Jean Dubé

Plasma concentrations of free and non-TeBG bound testosterone in women on oral contraceptives

Differential precipitation of plasma proteins after equilibration of the plasma with tracer amounts of testosterone (T) was used to measure free and not-T-estradiol binding globulin (TeBG)-bound T in 20 women and 10 men. Tritiated T was the tracer used. Comparison of this method with the equilibration dialysis method produced a good agreement in accuracy. The method is described as simple and reproducible, allowing routine assay of 20 plasma samples in duplicate by 1 technician. Normal values for men were 497 ng/100 ml (total T), 42 ng/100 ml(free T), 137 ng/100 ml (non-TeBG bound), and .5 mcg/100 ml (TeBG). Womens values were 52 ng/100 ml (Total T), 2.6 ng/100 ml (free T), 5 ng/100 ml (nonTeBG) and .9 mcg/100 ml (TeBG). Significant increases in total (p less than .05), free (p less than .01), non-TeBG-bound (p less than .05), and TeBG-T (p less than .01) were found for women taking norethindrone and mestranol. Women taking norgestrel-ethinyl estradiol had significant increase (p less than .01) in non-TeBG-bound T. It is concluded that estrogens increase the binding capacity of TeBG, and this is inhibited by norgestrel.

High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate-specific antigen.

Using a synthetic oligonucleotide primer complementary to human prostate‐specific antigen mRNA, we found that an additional sequence possibly similar to human glandular kallikrein‐1 could be read by a primer‐extension sequencing technique. We were able to confirm the identity of that additional sequence with another oligonucleotide primer complementary to a specific region of the human glandular kallikrein‐1 mRNA sequence. Northern blot analysis with 2 oligonucleotide probes respectively specific for prostate‐specific antigen and human glandular kallikrein‐1 mRNAs showed that the length of both mRNAs was similar at 1.5 kb. The level of human glandular kallikrein‐1 mRNA relative to that of prostate‐specific antigen could be estimated as approx. 10–20%. This study constitutes the first evidence that the human glandular kallikrein‐1 gene is expressed at a high level in a human tissue.

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. Int. J.Cancer 71: 897‐899, 1997.

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.

Effect of sodium molybdate on cytosolic androgen receptors in rat prostate

Abstract The addition of 10 −2 M sodium molybdate either to the homogenization buffer or to the homogenate (immediately after the homogenization) results in higher yields of cytosolic rat prostate androgen receptor as determined with [ 3 H]-17β-hydroxy-17α-methyl-estra-4,9,11-triene-3-one (R1881) or [ 3 H]-dihydrotestosterone (DHT) at 0°C. The increase varies between 10 and 80%, depending upon the experiments and the physiological condition of the animals. It is observed both by charcoal assay and by sucrose density gradient centrifugation. Molybdate has no effect on association constant ( K a , on sedimentation profiles on sucrose gradients, on steroid specificity and on the rate of dissociation of [ 3 H]-steroid receptor complex in the presence of unlabelled ligand. Molybdate increases the stability of androgen receptor complex against physiochemical denaturation at 0°hC and thermal denaturation at 15° and 25°C. Protection afforded by molybdate appears to occur by a mechanism common to receptor protection by endogenous hormones.

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.

Estrogen binding proteins in rat skeletal and perineal muscles: in vitro and in vivo studies.

Specific vitro binding of [3H]-estradiol was demonstrated in the cytosolic fraction of levator ani/bulbocavernosus (LA/BC) muscles and thigh muscles (TM) of 50-day-old rats. Using a charcoal assay, the apparent association constant (Ka) for estradiol was found to be 1.6 × 109 M−1 in LA/BC and 0.9–2.0 × 109 M−1 in female and male TM. The Ka was not affected by 24 h of castration but the number of binding sites increased significantly (P < 0.05) from 7.8 ± 1.7 (SD) to 11.7 ± 2.8 (SD) fmol/mg protein in LA/BC and from 3.4 ± 1.6 (SD) to 5.8 ± 0.5 (SD) fmol/mg protein in male TM. In female TM, the level of binding sites of 24 h ovariectomized rats (4.3 ± 1.0 (SD) fmol/mg protein) was relatively similar to the one found in intact rats in diestrus phase (3.4 ± 0.8 (SD) fmol/mg protein) but higher than in proestrus phase (0.8 ±0.15 (SD) fmol/mg protein). In both types of muscles, this cytosol estradiol binding protein sedimented in the 9–11S region of sucrose density gradients. Competition studies with androgens, progestins and corticosteroids showed that the binding was highly specific for estradiol and was only partially decreased by 5α-androstan-3β, 17β-diol. In vivo binding of estradiol in muscles was also found after intravenous perfusion of [3H]-estradiol in castrated and functionally hepatectomized rats. Specific 8–10S binding peaks on sucrose gradients were present in LA/BC and in TM cytosols. Moreover in LA/BC muscles, in vivo nuclear estradiol binding proteins could be detected in a 4–5S binding peak on sucrose gradients obtained with 0.4 M KCl nuclear extract. These results show unequivocally the presence of cytosol estrogen binding proteins in skeletal and perineal muscles and suggest also the existence of nuclear estrogen-binding proteins complexes in LA/BC muscles.

Airway inflammation and structural changes in airway hyper-responsiveness and asthma: an overview.

Asthma treatment has moved from bronchodilator therapy to an emphasis on anti-inflammatory therapy. Airway inflammation is believed to induce airway hyper-responsiveness (AHR) through the release of mediators that increase the airway response to agonists. However, the exact contribution of airway inflammation in the physiology of airway hyper-responsiveness remains undefined. Structural modifications in airways resulting from inflammation may contribute to the development and persistence of AHR and the development of asthma. This paper reviews some of the main components of airway inflammation and structural changes in asthma, and discusses how these processes may interact to modify airway function and induce respiratory symptoms.

Paving the way for invasive species: road type and the spread of common ragweed (Ambrosia artemisiifolia).

Roads function as prime habitats and corridors for invasive plant species. Yet despite the diversity of road types, there is little research on the influence of these types on the spread of invaders. Common ragweed (Ambrosia artemisiifolia), a plant producing large amounts of allergenic pollen, was selected as a species model for examining the impact of road type on the spread of invasive plants. We examined this relationship in an agricultural region of Quebec, Canada. We mapped plant distribution along different road types, and constructed a model of species presence. Common ragweed was found in almost all sampling sites located along regional (97%) and local paved (81%) roads. However, verges of unpaved local roads were rarely (13%) colonized by the plant. A model (53% of variance explained), constructed with only four variables (paved regional roads, paved local roads, recently mown road verges, forest cover), correctly predicted (success rate: 89%) the spatial distribution of common ragweed. Results support the hypothesis that attributes associated with paved roads strongly favour the spread of an opportunistic invasive plant species. Specifically, larger verges and greater disturbance associated with higher traffic volume create propitious conditions for common ragweed. To date, emphasis has been placed on controlling the plant in agricultural fields, even though roadsides are probably a much larger seed source. Strategies for controlling the weed along roads have only focused on major highways, even though the considerable populations along local roads also contribute to the production of pollen. Management prioritizations developed to control common ragweed are thus questionable.

Estradiol and progesterone receptors in dog prostate cytosol

Abstract When intact or castrated dog prostate cytosol is incubated with tritiated estradiol, high levels of saturable binding can be observed in the 3–4S region of sucrose gradients. Occasionally a shoulder is also seen in the 8S region. The estradiol binding is specific for natural and synthetic estrogens. It is of high affinity ( K A = 2 × 10 9 M −1 ). The binding component also has a relatively high affinity for 5α-androstane-3β,17β-diol, a major metabolite of 5α-dihydrotestosterone in dog prostate cytosol. Estradiol and 5α-androstane-3β,17β-diol can displace each other in competition experiments and they both yield identical concentration of sites by Scatchard analysis. The concentration of estradiol binding sites is quite variable in prostates from 24 h castrated dogs and no apparent correlation can be found between the concentration of sites and prostate wt. By contrast in the prostates from intact dogs there is a highly significant inverse relationship between prostate wt. and concentration of estradiol binding sites. The presence of progesterone receptors in dog prostate can also be demonstrated by sucrose density gradient analysis using the synthetic steroids R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) and R1881 (17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one). The binding component sediments both in the 8S and 4S regions of sucrose gradients. It is present in low amounts under basal conditions. Five days after a single injection of estradiol valerate, the binding is increased about 10-fold. It is almost completely abolished by the addition of progesterone and various unlabeled synthetic progestins but only partly decreased by 5α-dihydrotestosterone. Relatively low and constant levels of progesterone receptors are observed in normal and hyperplastic prostates from adult dogs (30 ± 1 [S.E.M.] fmol/mg0. prot.)