RESUMO
The interconversion and extraction of testosterone and androstenedione across and within different tissues or areas have been studied by the constant infusion technique. The results were calculated using the (3)H/(14)C ratios and radioactive concentrations of testosterone and androstenedione obtained from afferent and efferent blood and tissues at equilibrium. In each tissue studied, the interconversion between testosterone and androstenedione inside the tissue was significantly higher than the corresponding interconversion across the tissue. The pulmonary contribution to the total interconversion between testosterone and androstenedione was far more important than that of any of the other tissues studied. The hepatic metabolic clearance rates of testosterone and androstenedione were not different from their metabolic clearance rates in the mesenteric area. The extraction of each of these compounds, although not negligible, was lower in the kidney and the femoral bed compared with the extraction in the liver and the mesenteric area. Finally, with the possible exception of the liver, testosterone and androstenedione were more completely metabolized when they originated from the cells than from afferent blood. The evaluation of these different tissue transfer constants provides more precise information concerning the relative importance of different sites in the metabolism of these interconverting hormones.
Assuntos
Androgênios/metabolismo , Testosterona/metabolismo , Androgênios/sangue , Androstanos/metabolismo , Animais , Medula Óssea/metabolismo , Isótopos de Carbono , Cães , Feminino , Cetonas/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Matemática , Mesentério/metabolismo , Taxa de Depuração Metabólica , Testosterona/sangue , TrítioRESUMO
Using the substrate poly[Glu80Na,Tyr20] [poly(GT)] and the autoradiographic detection of alkali-resistant phosphoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tyrosine protein kinase (TPK) has been evidenced in human hyperplastic prostates (BPH) and the prostatic carcinoma cell lines PC3 and DU145. The enzyme was mainly found in the soluble fractions from hyperplastic tissues and in Triton extracts from the cell lines. However, its specific activity in tissues was 1.5- to 4.5-fold times higher in particulate than in soluble fractions and it was of the same order of magnitude as that of neoplastic cells. Under these conditions, no activity was detected in human seminal plasma and in sera from normal adult males or patients with BPH and/or prostatic carcinoma. On the other hand, some TPK activity was associated with human spermatozoa, with a specific activity 4- to 6-fold lower than in BPH tissue fractions and a total activity, per 10(6) cells, 5- to 20-fold lower than that in prostatic carcinoma cells. The activity of prostatic TPK was dependent upon the presence of the divalent cations Mn2+ or Mg2+ and it was completely abolished by heat denaturation. Angiotensin II, casein, and histone H2B were poor substrates compared to poly(GT). The TPK activities towards poly(GT) as well as endogenous proteins were not stimulated by epidermal growth factor and insulin or by dihydrotestosterone and estradiol. The autoradiography of alkali-resistant phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several bands in both BPH tissues and neoplastic cells (molecular weight ranging from 17,000 to 200,000). Preliminary characterization of TPK by gel filtration on Sephacryl S-300 showed that the soluble enzyme from BPH tissues had a molecular weight of 50,000, while the particulate-associated TPK, when assayed on poly(GT), eluted with proteins of Mr 210,000. When these peak fractions were used for endogenous phosphorylation, several major alkali-resistant phosphoproteins in the range of Mr 40,000-60,000 were evidenced, together with a Mr 110,000 band phosphorylated by the particulate TPK of Mr 210,000. In similar conditions, the TPK solubilized from rat liver membranes and partially purified by gel filtration was associated with a Mr 170,000 alkali-resistant phosphoprotein. Thus, TPKs are expressed in BPH tissues and carcinoma cell lines. In BPH tissues, two forms of TPK are expressed and the predominant enzyme is soluble and of low molecular weight (Mr 40,000-60,000).
Assuntos
Hiperplasia Prostática/enzimologia , Neoplasias da Próstata/enzimologia , Proteínas Tirosina Quinases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Cromatografia em Gel , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Pessoa de Meia-Idade , Peso Molecular , Peptídeos/metabolismo , Fosfoproteínas/biossíntese , Fosforilação , Espermatozoides/enzimologia , Especificidade por SubstratoRESUMO
Scanning electron microscopic studies demonstrate that the normal biconcave shape of the human erythrocyte is maintained in hypotonic saline when physiological levels (10 minus 5M) of cholesterol sulfate are added to the solutions. Cholesterol sulfate is a naturally occurring sterol conjugate in plasma and erythrocyte membranes and we propose that it may belong to that class of amphipathic molecules responsible for the maintenance of structure of the erythrocyte by interaction with membrane components.
Assuntos
Membrana Celular/ultraestrutura , Eritrócitos/ultraestrutura , Colesterol , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , SulfatosRESUMO
Previous in vitro studies to evaluate the possible role of cholesterol sulfate in the stabilization of the human erythrocyte membrane have been extended to the dog in vivo. Thus, following the injection of labelled cholesterol sulfate, a large fraction of the administered sterol conjugate is taken up by the membrane of the canine erythrocyte. Peak membrane levels were obtained within 30-60 min. Measurement of radioactivity associated with the plasma and red cell fractions in serial samples allowed the calculation of the half-life of cholesterol sulfate in each fraction. From the data obtained from the plasma of four dogs, the half-life was calculated to 5.8 plus or minus 0.9 h. The half-life of chlesterol sulfate associated with the erythrocyte membrane was calculated to be 6.7 plus or minus 1.2 h. In addition, following the intravenous administration of 0.2-0.7 mg of cholesterol sulfate/kg of body weight and withdrawal of serial blood samples, a significant diminution in the degree of hemolysis was observed when the red cells were exposed to hypotonic saline solutions. Maximal stabilization effects were observed at approx. 6-7 h after the administration of the sterol conjugate. Hemolytic properties returned to normal at approx. 24 h following the injection.
Assuntos
Colesterol/sangue , Eritrócitos/metabolismo , Ácidos Sulfúricos/sangue , Animais , Membrana Celular/metabolismo , Cães , Cinética , Receptores de Droga , Fatores de TempoRESUMO
Whole microsomal membrane preparations and Triton X-100-solubilized human placental steroid sulfatase were subjected to radiation inactivation analysis using gamma rays from a 60Co irradiator in order to assess the size of the physiological form of the enzyme. The data indicate that the enzyme exists as a monomer of molecular weight 78600 in Triton-containing buffers and as a polymer of molecular weight 533000 within the microsomal membrane.
Assuntos
Microssomos/efeitos da radiação , Placenta/enzimologia , Sulfatases/efeitos da radiação , Feminino , Humanos , Cinética , Microssomos/enzimologia , Peso Molecular , Gravidez , Esteril-Sulfatase , Sulfatases/metabolismoRESUMO
Focal adhesion kinase (pp125FAK) is a nonreceptor protein tyrosine kinase transducing signals initiated through integrin activation triggered by cell/extracellular matrix (ECM) interactions. To examine its role in epithelial cell adhesion, proliferation, and differentiation, we have studied pp125FAK expression, activity, and association with paxillin in two canine prostate models in which these functions can be selectively regulated: in vitro by vitronectin (VN) and serum factors, and in vivo by sex steroids. Kinetic studies revealed that the adhesion and spreading of prostatic epithelial cells in primary culture was regulated by serum VN and a natural ECM containing VN produced by prostate cells. While barely detectable in freshly isolated prostate cells, proliferating cells, after 72 h in culture, expressed higher levels of FAK mRNA (8-fold), pp125FAK (50-fold), and paxillin (50-fold). In prostate cells with a reduced growth rate after 2 weeks in culture, we observed a decrease in pp125FAK (4-fold) and its transcript (3-fold), but no change in paxillin. In vivo, both proteins were undetectable in normal and hyperplastic glands composed of a well differentiated epithelium, and in prostates restored by androgen supplementation. In contrast, pp125FAK and paxillin were up-regulated by androgen deprivation (castration) and further increased by estrogen treatment, which yielded metaplastic prostates mostly composed of proliferating basal epithelial cells. Moreover, both proteins were constitutively phosphorylated on tyrosine in the metaplastic prostate, as well as in proliferating cultured cells. Together, these results demonstrate that pp125FAK expression is regulated at the protein and mRNA levels and forms active signaling complexes with paxillin when epithelial cells in contact with ECM proteins are induced to proliferate in vivo and in vitro.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Diferenciação Celular , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Próstata/citologia , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Células Cultivadas , Cães , Células Epiteliais , Epitélio/metabolismo , Matriz Extracelular/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Hormônios Esteroides Gonadais/farmacologia , Humanos , Masculino , Dados de Sequência Molecular , Paxilina , Próstata/efeitos dos fármacos , Próstata/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , RNA Mensageiro/metabolismo , Homologia de Sequência , Vitronectina/farmacologiaRESUMO
The contribution of the vas deferens to the metabolism of steroids was investigated in the human and in the dog by perfusion experiments and in vitro incubation. Perfusion of testosterone or androstenedione through the canine vas deferens resulted in the formation of dihydrotestosterone; no significant formation of estrogens could be detected. When dihydrotestosterone was perfused, 3alpha- and 3beta-androstanediols were isolated. In vitro incubation experiments with human was deferencs have shown an active metabolism of androgens by this time and, therefore, confer a new role to the vas deferens in the male.
Assuntos
Androgênios/metabolismo , Estrogênios/metabolismo , Ducto Deferente/metabolismo , Animais , Radioisótopos de Carbono , Cães , Humanos , Marcação por Isótopo , Masculino , TrítioRESUMO
We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X-100-solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of Mr 44,000 [(1991) Biochem. Cell. Biol. 69, 146-153]. Herein we demonstrate by immunoprecipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches: (1) a two-dimensional electrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [gamma-32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.
Assuntos
Próstata/enzimologia , Proteínas Tirosina Quinases/metabolismo , Animais , Citosol/enzimologia , Cães , Eletroforese em Gel Bidimensional , Ponto Isoelétrico , Masculino , Peso Molecular , Fosforilação , Fosfotirosina , Desnaturação Proteica , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/isolamento & purificação , Tirosina/análogos & derivados , Tirosina/imunologiaRESUMO
A method was developed to prepare doubly labeled antibodies whereby fluorescein-labeled antibodies were reacted with N-[ethyl-2-3H]ethylmaleimide. This tritiated conjugate exhibited the same immunoreactivity as the non-derivatized fluorescein-labeled antibody. This method provides a marker for immunocytochemical studies which takes advantage of the rapidity of fluorescent tracing combined with the stability and precision of the radioautography technique.
Assuntos
Etilmaleimida , Fluoresceínas , Imunoglobulina G , Tiocianatos , Trítio , Animais , Reações Antígeno-Anticorpo , Autorradiografia , Cricetinae , Fluoresceína-5-Isotiocianato , Imunofluorescência , Humanos , Camundongos , Óvulo/imunologia , Testes de Precipitina , Coloração e RotulagemRESUMO
Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microencapsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.
Assuntos
Alginatos , Anticorpos/metabolismo , Ilhotas Pancreáticas/imunologia , Membranas Artificiais , Polilisina , Animais , Técnicas In Vitro , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas , Camundongos , Peso Molecular , Permeabilidade , Ratos , Ratos WistarRESUMO
Sex steroid concentrations in plasma collected from the canine deferential vein were measured, after separation, by radioimmunoassay. The concentrations found were compared with those in the peripheral plasma. The mean concentrations of androstenedione, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol and oestradiol-17 beta were 13.2, 14.7, 8.9, 4.6, 8.0 and 7.5 fold higher respectively in plasma from the deferential vein than in peripheral plasma. A close anatomical relationship was found between the vasa deferentia, the deferential vein and the peripheral plasma as well as between the deferential vein and the prostate gland. These findings emphasize and extend earlier conclusions that high levels of sex steroids present in the deferential vein could have a local influence on the growth of the prostate.
Assuntos
Hormônios Esteroides Gonadais/sangue , Ducto Deferente/irrigação sanguínea , Androstano-3,17-diol/sangue , Androstenodiona/sangue , Animais , Di-Hidrotestosterona/sangue , Cães , Estradiol/sangue , Masculino , Próstata/irrigação sanguínea , Estereoisomerismo , Testosterona/sangueRESUMO
Radioimmunoassays were used for the measurement of several androgens in canine plasma and in the liquid of the vas deferens. Large variations in the plasma concentrations of androstenedione, testosterone and 5 alpha-dihydrotestosterone occurred during a period of 24 h, but there was no evidence of a circadian rhythm. The ratios of the androgen concentration in the liquid of the vas deferens compared with that in the peripheral plasma were: androstenedione, 4.6; testosterone, 1.9; 5 alpha-dihydrotestosterone, 13.6; 5 alpha-androstane-3 alpha, 17 beta-diol, 17.0; 5 alpha-diol, 22.4. These high levels of androgens in the liquid of the vas deferens could play a role in the development of prostatic hypertrophy.
Assuntos
Androgênios/metabolismo , Ducto Deferente/metabolismo , Androgênios/sangue , Androstenodiona/metabolismo , Animais , Di-Hidrotestosterona/metabolismo , Cães , Masculino , Radioimunoensaio , Testosterona/metabolismoRESUMO
To determine how functional changes such as growth and differentiation in the prostatic epithelium would alter protein phosphorylation on tyrosyl residues, differentiated (secretory) and basal (non-secretory) prostatic epithelial cells from normal and from metaplastic canine prostates were labeled with [32P]phosphate and their alkali-resistant phosphoproteins were analyzed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tyrosine protein kinase activity of the cells was also assayed by phosphorylation of a synthetic substrate, poly(Glu80-NaTyr20), in the presence of [gamma-32P]ATP. The prostates were characterized by tissue morphology and the cells by their density on Percoll gradients and [3H]thymidine incorporation into DNA. In prostates from intact dogs, tall columnar secretory epithelial cells were predominant and the non-secretory or basal epithelial cells were quiescent and did not incorporate [3H]thymidine. In metaplastic glands obtained from estrogen-treated castrated dogs, most of the isolated cells were non-secretory and synthesized DNA. Alkali-resistant phosphoproteins were present in all cell types. Except for two common phosphoproteins, p44 and p82, the relative phosphoprotein distribution within the gel differed when normal prostatic cells were compared to the metaplastic cells. However, the phosphoprotein patterns were the same in secretory and non-secretory cells from the same prostate tissue. On the other hand, the relative labeling intensity of phosphoproteins was always higher in non-secretory cells compared to corresponding secretory cells. Accordingly, tyrosine protein kinase (TPK) activity, expressed on a cell basis, was also higher in non-secretory cells; the ratios of TPK activities, non-secretory over corresponding secretory cells, was always higher than unity for all preparations but overlapped when cells from metaplastic glands were compared to those isolated from normal prostates. Thus, non-secretory epithelial cells isolated from either normal or metaplastic glands have a higher ability than corresponding secretory cells to phosphorylate endogenous alkali-resistant phosphoproteins and their TPK activity, measured with the synthetic substrate poly(Glu80-NaTyr20), is also higher.
Assuntos
Fosfoproteínas/biossíntese , Próstata/metabolismo , Proteínas Tirosina Quinases/metabolismo , Álcalis/farmacologia , Aminoácidos/metabolismo , Animais , DNA/biossíntese , Cães , Células Epiteliais , Epitélio/metabolismo , Masculino , Metaplasia , Fosforilação , Próstata/enzimologia , Próstata/patologiaRESUMO
To evaluate the possible role of intracellular phosphatases in the local regulation of prostatic functions, the effect of sodium orthovanadate (VO4), an inhibitor of phosphotyrosyl protein phosphatases, was studied on both protein phosphorylation and acid phosphatase activity. Secretory and non-secretory epithelial cells were isolated from normal and metaplastic prostates and incubated with [32P]phosphate in the presence and in the absence of VO4; the phosphoproteins were separated by electrophoresis and the gels were either directly submitted to autoradiography or after an alkali treatment to reveal those proteins enriched in phosphotyrosine. Prior to alkali treatment, several phosphoproteins were evidenced and in less than half of the cell preparations a slight increase in labeling intensity under vanadate (less than 75%) was observed in two phosphoproteins, p57 and p44. After alkali treatment: (1) the effect of VO4 on p57 remained in the order of 44-45% and it was restricted to less than half of non-secretory cell preparations; (2) its effect on p44 was intensified (134-207%) and observed in all cell types and in more than 80% of all preparations; and (3) in half of non-secretory cell preparations from metaplastic glands, an effect of VO4 on p35 (127%) became evident. In all instances, with normal and/or metaplastic prostates, protein phosphorylation activity, either total or alkali-resistant and in the presence or in the absence of VO4, was always higher in non-secretory epithelial cells as compared to secretory cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fosfatase Ácida/metabolismo , Próstata/efeitos dos fármacos , Proteínas/metabolismo , Vanadatos/farmacologia , Animais , Cães , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Masculino , Fosfoproteínas/metabolismo , Fosforilação , Próstata/enzimologia , Próstata/metabolismoRESUMO
Secretory and non-secretory epithelial cells and fibroblasts obtained from normal and hyperplastic canine prostate glands and from prostates of 6-week castrated dogs are cultured in monolayers. Prostatic fibroblasts are grown in non-selective culture medium and found at densities of 1.040-1.045 g/ml in Percoll gradients. Enriched populations of each epithelial cell type area obtained by varying the duration of the culture combined with the use of selective MEM D-Val mixture. When separated by centrifugation in Percoll density gradients, the secretory cells (high A.P.) are found at densities of 1.02-1.03 g/ml whereas the non-secretory cells (low A.P.) have densities of 1.05-1.06 g/ml. Both epithelial cell types are present in the normal and hyperplastic glands at the time of explantation. There is no correlation between the prostatic weight and the proportion of each cell type present in the tissue. On the basis of cell density in Percoll gradients and A.P. activity, those prostates with a high percentage of non-secretory epithelial cells yield better attachment and overall cultures than glands consisting mainly of secretory cells. Our results strongly suggest that non-secretory cells are precursors of the secretory type. In addition, the cells involved in the aging process of the culture are the secretory epithelial cells.
Assuntos
Próstata/citologia , Hiperplasia Prostática/patologia , Fosfatase Ácida/análise , Animais , Separação Celular , Sobrevivência Celular , Centrifugação com Gradiente de Concentração , Cães , Histocitoquímica , MasculinoRESUMO
Protein tyrosine kinases (PTKs) are key enzymes implicated in signal transduction pathways regulated by growth factors (GFs). We have previously shown by immunohistochemistry that the level of phosphotyrosine (pY) proteins is increased in prostatic basal epithelial cells following estrogen treatment in castrated dogs. In this study, we investigated if this treatment increases the level and distribution of prostatic PTK activity, and more specifically, if it alters the expression and/or activity of the Src family members p60src and p53/56lyn. Prostates from normal and hyperplastic dog prostates, as well as those from castrated dogs treated with androgens, were also examined. Only the glands obtained from estrogen-treated dogs had a significantly increased total and specific PTK activity, observed uniquely in the particulate extract, as compared to the other types of prostates studied. In addition, this increased activity was correlated upon gel filtration chromatography with the presence of an additional peak of activity with an apparent molecular weight of 130 kDa, which was absent in other prostate fractions presenting only 50 kDa peaks. Using antibodies, we demonstrate that active p60src and pp53/56lyn kinases accounted for 81% of the activity in this 130 kDa peak. On the other hand, in situ renaturation also revealed the presence of still uncharacterized 50/55 kDa PTKs in the 130 kDa peak. Altogether, these findings raise the possibility that these PTKs contribute to the transmission of mitogenic signals originating directly or indirectly from estrogen stimulation of the basal cell layer of the prostate.
Assuntos
Estradiol/farmacologia , Proteína Oncogênica pp60(v-src)/metabolismo , Próstata/enzimologia , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Cães , Ativação Enzimática/efeitos dos fármacos , Masculino , Peso Molecular , Desnaturação ProteicaRESUMO
When cultured in monolayers, non-secretory epithelial cells from canine prostates actively synthesize DNA, RNA and proteins; subsequently, mitotic figures and an increase in cell number are observed. During this culture period, cell size and acid phosphatase activity remain constant. As the culture proceeds, these cells mature into secretory cells as evidenced by a gradual shift in their density in Percoll gradients to the density of secretory cells and by an increase in the cellular content of acid phosphatase. During maturation, the size of the non-secretory cells increases and their morphology changes and becomes similar to that of the secretory cells. When a homogeneous population of secretory cells is cultured, DNA synthesis is minimal and few mitotic figures may be observed while cell number and cell density remain constant. Early in the culture period, their size increases and by 2 weeks their acid phosphatase activity is 2--3-fold higher than that of the non-secretory cells. Thus, upon culture, the non-secretory epithelial cells enter and proceed through the cell cycle with evidence of DNA synthesis and mitosis. Those cells leaving the cycle undergo maturation into secretory cells which further differentiate with the concomitant appearance of acid phosphatase activity. This model will be useful to study prostatic hyperplasia and hypertrophy and the control mechanisms involved in these phenomena.
Assuntos
Próstata/citologia , Fosfatase Ácida/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , DNA/biossíntese , Cães , Células Epiteliais , Masculino , Mitose , Biossíntese de Proteínas , RNA/biossínteseRESUMO
The increase in acid phosphatase (AP) activity in cultured canine prostatic epithelial cells was investigated as a biochemical marker of in vitro cellular differentiation. The enzyme was studied in secretory and non-secretory epithelial cell populations obtained from control and cycloheximide-treated cultures over a period of 3 weeks and compared to the AP present in tissue and cellular extracts from normal canine prostates. The progressive increase in AP activity with the duration of culture was strongly inhibited by cycloheximide in both cell populations. The degree of inhibition was more pronounced late in the culture when AP activity increased at a faster rate in secretory cells. Cycloheximide inhibited protein biosynthesis by 70-80% as evidenced by a reduction in the incorporation of amino acids into acid-insoluble material. However, the specific activities of AP in the cellular extracts were similar in control and cycloheximide-treated cultures and increased sharply by 3-4-fold in the secretory cells after 12 days of culture. When extracts derived from control and cycloheximide-treated cells of various duration were submitted to electrophoresis in polyacrylamide gels (PAGE), a unique pattern of three bands of AP activity with Rf values of 0.18, 0.27 and 0.38 was obtained. In controls the AP activity in the band with an Rf of 0.18 increased preferentially during the culture period and was more important quantitatively in secretory cells. In cycloheximide-treated cultures the increase of AP activity associated with the band with an Rf of 0.18 was more strongly inhibited. The addition of tartrate to the staining mixture inhibited all three bands of AP activity. Similar results were obtained when extracts derived from freshly dispersed cells as well as from normal canine prostatic tissue were submitted to PAGE; the AP activity was resolved into 3 bands with Rf values of 0.15-0.18, 0.23-0.27 and 0.33-0.38; all three bands were inhibited by the addition of tartrate and the first band was predominant. Thus, the increase in AP activity in prostatic epithelial cells in a culture medium supplemented with serum and deprived of sex steroids is due to the de novo synthesis of a major form of the enzyme by the secretory cells.
Assuntos
Fosfatase Ácida/metabolismo , Próstata/enzimologia , Animais , Diferenciação Celular , Células Cultivadas , Cicloeximida/farmacologia , Cães , Masculino , Próstata/citologia , Tartaratos/farmacologiaRESUMO
We report on a patient with Klinefelter syndrome (KS) and the homogeneous aneuploidy 47,Xi(Xq)Y, or male trisomy Xq. He had many characteristics of classical KS: small testes, azoospermia, elevated FSH and LH, average intelligence, and normal androgenization, but his stature was not increased, compared with his father's and brothers'. The i(Xq), found in all cells analyzed, was late-replicating, monocentric, and also asymmetric for the RBG-banding of the two arms, indicating a different chronology of DNA synthesis in each arm. When indicated, in the seven previously reported cases, the level of plasma testosterone was always subnormal; it was normal (650 ng/100 ml) in our patient, who had normal masculinization. Thus the level of testosterone among patients with KS is not necessarily lower with an extra Xq. Furthermore, the sharp contrast in the height of KS patients with or without an i(Xq) is striking. It appears definitely possible to associate the isochromosome Xq Klinefelter syndrome with a lack of height increase.
Assuntos
Síndrome de Klinefelter/genética , Trissomia , Cromossomo X , Adulto , Aneuploidia , Estatura , Bandeamento Cromossômico , Humanos , Cariotipagem , Masculino , Testosterona/sangueRESUMO
The influence of menstrual cycle phases and hormonal contraception on serum lipid and apolipoprotein (apo) levels was investigated in a group of normally menstruating young women. The study period covered a normal menstrual cycle (pretherapy), the fourth cycle of treatment with a triphasic oral contraceptive (OC) preparation, and the cycle immediately following interruption of therapy (cycle 5, posttherapy). Cycle phases were defined on the basis of serum hormone levels and basal body temperature determinations. Significant differences in cholesterol (free and esterified) levels were observed during the menstrual phase of both the normal menstrual cycle (lower) and the OC cycle (higher), when compared with the other phases. Triglycerides, which were higher under OCs, fluctuated similarly throughout the two cycles, but phase differences did not reach statistical significance. Apo AI and apo B were both higher under OCs, and apo B followed a trend similar to cholesterol during the two cycles. During the first month after discontinuation of OCs, cholesterol levels returned progressively to baseline values, while triglycerides were only partially decreased. We conclude that cyclic fluctuations in lipid levels do occur under the influence of both endogenous and exogenous sex hormones.
PIP: Total and free cholesterol, triglycerides, and apolipoproteins apo-A1 and B were determined at precise phases of a pre-therapy menstrual cycle, the 4th cycle on a triphasic oral contraceptive, and in the 1st post-therapy menstrual cycle in 18 women. The triphasic pill contained 5 mcg ethinyl estradiol and 180, 215 and 250 mcg norgestimate for 7 days each (ORF 10131 Triphasic, Ortho Pharmaceuticals, Raritan, NJ). Total cholesterol and triglycerides were measured enzymatically by autoanalyzer (Abbott Bichromatic Analyzer 100), free cholesterol by commercial kit (Boehringer-Mannheim, Mannhein, Germany), and apolipoproteins by electroimmunoassay (Hydragel Apo A1/B, Sebia, Issy- les-Moulineaux, France), with strict quality control using commercial standards. Sera were sampled in 4 phases: Days 3, 4 or 5 of menses, in the follicular phase during rising or peak estradiol levels, at ovulation at peak or highest LH level, and in luteal phase at peak progesterone level. In pill cycles, sera were sampled during each week. Total cholesterol was significantly lower in the menstrual phase, rose on average 9.2% in follicular phase (range -6.8% to +34.4%, in 13 of 18 women), and declined only slightly in luteal phase. Free and esterified cholesterol showed a similar pattern. Triglycerides similarly were lowest in menstrual phase, but were not significantly higher during menstrual cycles. In oral contraceptive cycles, total cholesterol fell an average of 10.7% in the 1st week, and remained at that level until the next pill-free interval or upon discontinuation, when cholesterol rose 11.2%. After discontinuation of the pill, all women resumed normal ovulatory cycles and showed stepwise normalization of cholesterol. Apo-A1 was significantly higher in pill cycles and pill-free intervals than in normal menstrual cycles (p0.001 at all 4 sample points); apo-B was also significantly higher in all samples form pill cycles (p0.05-0.001). There was no correlation between cholesterol levels and any of the hormone levels measured, estradiol, progesterone, LH or FSH.