryocytes were prepared on BSA or Percoll gradients to purities of 70-95%. 3H-leucine incorporation was measured after a 15 hr incubation of the megakaryocytes in medium containing 10% thrombopoietin or control preparations of normal plasma or phosphate-buffered saline. Utilization of isotope increased over a 24 hr period and was higher in the thrombopoietin-supplemented cultures. In addition, synthesis of specific proteins was analyzed by using SDS- polyacrylamide gel electrophoresis and quantitation was achieved by employing rocket immunoelectrophoresis. The results indicate that thrombopoietin stimulates endoredu- plication and protein synthesis in megakaryocytes in vitro and that this system may serve as a model for studying the mechanism of action of thrombopoietin in megakaryocytopoiesis.

scholarly journals Effects of Progesterone on the Oestrogen-Stimulated Uterus: a Comparative Study of the Mouse, Guinea Pig, Rabbit and Sheep

1979 ◽  
Vol 32 (6) ◽  
pp. 549 ◽  
Author(s):  
BG Miller ◽  
Jennifer Wild ◽  
GM Stone
Keyword(s):  
Protein Synthesis ◽  
Guinea Pig ◽  
Comparative Study ◽  
Peroxidase Activity ◽  
Species Differences ◽  
Inhibitory Effects ◽  
Dna And Rna ◽  
Wet Weight ◽  
Vitro Protein

To examine more closely the anti-oestrogenic action of progesterone (P), its effect on various parameters in the 17 fl-oestradiol (E2)-primed uterus of the mouse, guinea pig, rabbit and ewe was studied. Changes in uterine wet weight, rate of in vitro protein synthesis, protein: DNA and RNA :. DNA ratios, peroxidase activity and the level of cytosol receptors for E2 and P were measured. Considerable between-species differences in the effect of P on these parameters were observed. The anti-uterotrophic action was greater in the mouse than in the guinea pig and was not seen in the rabbit or ewe. P inhibited protein synthesis in the mouse, was without significant effect in the guinea pig and was mildly stimulatory in the rabbit and ewe. Inhibitory effects on protein: DNA and RNA: DNA ratios were substantial in the mouse, minor in the guinea pig and absent in the rabbit and ewe. Peroxidase activity was decreased in the mouse and guinea pig, essentially lacking in the rabbit and not detectable in the ewe. In all species the level of both oestrogen and progesterone cytosol receptors was decreased, although the effect on the E2 receptor was less marked in the ewe.

The initial synthesis of haemoglobin in de-embryonated chick blastoderms.

Development ◽  
1964 ◽  
Vol 12 (4) ◽  
pp. 609-619
Author(s):  
Anna Hell
Keyword(s):  
Protein Synthesis ◽  
Deoxyribonucleic Acid ◽  
Primitive Streak ◽  
Specific Protein ◽  
In Vitro System ◽  
Embryonic Tissues ◽  
Different Types ◽  
Excellent Tool ◽  
Made In

Enormous progress has been made in the last few years towards the elucidation of the mechanism of protein synthesis, and great interest is centred on the steps leading to cellular differentiation and specific protein synthesis. We know that genetic information is passed on from one generation of cells to the next by deoxyribonucleic acid (DNA), and that this material directs all protein synthesis by the intermediary of the different types of ribonucleic acid (RNA). A simple in vitro system described by O'Brien (1959) seemed to offer an excellent tool for the study of the differentiation of the blood islands, and the initial formation of a well-known protein, haemoglobin (Hb), in chick embryonic tissues. After de-embryonation, chick blastoderms, from the stage of primitive streak onwards, can be cultured in vitro on a saline agar medium supplemented with glucose.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

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.

Effects of oestradiol, the oestrous cycle and pregnancy on weight, metabolism and cytosol receptors in the uterus of the brush-tail possum (Trichosurus vulpecula)

1986 ◽  
Vol 108 (2) ◽  
pp. 201-210 ◽  
Author(s):  
J. D. Curlewis ◽  
G. M. Stone
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Corpus Luteum ◽  
Metabolic Activity ◽  
Weight Ratio ◽  
Oestrous Cycle ◽  
Uterine Weight ◽  
In Vitro Protein Synthesis ◽  
Vitro Protein

ABSTRACT Uterine weight, RNA, DNA, protein content, in-vitro rate of protein synthesis, cytosol oestrogen and progesterone receptors were examined after administration of oestradiol to ovariectomized animals and on days 0, 5, 9 and 13 of the non-pregnant cycle and day 13 of pregnancy. In ovariectomized animals, oestradiol increased uterine weight, RNA: DNA and protein: DNA ratios and the concentration of cytosol receptors for oestradiol and progesterone. During the oestrous cycle there was a linear increase in uterine weight and a significant effect of the corpus luteum on the weight of the ipsilateral uterus. Changes in RNA, DNA and protein content between days 0 and 5 were not observed, but between days 5 and 13 RNA: DNA and protein: DNA ratios increased and the DNA: tissue weight ratio decreased. Thus, cellular hypertrophy and/or increased metabolic activity rather than hyperplasia occur over this period, which is coincident with the known rise in plasma progesterone levels. The rate of in-vitro protein synthesis (per unit tissue protein) during the non-pregnant cycle was greatest at day 0. These changes in uterine metabolic activity were associated with alterations in cytosol receptor concentrations for both steroids. Cytosol progesterone receptor concentrations were highest at day 0 after which they declined to a minimum at day 13. Cytosol oestradiol receptor concentrations, however, rose between days 0 and 5 and then declined. Although lutectomy on day 8 of the cycle does not interfere with the development of a histologically normal luteal phase, high peripheral progesterone levels which occur after day 8 in intact animals are associated with major increases in uterine metabolic activity. The unilateral effect of the corpus luteum on uterine weight was associated with a decrease in DNA: g tissue ratio and an increase in rate of in-vitro protein synthesis indicating hypertrophy and/or extracellular accumulation of secreted material as well as enhanced metabolic activity. There was a significant effect of pregnancy on uterine weight at day 13 and this was associated with an increase in DNA content of both uteri. There was a unilateral effect of pregnancy on RNA: DNA ratio and in-vitro rate of protein synthesis, but not on uterine weight. J. Endocr. (1986) 108, 201–210

scholarly journals Cardiac Pacemaker Cells Generate Cardiomyocytes from Fibroblasts in Long-Term Cultures

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shigeki Kiuchi ◽  
Akino Usami ◽  
Tae Shimoyama ◽  
Fuminori Otsuka ◽  
Sachiko Yamaguchi ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Cardiac Fibroblasts ◽  
Cluster Formation ◽  
Cardiac Pacemaker ◽  
Pacemaker Cells ◽  
Intracellular Ca ◽  
Specific Proteins ◽  
Cell Densities

Abstract Because cardiomyocyte generation is limited, the turnover of cardiomyocytes in adult heart tissues is much debated. We report here that cardiac pacemaker cells can generate cardiomyocytes from fibroblasts in vitro. Sinoatrial node cells (SANCs) were isolated from adult guinea pig hearts and were cultured at relatively low cell densities. Within a week, a number of fibroblast-like cells were observed to gather around SANCs, and these formed spontaneously beating clusters with cardiomyocyte structures. The clusters expressed genes and proteins that are characteristic of atrial cardiomyocytes. Pharmacological blocking of pacemaker currents inhibited generation of action potentials, and the spontaneous beating were ceased by physically destroying a few central cells. Inhibition of beating during culture also hampered the cluster formation. Moreover, purified guinea pig cardiac fibroblasts (GCFs) expressed cardiac-specific proteins in co-culture with SANCs or in SANC-preconditioned culture medium under electrical stimulation. These results indicate that SANCs can generate cardiomyocytes from cardiac fibroblasts through the influence of humoral factor(s) and electrophysiological activities followed by intracellular Ca2+ oscillations. This potential of SANCs to generate cardiomyocytes indicates a novel mechanism by which cardiomyocytes turns over in the vicinity of pacemaker cells and could be exploited in the development of strategies for cardiac regenerative therapy in adult hearts.

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