Biomedical subjects

G Frenette

Publications and source records attributed to G Frenette.

At least 19 recordsLinked to original sources

PubMed1995

Mediation of glucocorticoid receptor function by transforming growth factor beta I expression in human PC-3 prostate cancer cells.

We investigated the role of glucocorticoids in controlling the proliferation of androgen-independent PC-3 human prostate cancer cells via the action of transforming growth factor beta 1 (TGF beta 1). The presence of glucocorticoid receptor (GR) in PC-3 cells was detected by immunoblotting analysis using a rabbit anti-GR polyclonal antibody against the synthetic human GR peptide (hGR383-393). In PC-3 cells, GR bound radiolabeled dexamethasone with an affinity similar to wild-type GR. In addition, GR-ligand complex bound radiolabeled DNA as detected by DNA band-shift analysis on gel electrophoresis and trans-activated the mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase chimeric gene in transiently transfected PC-3 cells. Dexamethasone (0.1 up to 100 nM) and TGF beta 1 (0.5 up to 50 ng/ml) inhibited PC-3 cell proliferation. TGF beta 1 and dexamethasone both increased the distribution of PC-3 cells into the G1/G0 phase of the cell cycle. Platelet-derived growth factor (PDGF) stimulated the proliferation of PC-3 cells and overcame dexamethasone's inhibition of PC-3 cell growth. Dexamethasone's inhibition (10(-7) M) of PC-3 cell growth was completely neutralized by RU 486 (10(-6)M) and partly neutralized by anti-TGF beta 1 polyclonal antibody. Furthermore, dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells. Because dexamethasone's inhibition was neutralized at least in part by an anti-TGF beta 1 polyclonal antibody and dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells, we conclude that GR function in human PC-3 prostate cancer cells is mediated at least in part by TGF beta 1 expression.

Cell Division↗

PubMed1995

N-terminal truncated forms of insulin-like growth factor binding protein-3 in the peritoneal fluid of women without laparoscopic evidence of endometriosis. Le groupe d'investigation en gynécologie.

OBJECTIVE: To characterize and purify peritoneal mitogens able to stimulate the proliferation of human endometrial cells in vitro. DESIGN: Peritoneal fluids (PFs) from 50 patients were collected at laparoscopy and pooled (270 mL) for purification of mitogenic activity. SETTING: University infertility clinic and endocrinology of reproduction and molecular endocrinology laboratories. PATIENTS: Fifty subjects presenting for tubal ligation, pelvic pain, mass, or infertility but otherwise having no evidence of endometriosis inflammation, infection, or tumor. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Assessment of mitogenic activity by 3H-thymidine incorporation into mouse embryo fibroblasts and into primary cultures of isolated epithelial and stromal cells of human endometrium. RESULTS: The PF mitogens were purified successively on carboxymethyl-sepharose and heparin-sepharose columns followed by fractionation on cartridges of C18 silica and reverse-phase high-performance liquid chromatography (HPLC). Four distinct bands were eluted from Sep-Pak, (Mississauga, Ontario, Canada) with molecular weights of 17 to 18, 20, 25, and 29 to 30 kd. The eluted fractions of Sep-Pak exerted preferential mitogenic activity on epithelial-derived human endometrial cells at an equimolar ratio with epidermal growth factor. Microsequencing of the 17 to 18, 20, 25, and 29 to 30 kd bands showed a homologous sequence with N-terminal amino acid sequences of insulin-like growth factor binding protein-3 (IGFBP-3). CONCLUSION: These data indicate that the PF of normal women without evidence of endometriosis contains N-terminal truncated forms of IGFBP-3 that mediate an apparent preferential mitogenic action on epithelial-derived endometrial cells. Therefore, they could play a role in the ectopic growth of endometrial cells.

Amino Acid Sequence↗

PubMed1991

Variations in 3H-diisopropylfluorophosphate binding proteins in human seminal plasma.

We have characterized the electrophoretic pattern and variations in 3H-diisopropylfluorophosphate (DFP)-binding proteins in human seminal plasma from normal men and from 103 patients attending the infertility clinic of our hospital. This study shows that 34 kDa prostate-specific antigen (PSA) is the major 3H-DFP-binding protein and that two other ubiquitous bands of 100 and 60 kDa are also present in seminal plasma from all the men studied. Additional bands of 92, 50-54 (doublet) and 38 kDa were also observed in some patients. The 38 kDa band was shown to be a highly glycosylated form of PSA. Further complexity was demonstrated by two-dimensional gel electrophoresis in the 27-30 kDa range of the gels since at least 10 major spots and rows of spots were seen. The concentration of these spots, including PSA, was extremely variable, as was their pattern of inhibition by various active site inhibitors of serine-proteases; these variations were not correlated with any specific sperm characteristics. With the exception of PSA, the proteins have not been identified. Their distribution suggests that most of them are exclusively of prostatic origin although a few could also derive from the seminal vesicles or blood. Future studies will be aimed at determining the nature of these proteins and their potential usefulness in andrology.

Antigens, Neoplasm↗

PubMed1991

Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma.

As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.

Carcinoma, Renal Cell↗

PubMed1990

Effects of androgen and antiandrogen treatment on canine prostatic arginine esterase.

A series of experiments were conducted to investigate the regulation of the primary secretory protein of the canine prostate, arginine esterase, by androgens and/or new antiandrogen under development. In the first experiment, castration decreased (P less than 0.05) prostatic arginine esterase levels relative to intact controls (0.26 +/- 0.1 and 17.0 +/- 0.1 mumole/min/mg protein, respectively). Treatment of castrate dogs with either 5, 10, or 20 silastic capsules (8 cm length) containing the androgen 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) plus 1 capsule containing estradiol-17 beta (E2) or the i.m. injection of 25 mg 3 alpha-diol and 0.25 mg E2 for 12 weeks resulted in a dose-dependent increase (P less than 0.05) in prostatic arginine esterase activity (6.8 +/- 1.7, 19.0 +/- 3.6, 21.3 +/- 0.9, and 14.2 +/- 0.7 mumole/min/mg protein, respectively). In the second experiment, steroid treatment (10 3 alpha-diol plus 1 E2 silastic capsules) of castrate dogs for 12 weeks resulted in prostatic arginine esterase activity of 17.8 +/- 2.3 mu mole/min/mg. Co-administration of the steroidal androgen receptor antagonist. Win 49,596 (WIN) at doses of 0.625, 2.5, 10, or 40 mg/kg/day p.o., dose-dependently inhibited (P less than 0.05) prostatic arginine esterase activity (14.9 +/- 1.1, 14.3 +/- 1.3, 3.4 +/- 1.9, and 0.21 +/- 0.1 mumole/min/mg, respectively) to levels similar to that observed in castrate controls (0.14 +/- 0.03 mumole/min/mg). Administration of the nonsteroidal androgen receptor antagonist flutamide at 10 mg/kg/day p.o. to steroid-induced dogs also inhibited (P less than 0.05) arginine esterase activity (0.07 +/- 0.02 mumole/min/mg). In the last experiment, treatment of intact dogs with WIN at 0.625, 2.5, 10, and 40 mg/kg/day for 16 weeks dose-dependently reduced (P less than 0.05) arginine esterase levels (17.0 +/- 1.0, 16.3 +/- 1.5, 10.2 +/- 1.2, and 3.9 +/- 2.5 mumole/min/mg, respectively) compared to intact controls (14.4 +/- 1.2 mumole/min/mg). Histomorphologic and ultrastructural evaluation of prostates from dogs indicated that antiandrogen treatment resulted in glandular epithelial atrophy as well as a reduction in the number of secretory granules. The results of these experiments support that canine prostatic arginine esterase activity is under androgenic control, can be inhibited by antiandrogen treatment and may serve as a functional marker of the androgenic state of the prostate. Whether the effects of androgen and antiandrogens on prostatic arginine esterase is direct or indirect due to a general inhibitory effect on secretory epithelial cell function requires additional study. Furthermore, subject to further evaluation, the steroidal androgen receptor antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)

5-alpha Reductase Inhibitors↗

PubMed1990

Fate of alpha-1,4-glucosidases and cathepsin D in the rat epididymis after vasectomy.

Short-term vasectomy was studied in adult male rats in order to ascertain whether cytosolic or lysosomal hydrolases were differently affected 100 days after vas ligation. The secretory form of alpha-1,4-glucosidase remained unchanged while the lysosomal form of the enzyme and also cathepsin D increased in the cytosol of both caput and cauda epididymis. This set of data demonstrates for the first time that a triggering mechanism which stimulates lysosomal activity is present all along the rat epididymis. Disposal of the continuous influx of spermatozoa from the testis could therefore require both an active and a passive process.

Animals↗

PubMed1989

Zinc binding to major human seminal coagulum proteins.

In vitro binding of zinc to proteins of the human ejaculate and of the various male accessory gland secretions was evaluated. The proteins were separated by sodium dodecyl sulfate gel electrophoresis and transferred to nitrocellulose filters that were subsequently incubated with 65ZnCl2. High levels of zinc binding were observed to approximately 20 protein bands (14 to 70 kDa) of the coagulated seminal plasma. There was only low binding to proteins of the spermatozoa and virtually no binding to any protein of the epididymal and prostatic fluids. When sperm liquefaction was allowed to occur, 65ZnCl2 binding to high-molecular weight proteins decreased rapidly, and after 15 min only the binding to proteins of molecular weights less than 25 kDa remained. In addition, zinc concentration was determined both in the centrifugate and in the supernatant after centrifugation of the coagulum. Zinc concentrations in the centrifugate and the supernatant were, respectively, 147 +/- 72 micrograms/g and 31 +/- 22 micrograms/g. The whole supernatant contained only 12% +/- 4% of total sperm zinc. Finally, in highly viscous sperm samples the concentration of zinc was not significantly different from that in normally liquefying sperm (167 +/- 87 micrograms/ml compared to 188 +/- 107 micrograms/ml). The main extracellular targets of prostatic zinc in humans are the secreted seminal vesicle proteins. The role of this binding remains unknown, however, because no direct relationship could be established between the concentrations of this metal and the phenomena of coagulation and liquefaction.

Humans↗

PubMed1987

Radioimmunoassay in blood plasma of arginine esterase: the major secretory product of dog prostate.

We have developed a radioimmunoassay to determine serum levels of arginine esterase, the major secretory product of the canine prostate. Although the antibodies recognized other related proteins in immunoblotting conditions, particularly in the pancreas, the radioimmunoassay appeared specific for arginine esterase of prostatic origin and reacted only slightly or not at all with cytosolic proteins from salivary glands, liver, skeletal muscles, and kidney. In serum samples, the sensitivity limit of the assay was about 2.5 ng per ml. The interassay and intraassay variations for serum samples that contained concentrations of arginine esterase ranging from 6 to 21 ng per ml were, respectively, 14% and 24%. Sera from 30 adult males and 14 adult females contained, respectively, 71 +/- 9 and 5.3 +/- 0.4 ng per ml. In a group of 12 dogs treated daily for 36-39 days with 50 micrograms of D-Trp6-luteinizing hormone-releasing hormone ethylamide, the concentrations of arginine esterase in the serum decreased progressively and became identical with those observed in the females. These results constitute the first evidence for the presence of a serum marker of the normal prostate in an experimental model. It should be particularly useful to test the effects of various agents on the prostate.

Animals↗

PubMed1987

Excretion of three major prostatic secretory proteins in the urine of normal men and patients with benign prostatic hypertrophy or prostate cancer.

We have studied the mode of excretion of three prostatic secretory proteins, namely acid phosphatase (PAP), prostate-specific antigen (PSA) and beta-inhibin, in the urine of normal adult men, and we have determined the urinary levels of these proteins in men with benign prostatic hypertrophy (BPH) or adenocarcinoma. The output of the three proteins was highly variable during the day. In order to minimize these variations, 24-hour urine samples were collected thereafter. Our study showed that PAP concentrations in 50% of men with or without symptomatic BPH were similar to those of normal young men. In the remaining 50%, PAP was undetectable. In contrast, average PSA and beta-inhibin concentrations were higher in patients with BPH than in young men (p less than 0.05). The three markers were decreased or nondetectable in about half of the patients with untreated prostatic cancer. This phenomenon was even more pronounced in patients receiving hormonal treatment (castration or diethylstilbestrol). However, some of these patients still excreted normal amounts of PAP, PSA, and beta-inhibin. Urinary and serum PAP levels showed no correlation. These results indicate that urinary prostatic markers provide an easy means to study the behavior of the primary prostatic tumor. This information may be of potential value since it is not obtained with serum markers which originate mostly from metastatic cells.

Acid Phosphatase↗

PubMed1987

The major 40-kDa glycoprotein in human prostatic fluid is identical to Zn-alpha 2-glycoprotein.

A major 40-KDA protein secreted by human prostate was isolated from whole seminal plasma by sequential column chromatography on DEAE-Sepharose CL-6B, concanavalin A(Con A)-Sepharose, and Sephadex G-100. Although the purified preparation still contained minor contaminants, its amino acid composition was found to be identical to the one of a glycoprotein isolated previously from seminal plasma by Lin et al (1983). Antibodies against this protein were produced in rabbits and their use in immunoblotting experiments revealed the presence of the antigen in several tissues including the prostate, the liver, the heart, the kidney, the epididymis, and the testis. A radioimmunoassay confirmed these results and showed that blood serum concentrations of this protein were relatively high in men (81 +/- 3 micrograms/ml), women (68 +/- 3 micrograms/ml), and cord blood of newborns (32 +/- 1 micrograms/ml). The serum concentrations of this protein along with its physicochemical characteristics suggested that it could be identical to Zn-alpha 2-glycoprotein, a human serum protein previously isolated by Burgi and Schmid (1961). This hypothesis was confirmed by a double immunodiffusion analysis using a commercial anti-Zn-alpha 2-glycoprotein antiserum. Finally, in vitro translation of prostatic poly(A) + RNA in rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes resulted in the formation of an immunoprecipitable 42-kDa band. These results show that Zn-alpha 2-glycoprotein can be synthesized in the prostate itself. The demonstration of high concentrations of this protein in prostatic tissue and prostatic secretion should facilitate the elucidation of its role in the prostate and in other tissues.

Amino Acids↗

PubMed1986

Proteolytic activity of arginine esterase from dog seminal plasma towards actin and other structural proteins. Comparison with trypsin and kallikrein.

At equimolar ratio of enzyme/substrate, actin, tropomyosin, fibronectin and myosin were extensively hydrolyzed during an incubation of one hour at 37 degrees C. Dog serum albumin, ovalbumin, bovine gamma-globulin and human prostatic acid phosphatase were not hydrolyzed. The activity of arginine esterase towards actin at pHs 6.5, 7.1 and 7.6 was respectively 60, 74 and 84% of the one found at optimum pH 8.2. The cleavage products of actin by arginine esterase and trypsin were similar although trypsin activity was 5000-fold higher. Kallikrein produced a major fragment of actin not observed with arginine esterase and trypsin. It is concluded that arginine esterase has a low trypsin-like activity towards structural proteins and that this activity may have a physiological significance.

Actins↗

PubMed1986

Origin of alkaline phosphatase of canine seminal plasma.

The tissular origin of alkaline phosphatase was evaluated in canine seminal plasma. Alkaline phosphatase activity was most concentrated in the first fraction of the split ejaculate and was virtually undetectable in the third and fourth fractions. By contrast, arginine esterase, a known marker of dog prostatic secretion, was present in similar concentrations in all fractions of the split ejaculates analyzed by SDS gel electrophoresis. Similarly, arginine esterase was very abundant in secretory granules prepared from dog prostate homogenates, whereas these granules contained virtually no alkaline phosphatase. Among male sex accessory organs, alkaline phosphatase activity was very high in the epididymis and much lower in the testis and prostate. Furthermore, the specific activity in epididymal fluid collected from the cauda epididymis was about 10 times higher than in the corresponding epididymal homogenates. These results show that the major portion of alkaline phosphatase in dog seminal plasma does not come from the prostate but from the epididymis.

Alkaline Phosphatase↗

PubMed1985

Enzymatic characterization of arginine esterase from dog seminal plasma.

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.

Animals↗

PubMed1985

In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product.

Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.

Animals↗

PubMed1985

Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically.

We have isolated secretory granules from dog prostate homogenates and have determined whether a major portion of arginine esterase was localized in this fraction and if it was enzymatically active. Secretory granules were purified by density gradient centrifugation on sucrose, metrizamide, or Percoll. A major proportion of whole prostate homogenate arginine esterase was found in the granule fractions. Furthermore, the specific enzymatic activity in the granules was similar to the one observed in seminal plasma. No evidence could be found for the existence of significant amount of a zymogen inactive form of arginine esterase. These results suggest that arginine esterase could be active within the secretory granules in vivo and that it could hydrolyze protein substrates contained in this organelle.

Acid Phosphatase↗

PubMed1985

Biochemical characteristics of the proteins secreted by dog prostate, a review.

Proteins secreted by dog prostate differ from those of other species and in particular from those of man, both in terms of quantity and quality. These differences can be observed in the electrophoretic pattern of proteins in polyacrylamide gels and in enzymes such as phosphatases, glycohydrolases and proteases. For instance, canine acid phosphatase, although quite similar biochemically to the human enzyme, is about one hundred fold less concentrated in both prostatic tissue and seminal plasma than in the human. By contrast, arginine esterase, which is virtually absent in human prostatic secretion, comprises more than 90% of total proteins secreted by dog prostate. The most recent data on each of these enzymes, enzyme classes and proteins will be reviewed. Despite recent advancement of knowledge on the biochemistry of these constituents, their biological function as well as their contribution to the pathogenesis of prostatic diseases still remain a matter of speculation. For both of these aspects, dog appears to be a particularly interesting model.

Acid Phosphatase↗

PubMed1984

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing.

Canine prostate fluids and seminal plasma contain a major androgen-dependent protein which was identified as a proteolytic enzyme exhibiting an Arg-esterase activity. This protease, as characterized, is shown to be present as a two-chain structure held together by at least one disulfide bridge and composed of approximately 220 amino acids. Amino acid sequence determination of both chains has revealed a clear homology to other known amino acid sequences of serine proteases. Furthermore, the comparison of the presented 58 amino acids of the Arg-esterase with the other sequences revealed a very strong homology (larger than 50%) to members of the kallikrein family. The two chain structure could thus result from autolysis of a single chain enzyme in the 'kallikrein autolysis loop'. Amino acid composition of the canine prostatic enzyme suggests that it is related, but not identical, to pancreatic canine kallikrein.

Amino Acid Sequence↗

PubMed1984

Involution of spontaneous benign prostatic hyperplasia in the dog under the influence of chronic treatment with a LHRH agonist.

Eight dogs with spontaneous benign prostatic hyperplasia were treated with daily subcutaneous injections of 25 micrograms of Buserelin, (D-Ser(TBU)6,des-Gly-NH2(10))ethylamide during 3 months. Prostate weight before treatment was estimated by tridimensional measurements during open surgery. After 3 months of treatment, prostate weight was decreased by 50-60% In two castrated control animals, prostate weight decreased about 70% after 3 months. Histological pattern in prostates from castrated or Buserelin-treated animals was similar and indicated considerable atrophy of glandular epithelium and predominance of stroma. At the same time, testes weight decreased with complete disappearance of spermatogenic activity. Acid phosphatase and arginine esterase activity levels, two markers of androgenic action in the prostate, were similar after Buserelin or castration. Steroid receptors for androgens, estrogens, and progesterone also behaved similarly under the influence of Buserelin and castration.

Animals↗