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Biomedical subjects

J Y Dubé

Publications and source records attributed to J Y Dubé.

At least 19 recordsLinked to original sources
PubMed1999

Epitope analysis of a prostate-specific antigen (PSA) C-terminal-specific monoclonal antibody and new aspects for the discrepancy between equimolar and skewed PSA assays.

BACKGROUND: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum. METHODS: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule. RESULTS: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with alpha1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by alphaACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA. CONCLUSION: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

Amino Acid Sequence↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1993

Characterization of rhesus monkey prostate specific antigen cDNA.

Using a RT-PCR approach, we obtained two overlapping cDNA clones containing the entire 1.5 kb sequence of rhesus monkey prostate specific antigen (rmPSA). The sequence obtained revealed an open reading frame of 261 amino acids. One potential N-glycosylation site was identified at Asn-78. The calculated molecular mass for the unglycosylated mature protein was 26,147 Da. Extensive amino acid homology (89%) was observed between rmPSA and its human counterpart. These results demonstrate that rhesus monkey and man prostate share a major biochemical component, and suggest that this animal species might be useful to answer specific questions related to human prostatic function and pathology.

Amino Acid Sequence↗
PubMed1993

Transcriptional regulation of dog prostate arginine esterase gene by androgens.

These studies were designed to define the molecular events involved in the modulation of dog prostate arginine esterase gene expression following short castration intervals and androgen treatment. Arginine esterase enzymatic activity and protein levels decreased about 50% 24 h after castration. Thereafter, a more progressive decrease was observed, resulting in 2-4-fold lower levels in 12-day castrates than in the intact controls. Total prostatic arginine esterase mRNA levels slowly decreased during the first five days after castration but more abruptly thereafter and were about 150-fold lower in 12-day castrated animals. By contrast, in isolated prostatic nuclei, levels of arginine esterase RNA precursors and mature transcripts rapidly fell following orchiectomy, with a 50-70% decrease 24 h after castration. Nuclear run-on experiments confirmed that the latter effects were the result of decreased arginine esterase gene transcription. All these changes could be at least partially reversed by administration of testosterone cypionate. Furthermore, no striking modifications in the proportion of epithelial/stromal cells in the prostatic tissue were observed following orchiectomy. These results show that castration and androgens exert very rapid effects on the gene expression of arginine esterase, and that the regulation occurs at the transcriptional level.

Androgens↗
PubMed1992

Demonstration of DNA binding factors interacting with a fragment of the canine prostate arginine esterase gene promoter.

We have studied, by the gel mobility shift assay, the interaction of DNA binding proteins with a fragment of the proximal promoter (from nucleotides -177 to -47) of the androgen-regulated canine prostate arginine esterase gene. Several shifted bands were obtained using nuclear extracts from various tissues. In the case of the prostate, the intensity of some of the shifted bands was decreased or increased when the extracts were prepared from animals that had been castrated 12 days earlier. Several of the DNA-protein complexes could be assigned to an interaction with part or all of the sequence GGGGGTGGGGG from-124 to -114. We also obtained evidence for the presence of protein(s) interacting with an Sp1 motif present in the same fragment. These results suggest that some ubiquitous factors different from the androgen receptors could be involved in the regulation of the arginine esterase gene.

Animals↗
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↗