Biomedical subjects

G Frenette

Publications and source records attributed to G Frenette.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1984

Single case report of prostate adenocarcinoma in a dog castrated three months previously. Morphological, biochemical, and endocrine determinations.

During the course of another investigation, three dogs had been castrated 3 months previously. Upon completion of the experiment, it was discovered that one dog presented a spontaneous prostatic adenocarcinoma of intraalveolar proliferative type at histology. Prostate weight of this dog before castration was estimated to be 22 g by tridimensional measurement at laparotomy and remained relatively constant (19 g) 3 months after castration. These results indicate that if regression had occurred in some cell populations (androgen-dependent) it was only partial and masked by growth of androgen-independent cells. Analysis of 12 individual steroids in peripheral blood and in prostatic tissue attested of a normal adrenal secretory activity. A series of 15 hydrolytic enzymes along with receptors for androgen, estrogen, and progesterone, were determined in prostatic tissue obtained at sacrifice. Enzymatic activities were those of typical epithelial cells, and most of them remained relatively high despite low levels of circulating testosterone. However, two markers of androgen action in dog prostate, acid phosphatase and arginine esterase, were significantly reduced. Receptor levels were similar to those of castrated animals. Thus, cancer cells had probably retained some androgen sensitivity.

Adenocarcinoma↗

PubMed1984

Characteristics of the inhibitory effect of chronic treatment with an LHRH agonist on testicular steroidogenesis in the dog.

Daily subcutaneous administration for 3 months of the potent LHRH agonist (D-Ser(TBU)6, des-Gly-NH2(10] LHRH ethylamide (25 micrograms) to adult dogs having spontaneous benign prostate hyperplasia (BPH) causes a marked inhibition of testicular androstenedione and testosterone secretion. This inhibition of delta 4-androgen secretion is accompanied by a decrease of testicular progestin precursors and 5 alpha-androgen metabolites, thus suggesting that, in dog, the loss of testicular steroidogenic activity, induced by the administration of an LHRH agonist, is due to a total inhibition of testicular steroidogenesis. In plasma, the concentration of both testosterone and dihydrotestosterone is also markedly depressed while androstane-3 alpha, 17 beta-diol levels remain unchanged. Measurement of prostatic steroid content has shown that administration of the LHRH agonist as well as castration is associated with a marked decrease in androstenedione, testosterone, and dihydrotestosterone levels in prostate while there is a small inhibition of androst-5-ene-3 beta, 17 beta-diol, androstane-3 beta, 17 beta-diol, dehydroepiandrosterone, and estrone concentrations in this tissue. The present data show that treatment with an LHRH agonist in the dog causes a marked inhibition of testicular steroid secretion similar to the one observed in adult men, and suggest that steroids from adrenal origin may also be involved in prostatic function.

17-alpha-Hydroxyprogesterone↗

PubMed1983

Effect of castration and steroid treatments on the activity of some hydrolytic enzymes in dog prostate.

Adult mongrel dogs were castrated and treated by intramuscular injections of 5 alpha-androstane-3 alpha,17 beta-diol (androstanediol) alone or in combination with estradiol in order to find convenient enzymatic markers of hormone action in prostate. The activities of 15 hydrolytic enzymes were determined. Arginine esterase, acid sulfatase, and acid phosphatase were found to be the most sensitive markers of testicular hormones since they were decreased 18-, 5- and 5-fold respectively after 1 month of castration. The enzyme activities returned to precastration levels after 2 weeks of injection of androstanediol to castrated animals. The effect of androstanediol on the majority of the remaining enzymes was small. In general, the activities obtained after androstanediol treatment in combination with estradiol were similar to those obtained with androstanediol alone. Finally, beta-glucuronidase and neutral sulfatase were increased after castration, a finding that suggests that these enzymes are constituents of stromal cells. These studies will provide a basis for future studies of hormone action in the dog prostate.

Acid Phosphatase↗

PubMed1982

Effect of hormone injections on levels of cytosolic receptors for estrogen, androgen and progesterone in dog prostate.

We have studied the effects of estradiol injections on cytosolic estrogen, androgen and progesterone receptor levels in order to understand the role of this steroid in the induction of prostatic hyperplasia in the dog. Adult mongrel dogs were castrated and were then given injections of estradiol (0.25, 0.8 or 2.5 mg) dissolved in olive oil containing 5% benzyl alcohol on days 0, 2, 5 and 7 after castration. Steroid receptor levels were determined by Scatchard analysis using charcoal assay one day after the last injection. All three receptors were increased maximally with the 0.8 mg dosage when compared with castrated controls. Estradiol binding increased from 89 +/- 10 (mean +/- SEM) to 361 +/- 37 fmol per mg prot., androgen binding from 47 +/- 5 to 123 +/- 11 fmol per mg prot. and progesterone binding from 26 +/- 3 to 211 +/- 36 fmol per mg prot. The small dose of estradiol. (0.25 mg) produced a significant (P less than 0.05) increase of the progesterone receptor levels from 25 +/- 3 to 48 +/- 14 fmol/mg prot. Substitution of estradiol by 5 alpha-androstan-3 beta,17 beta-diol (2.5 or 25 mg) resulted in receptor levels similar to castrated animals. However treatment with 5 alpha-androstan-3 alpha,17 beta-diol (25 mg) alone or in combination with estradiol (0.25 mg) increased significantly the androgen receptor while it decreased the estrogen receptor. These results show that the administration of estradiol at doses used to induce experimental prostate hyperplasia produce measurable effects in the prostate and suggest that the estradiol receptor may be implicated in this phenomenon.

Androstane-3,17-diol↗

PubMed1981

Effect of endocrine manipulations on the levels of cytosolic and nuclear receptors for androgens in dog prostate.

Cytosolic and nuclear androgen receptor levels were determined in dog prostates after castration and after treatment with steroids. The cytosolic androgen receptor levels 1, 5, and 13 days after castration were reduced to 32, 47, and 34 per cent of intact dog levels. We also initiated steroid treatments immediately after castration. The administration three times weekly of either dihydrotestosterone, 5 alpha-androstane-2 alpha, 17 beta-diol (3 alpha-diol), or of a combination of 3 alpha-diol and estradiol resulted after 13 days in a 2 fold increase of cytosolic androgen receptors relative to intact untreated dogs. Androgen receptor levels in 0.5 M KCl nuclear extracts were significantly (P less than 0.01) increased in spontaneously hyperplastic prostates relative to normal prostates. They were also significantly (P less than 0.05) increased in dogs treated with either dihydrotestosterone, 3 alpha-diol, or with a combination of 3 alpha-diol and estradiol but were highly depressed after castration. These results suggest that experimental prostatic hyperplasia produced by the injection of androgens probably occurs via increased androgen receptor levels.

Androstane-3,17-diol↗