Bioavailable testosterone as index of androgenicity (from 1973 to 2003).
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Aging↗
Biomedical subjects
R R Tremblay
Publications and source records attributed to R R Tremblay.
The "climacterium virile": from Lagueur's hesitations to our legitimate hedging in 2003.
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Aging↗
Simple purification procedure for human prostatic kallikrein hK2 in its active form.
Kallikrein hK2 is a new potential marker of prostate cancer. It is the last member of the human kallikrein gene family to be isolated. We propose a simple purification procedure permitting us to obtain the active form of hK2 starting from human seminal plasma and using commonly available chromatography matrices. In contrast to recently published papers, this procedure is carried out without any immunoaffinity chromatography step and without the need for any antibody to follow the purification. Furthermore, it does not require any recombinant DNA technology nor sophisticated instruments.
Biomarkers, Tumor↗
Assessment of the trypsin-like human prostatic kallikrein, also known as hK2, in the seminal plasma of infertile men: respective contributions of an ELISA procedure and of Western blotting.
Human seminal plasma (SP) is a unique source of kallikreins. Prostate-specific antigen (hK3), which is a chymotrypsin-like human prostatic kallikrein (CHPK), and its cousin protein (hK2), which is recognized as a trypsin-like human prostatic kallikrein (THPK), have been assessed in infertility disorders to test the hypothesis that oligoasthenoteratozoospermia (OAT) is associated with an abnormal prostatic function. Monoclonal antibodies specific for THPK (hK2) were produced by Immunova, Canada, and used to develop a new enzyme-linked immunosorbent assay procedure and to perform Western blot analyses in SP. The immunoradiometric assay from Hybritech Inc., San Diego, Calif., was selected for CHPK (hK3) measurements in SP. Determinations of the THPK and of CHPK contents in SP from four groups of subjects were performed after validation of the assays. The concentration of both kallikreins was similar in three groups of infertile men, and no statistical difference from the control group was recorded. Western blot analysis confirmed the existence of different molecular forms of both kallikreins in SP. Generally, these molecular forms were not affected by infertility disorders except when obstructive azoospermia led to the exclusion of seminal vesicles, which are the sources of protein C inhibitor (PCI). No THPK-PCI complex was observed because THPK, unlike CHPK, is bound mainly to PCI within a few minutes after ejaculation. These data suggest that measurements of kallikreins in the SP of infertile men are much less useful than evaluation of their different molecular forms. Specifically, the absence of THPK-PCI appears to be a reliable feature of obstructive azoospermia, and this test should be routinely practiced in andrology laboratories.
Blotting, Western↗
Contamination of purified prostate-specific antigen preparations by kallikrein hK2.
This paper ascertained the contamination by hK2 of two types of PSA preparation, that of Sensabaugh and Blake (J. Urol., 144: 1523, 1990) and that of Deperthes et al (J. Androl., 17: 659, 1996). In the first procedure, the free forms of hK2 co-migrated with PSA during the CM-Sephadex and the Sephacryl S-200 steps. By contrast, in the second procedure a very high proportion of hK2 was separated from PSA. In two different Sensabaugh and Blake procedures, the hK2 contamination per microg. of PSA was found to be respectively 0.3 and 1.0 ng. We conclude that hK2 is a quantitatively minor contaminant of some PSA preparations. That contamination is probably of little consequence for PSA standardization but it could lead to erroneous conclusions in enzymatic studies of PSA.
Chromatography↗
Serpin-derived peptide substrates for investigating the substrate specificity of human tissue kallikreins hK1 and hK2.
The third human tissue kallikrein to be identified, hK2, could be an alternate or complementary marker to kallikrein hK3 (prostate-specific antigen) for prostate diseases. Most of the hK2 in seminal plasma forms an inactive complex with protein C inhibitor (PCI), a serpin secreted by seminal vesicles. As serpin inhibitors behave as suicide substrates that are cleaved early in the interaction with their target enzyme, and kallikreins have different sensitivities to serpin inhibitors, we prepared a series of substrates with intramolecularly quenched fluorescence based on the sequences of the serpin reactive loops. They were used to compare the substrate specificities of hK1 and hK2, which both have trypsin-like specificity, and thus differ from chymotrypsin-like hK3. The serpin-derived peptides behaved as kallikrein substrates whose sensitivities reflected the specificity of the parent inhibitory proteins. Substrates derived from PCI were the most sensitive for both hK1 and hK2 with specificity constants of about 10(7) M-1. s-1. Those derived from antithrombin III and alpha2-antiplasmin were more specific for hK2 while a kallistatin-derived substrate was specifically cleaved by hK1. hK1 and hK2 substrates of greater specificity were obtained using chimeric peptides based on the sequence of serpin reactive loops. The main difference between specificities of hK1 and hK2 arise because hK2 can accommodate positively charged as well as small residues at P2 and requires an arginyl residue at P1. Thus, unlike hK1, hK2 does not cleave kininogen-derived substrates overlapping the region of N-terminal insertion of bradykinin in human kininogens.
Amino Acid Sequence↗
Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none.
In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.
Animals↗
Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator.
Our work was undertaken to compare the relative efficiency of 2 purified prostatic kallikreins, namely, hK2 and prostate-specific antigen (PSA or hK3), in the activation of single-chain urokinase (scuPA). We found that hK2 converts scuPA into an active enzyme with an efficiency equal to approximately 1/50 that of plasmin. During the activation of scuPA by hK2, two fragments of 33 and 22 kDa were generated. The NH2-terminal amino acid sequence of the 33 kDa fragment showed that hK2 cleaved scuPA between Lys158 and Ile159. In contrast to a previous report by another group, our purified hK3 preparation containing no trypsin-like contaminants was totally unable to activate scuPA. Our results show that kallikrein hK2 has plasmin-like activity and suggest that it could be the initiator of a proteolytic cascade leading to prostatic cancer invasion.
Amino Acid Sequence↗
Purification of enzymatically active kallikrein hK2 from human seminal plasma.
Kallikrein hK2 is a member of the human glandular kallikrein family which includes prostate-specific antigen (PSA) and pancreatic-renal kallikrein. The purpose of this work was to isolate and characterize for the first time the enzymatically active form of the hK2 protein starting from the PCI-hK2 complex isolated from human seminal plasma (Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hébert, J., Lazure, C. and Dubé, J.Y. (1995) Biochim. Biophys. Acta 1245, 311-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The purified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyzed synthetic chromogenic substrates containing esters of lysine and arginine but not of phenylalanine. Furthermore, hK2 formed molecular complexes with alpha 2 -antiplasmin, alpha 1-antichymotrypsin, antithrombin III and alpha 2-macroglobulin but not with alpha 1-antitrypsin. In conlusion, the new findings of the present paper are that the PCI-hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that the recovered free hK2 is a trypsin-like enzyme and that it can form molecular complexes with many of the major serum proteinase inhibitors.
Amino Acid Sequence↗
Immunohistochemical study suggesting a complementary role of kallikreins hK2 and hK3 (prostate-specific antigen) in the functional analysis of human prostate tumors.
The development of monoclonal antibodies directed against prostatic kallikrein hK2 prompted us to evaluate its content, along with that of hK3 (prostate-specific antigen), in human prostate carcinoma. Seventy tumors categorized according to the M.D. Anderson Hospital classification (grade I to IV) were analyzed by immunohistochemistry. The staining intensity or the kallikrein content of benign prostatic hyperplasia glandular tissue (used as control) and of grade I tumors appeared similar. In grade II to IV tumors, histochemical data revealed highly variable hK2 or hK3 content in approximately 25% of tumors. Such patterns are consistent with a current observation related to heterogeneity of prostate tumors. In addition, a few tumors did not express hK3 (n = 3), hK2 (n = 3), or both (n = 3), indicating that some growth patterns of prostatic neoplasia are associated with a lack of secretion or storage of hK3 or hK2 for immunodetection. This statement also appears relevant to metastases. It was interesting to note that 4% of hK3-negative tumors had detectable hK2. Because of the importance of hK3 as a serum marker of prostate disorder, this study addresses for the first time the question of the relative importance of both hK3 and hK2 in the immunohistochemical diagnosis of prostatic tumors. We conclude that hK2 may add new information to prostate cancer diagnosis and characterization.
Carcinoma↗
Remodeling of the cytoskeletal lattice in denervated skeletal muscle.
The effect of denervation-induced atrophy on the cytoskeletal lattice in rat fast- and slow-twitch skeletal muscle has been investigated. Immunochemical analyses and immunofluorescence microscopy experiments employing monospecific antibodies to dystrophin, desmin, and alpha-tubulin were carried out on intact and denervated muscles. The relative cellular content of dystrophin and desmin were reduced in the soleus muscle (slow-twitch), while significant increases were shown in the gastrocnemius muscle (fast-twitch). In both muscles, alpha-tubulin levels increased up to 12-fold as a function of time compared to control values. Immunofluorescence microscopy revealed a distinct rearrangement of the microtubule network toward a predominantly longitudinal alignment, which was accompanied by an increase in the density of the fluorescence. It is concluded that the relative increase of the three structural proteins in the fast-twitch gastrocnemius muscle may be related to the apparent resistance of this muscle type to denervation-induced atrophy. The increased alpha-tubulin content in denervated slow- and fast-twitch muscles could be indicative of an adaptive mechanism designed to maintain the integrity of the muscle fiber in view of eventual regenerative activities.
Animals↗
Kallikreins expression in a mature cystic ovarian teratoma.
We report an example of benign cystic ovarian teratoma that was incidentally discovered in a nulliparous 24-year-old woman taking contraceptive pills. Histological examination of the cyst revealed the presence of prostatelike tissue in association with a wide variety of other tissues. The use of highly specific monoclonal antibodies developed against the two prostate-specific kallikreins (hK2 and hK3) in humans allowed the demonstration that the multiple islets of epithelial cells were prostatic tissue in nature and that only part of these cells had conserved their intrinsic property of producing kallikreins.
Adult↗
Clinical practice guidelines for screening and monitoring male patients receiving testosterone supplementation therapy.
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Humans↗
Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma.
To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.
Animals↗
Identification of glandular kallikrein in dog pancreas and determination of its tissue distribution.
In order to establish a formal link between previously purified canine urinary kallikrein and dog pancreatic kallikrein whose cDNA sequence has recently been published, we have isolated the pancreatic kallikrein from that animal species. Pancreatic cytosol proteins were sequentially subjected to chromatography on DEAE-Sepharose CL-6B and Concanavalin A-Sepharose, to an autolysis step and finally to two-dimensional gel electrophoresis. Kallikrein immunoreactive spots were identified with an antibody directed against canine urinary kallikrein. These proteins were isolated after electroblotting and the amino acid sequence of their NH2-terminal portion was determined by microsequencing. The sequence was found to be identical to the one deduced from pancreatic kallikrein cDNA. Using the same antibody and immunohistochemical procedures, kallikrein was found to be present in the pancreas, the salivary glands, the kidney, the colon, the lungs and the testis. These results thus confirm the molecular nature of a glandular kallikrein in the canine species.
Amino Acid Sequence↗
Characterization of canine pancreas kallikrein cDNA.
Using a combination of primer extension and RT-PCR, the cDNA encoding a canine tissue kallikrein expressed in the pancreas was cloned and sequenced. The cloned 0.85 kbp cDNA contained a complete open reading frame encoding a polypeptide of 261 amino acids. The calculated molecular mass of the processed, unglycosylated, 237 amino acid protein was 26,428 Da. Its mRNA was expressed at high levels in the pancreas, kidney and submaxillary gland. The sequence of the encoded protein was highly homologous with canine prostatic arginine esterase (66%) and human renal/pancreatic kallikrein (74%). Therefore, the cloned cDNA encoded a previously uncharacterized canine kallikrein enzyme which was named dog renal/pancreatic kallikrein or dK2 according to the new nomenclature for kallikrein gene family members. Because of its specific pattern of tissue expression and the presence of all the amino acid residues necessary for kininogenase activity, we suggest that dK2 is the canine true tissue kallikrein.
Amino Acid Sequence↗
Putative effects of nerve extract on carbonic anhydrase III expression in rat muscles.
Carbonic anhydrase III (CA III), the predominant CA isoform in skeletal muscle is very sensitive to neuronal influences. We aimed to determine whether CA III expression could be influenced by neurotrophic factor(s) present in sciatic nerve extract (SNE). Intact muscles were thus compared with denervated soleus (SOL), extensor digitorum longus (EDL), and tibialis anterior (TA) muscles injected daily for 7 days with saline solution (SS) or with SNE. CA III activity was significantly increased in SS-treated EDL and TA muscles compared to control (CTR), while SNE injections partially prevented this increase. There was no significant difference for CA III activity in the SOL between CTR, SS, and SNE groups. The CA III mRNA increase observed in response to denervation was reduced by 40% in SNE-treated EDL and TA muscles. While SOL CA III mRNA level was not affected by denervation, a 52% decrease was observed with SNE. We concluded that neuronal modulation of CA III expression in type II fibers may involve a neurotrophic component.
Analysis of Variance↗
Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial.
OBJECTIVE: To compare the clinical efficacy and safety of the pure antiandrogen flutamide and the steroidal derivative spironolactone in the treatment of hirsutism in women. DESIGN: Fifty-three premenopausal women suffering from moderate to severe hirsutism were randomized into two groups and received either flutamide or spironolactone in association with a triphasic oral contraceptive (OC) pill. Hirsutism, acne, seborrhea, alopecia, and side effects were monitored monthly for a treatment period of 9 months and a follow-up after treatment period of 6 months. Blood samples were taken at each visit for assessment of endocrine, biochemical, and hematologic parameters. RESULTS: After only 6 months of therapy, flutamide caused a maximal reduction in the hirsutism score to a value within almost normal range; during the same period, spironolactone caused only a 30% reduction of the hirsutism score. Whereas flutamide caused a dramatic (80%) decrease in total acne, seborrhea, and hair loss score after only 3 months of therapy, spironolactone caused only a 50% reduction in acne and seborrhea, with no significant effect on the hair loss score. Four patients in the spironolactone group but only one in the flutamide group stopped the medication because of adverse side effects. CONCLUSION: The present data obtained in a randomized prospective study clearly demonstrate that the pure antiandrogen flutamide is superior to spironolactone in the treatment of female hirsutism and its related androgen-dependent symptoms and signs in women.
Adult↗