Glandular Kallikrein Research Articles

Kininogenase activity of prostate-derived human glandular kallikrein (hK2) purified from seminal fluid.

Prostate-specific human glandular kallikrein (hK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of hK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PKa potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-hK2 complex, a serpin protease inhibitor that binds hK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the hK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that hK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other internal cleavage sites for hK2 in HMWK (Arg427, Arg437, and Arg457) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.

Characterization and processing of prostate specific antigen (hK3) and human glandular kallikrein (hK2) secreted by LNCaP cells.

Prostate specific antigen (PSA, hK3) in serum is predominantly complexed to alpha-1-antichymotrypsin (ACT), but a minor fraction remains in a free form despite the very large excess of serine protease inhibitors and alpha-2-macroglobulin. The fraction of free to total PSA is significantly lower in prostate cancer (CaP) compared to benign prostatic hyperplasia (BPH) which provides improved discrimination of these conditions. The molecular nature of free PSA in the circulation and the reason for its varying concentration in malignant and benign conditions is currently not known. The objective of the present investigation was to study the secretion of PSA and human glandular kallikrein 2 (hK2) by the LNCaP prostate cancer cell line, and to purify and characterize both proteins. LNCaP PSA was thoroughly characterized by immunological characterization, SDS-PAGE, isoelectric focusing, gel filtration, aminoterminal sequencing, reverse-phase chromatography, mass spectrometry and enzymatic activity measurements. LNCAP cells produced approximately equal amounts of zymogen (proPSA) and the one-chain mature form of PSA, whereas there was no evidence for the secretion of any internally cleaved forms. LNCaP cells secreted hK2 into the growth medium at about 3-5% of the amount of PSA. One-chain, mature PSA and hK2 obtained when LNCaP cells were grown in the presence of fetal bovine serum, had no enzymatic activity, but were active when the cells were grown in the absence of serum. Using enzymatically active recombinant hK2, it was possible to activate proPSA secreted by LNCaP cells. ProPSA formed two bands with high isoelectric points (8.2 and 8.4), which disappeared when proPSA was converted to mature PSA with hK2. Cancerous cells produce the zymogen forms of PSA, which by their isoelectric pI points seem to be found in serum of prostate cancer patients, but not BPH patients. Mature, one-chain PSA is inactive in the presence of serum. These findings may be highly relevant for the understanding of the generation of free and complexed PSA in the circulation.

Characterization and processing of prostate specific antigen (hK3) and human glandular kallikrein (hK2) secreted by LNCaP cells

Characterization and processing of prostate specific antigen (hK3) and human glandular kallikrein (hK2) secreted by LNCaP cells

Characterization of Monoclonal Antibodies for Prostate-specific Antigen and Development of Highly Sensitive Free Prostate-specific Antigen Assays

The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA. We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Ka values from 1.1 x 10(8) to 1.8 x 10(10)L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA. The analytical detection limit of these immunoassays is 0.001 microg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-alpha1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule. The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays.

Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Cross-Reactivity with Human Kallikrein-2

Immunoassays for prostate-specific antigen (PSA) using monoclonal or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA shares 80% sequence homology with human glandular kallikrein (hK2). In the present study, we have used SDS-PAGE and Western blotting of a recombinant form of hK2 to determine the degree of cross-reactivity in a panel of 83 antibodies submitted to the ISOBM TD-3 PSA Workshop. From this panel, 24 of the 83 antibodies showed cross-reactivity with hK2. The majority of these antibodies showed binding to conformationally independent or linear epitopes. Fourteen of these antibodies appear to map to the same epitope group, indicating the existence of a dominant and linear immunogenic domain shared by PSA and hK2.

Large-Scale Propagation of Recombinant Adherent Cells That Secrete a Stable Form of Human Glandular Kallikrein, hK2

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade. Therefore, to study biochemical and biological characteristics of hK2, a stable and enzymatically inactive mutant form of hK2, hK2A217V, was expressed in a hamster cell line, AV12-664 (AV12-hK2A217V). AV12-hK2A217Vcells secreted prohK2A217V(phK2A217V) in the spent medium at ∼2.5 μg/ml. Since AV12-hK2A217Vare adherent cells, it was necessary to develop an efficient system to propagate large numbers of cells to obtain significant quantities of phK2A217V. In this paper, we compared ceramic core bioreactor and microcarrier beads as alternatives to static culture to propagate adherent cells. Considering production levels, ease of operation, cost effectiveness, and labor, microcarrier beads were found to be a better alternative. Our findings led to the development of a general protocol for large-scale propagation of adherent cells on microcarrier beads eliminating the need for propagating AV12-hK2A217Vin culture flasks or bioreactors. Microcarrier beads coated with AV12-hK2A217Vcells could be propagated in 1- or 3-liter spinner flasks and were passed from one spinner to the next in a manner analogous to static culture or could be frozen and later used as inoculum for subsequent spinners. Using this protocol, >40 liters of spent medium was harvested within 30 days, which in turn was used to purify phK2A217V. phK2A217Vpurified from spent medium of cells grown either on microcarrier beads or in culture flasks were biochemically similar as indicated by HIC–HPLC profile followed by sequencing of relevant peaks.

Early Diagnosis and Staging.

This report reviews developments in the early diagnosis and staging of prostate cancer and updated on the incidence of postate cancer to discuss the pros and cons of population based screening. Refinements and reliability of various diagnosic procedures are described such as PSA testing, transrectal ultrasound, ratio beteen PSA-level and ultrasound measured prostate volume, rate of change of PSA-level, combination factor equations computed by neural network programs to predict likelihood of prostate cancer, artificial neural network analysis of subvisual transrectal ultrasound information, measurements of different PSA-forms of PSA (in particular percent free PSA), and glandular kallikrein 2.

Development of plasma kallikrein selective inhibitors.

During the course of the development of active center-directed plasmin inhibitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine-4-methoxycarb onylanilide inhibited plasma kallikrein more potently than other enzymes such as plasmin, urokinase, and thrombin, although the inhibitory activity was not as potent and enzyme selectivity not as high. Based on studies of structure-activity relationship, we designed and synthesized the plasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarbonyl)-phenylalanine-4-carboxy methyl- anilide (Tra-Phe-APAA). Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator, factor Xa, factor XIIa, and thrombin with Ki values of > 500, 390, 200, > 500, > 500 > 500, and > 500 microM, respectively. We designated Tra-Phe-APAA as PKSI-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycinehydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma.

Immunoregulation by the salivary glands

The evidence for the integration of the submandibular gland (SMG) into the neuroimmunoregulatory network is reviewed. In laboratory rodents, factors extracted from the SMG were shown to stimulate lymphocyte proliferation, to affect the weight of the thymus, spleen and lymph nodes and to induce immunosuppression in several in vivo and in vitro models. The SMG produces significant amount of nerve growth factor (NGF), epidermal growth factor (EGF), transforming growth factor-β, and kallikreins. These factors are secreted into the saliva and affect immune and mucosal tissues and nerve endings in the gastrointestinal tract. They may thus play a role in the regulation of mucosal immune/inflammatory responses. The major salivary glands also produce antimicrobial proteins and secretory IgA antibodies which are essential factors in mucosal host defense. SMG-derived NGF, EGF and glandular kallikrein are delivered into the bloodstream where they may act as important systemic immunoregulators and exert regulatory influences on the neuroendocrine system. Growth hormone, prolactin, androgens, thyroid hormone and corticosteroids regulate protein synthesis in the SMG, where as secretory activity is regulated by sympathetic, parasympathetic and peptidergic nerve fibers. Steroid hormones and cytokines as interleukin-1α, -1β, tumor necrosis factor-α, interferon-γ have major regulatory influence on protein secretion, including the secretion of immunoglobulin into the saliva. The SMG interacts with the mucosal and systemic compartments of the immune system, with the central and peripheral nervous systems, with the pituitary gland, and with peripheral endocrine organs. These interactions enable the SMG to exert regulatory influences on immune/inflammatory reactions in the gastrointestinal tract, in the lungs, and possibly elsewhere. It is suggested that these functions make this gland a key regulatory organ in the neuroimmunoregulatory network. Biomedical Reviews 1998; 9: 79-91.

The importance of human glandular kallikrein and its correlation with different prostate specific antigen serum forms in the detection of prostate carcinoma

Human glandular kallikrein (hK2), the prostate specific antigen (PSA) close homologue, possesses approximately 80% structure identity with PSA. The identification of PSA was an important step in the detection of prostate carcinoma (PCa). Thus, hK2 measurement in the serum has the potential to become another important diagnostic test for PCa. In the current study, the authors measured the serum concentrations of the hK2 with "in-house" immunofluorometric assays in different patient groups. The correlation between serum hK2 and different PSA forms was investigated. The prospectively collected serum samples were obtained preoperatively on admission from 311 consecutive male patients. Sixteen patients did not fulfill inclusion criteria; the remaining patients were divided into four groups (Groups I-III confirmed histologically): Group I: patients with PCa (n = 56); Group II: patients with benign prostatic hyperplasia (BPH) (n = 163); Group III: patients with BPH with a chronic in-dwelling catheter (BPH cat) (n = 44); and Group IV-control group (n = 32). The patients in Group IV had urolithiasis, varicocele, or kidney or bladder tumors). An experimental immunofluorometric assay with an analytic sensitivity of 0.01 ng/mL and a functional sensitivity of 0.05 ng/mL was used to determine serum hK2 concentrations. Total PSA, free PSA, and PSA complexed to alpha-1-antichymotrypsin (PSA-ACT) also were measured. hK2 concentrations equal to or above the functional sensitivity limit were correlated with each of these PSA serum forms. Free to total PSA, hK2 to total PSA, and hK2 to free PSA ratios were calculated and compared in different patient groups. The hK2 concentrations were equal to or above the functional sensitivity limit in 179 of 311 samples (57.6%). In these samples, hK2 correlated best with free PSA (correlation coefficient [r] = 0.79) and correlated well with total PSA (r = 0.72) and PSA-ACT (r = 0.74). Similar correlations also could be observed when each clinical group was analyzed separately. The median proportion of hK2 in relation to total PSA was 2.1%, 1.8%, and 1.4%, respectively, for PCa, BPH, and BPH cat patients. Both the free to total PSA ratio and the hK2 to free PSA ratio discriminated well between PCa and BPH patients. Within the range of total PSA of 4-10 ng/mL (PCa [n = 11] and BPH [n = 41]) the hK2 to free PSA ratio had a specificity of 63.4% and 90.9% sensitivity (area under the receiver operating characteristic [ROC] curve = 0.85) whereas the free to total PSA ratio had a 34.1% specificity at the same sensitivity level (area under ROC curve = 0.74). The hK2 serum level correlates well with all PSA serum forms in all clearly defined clinical groups. The preliminary finding that the hK2 to free PSA ratio appeared to improve the detection of PCa compared with the free to total PSA ratio in patients with total PSA within a 4-10 ng/mL range is of clinical interest. Combining human serine proteases in the multivariate regression analysis will be a tool to improve cancer detection. Further investigations with more sensitive hK2 assays and in larger patient populations are needed to confirm this finding.

The importance of human glandular kallikrein and its correlation with different prostate specific antigen serum forms in the detection of prostate carcinoma

BACKGROUND Human glandular kallikrein (hK2), the prostate specific antigen (PSA) close homologue, possesses approximately 80% structure identity with PSA. The identification of PSA was an important step in the detection of prostate carcinoma (PCa). Thus, hK2 measurement in the serum has the potential to become another important diagnostic test for PCa. In the current study, the authors measured the serum concentrations of the hK2 with "in-house" immunofluorometric assays in different patient groups. The correlation between serum hK2 and different PSA forms was investigated. METHODS The prospectively collected serum samples were obtained preoperatively on admission from 311 consecutive male patients. Sixteen patients did not fulfill inclusion criteria; the remaining patients were divided into four groups (Groups I-III confirmed histologically): Group I: patients with PCa (n = 56); Group II: patients with benign prostatic hyperplasia (BPH) (n = 163); Group III: patients with BPH with a chronic in-dwelling catheter (BPH cat) (n = 44); and Group IV-control group (n = 32). The patients in Group IV had urolithiasis, varicocele, or kidney or bladder tumors). An experimental immunofluorometric assay with an analytic sensitivity of 0.01 ng/mL and a functional sensitivity of 0.05 ng/mL was used to determine serum hK2 concentrations. Total PSA, free PSA, and PSA complexed to α-1-antichymotrypsin (PSA-ACT) also were measured. hK2 concentrations equal to or above the functional sensitivity limit were correlated with each of these PSA serum forms. Free to total PSA, hK2 to total PSA, and hK2 to free PSA ratios were calculated and compared in different patient groups. RESULTS The hK2 concentrations were equal to or above the functional sensitivity limit in 179 of 311 samples (57.6%). In these samples, hK2 correlated best with free PSA (correlation coefficient [r] = 0.79) and correlated well with total PSA (r = 0.72) and PSA-ACT (r = 0.74). Similar correlations also could be observed when each clinical group was analyzed separately. The median proportion of hK2 in relation to total PSA was 2.1%, 1.8%, and 1.4%, respectively, for PCa, BPH, and BPH cat patients. Both the free to total PSA ratio and the hK2 to free PSA ratio discriminated well between PCa and BPH patients. Within the range of total PSA of 4-10 ng/mL (PCa [n = 11] and BPH [n = 41]) the hK2 to free PSA ratio had a specificity of 63.4% and 90.9% sensitivity (area under the receiver operating characteristic [ROC] curve = 0.85) whereas the free to total PSA ratio had a 34.1% specificity at the same sensitivity level (area under ROC curve = 0.74). CONCLUSIONS The hK2 serum level correlates well with all PSA serum forms in all clearly defined clinical groups. The preliminary finding that the hK2 to free PSA ratio appeared to improve the detection of PCa compared with the free to total PSA ratio in patients with total PSA within a 4-10 ng/mL range is of clinical interest. Combining human serine proteases in the multivariate regression analysis will be a tool to improve cancer detection. Further investigations with more sensitive hK2 assays and in larger patient populations are needed to confirm this finding. Cancer 1998;83:2540-2547. © 1998 American Cancer Society.

α2-Macroglobulin and C1-Inactivator are Plasma Inhibitors of Human Glandular Kallikrein

ABSTRACT: Human glandular kallikrein (hK2) is a possible new marker for prostate cancer that is homologous to prostate specific antigen. Purified hK2 added to serum or plasma reacted with endogenous protease inhibitors to form complexes of >350, 135, and 80 kDa, and some hK2 remained free, as judged by immunoblotting. The former two complexes could be removed by specific antibodies to α2-macroglobulin and to C1- inactivator, respectively, and they comigrated on SDS-PAGE with complexes formed between hK2 and purified α2-macroglobulin or C1-inactivator. hK2 complexes of 80 kDa could not be completely removed with any anti-serpin antibody used. Thus, these may consist of more than one type of hK2 complex. In contrast, essentially all hK2 complexes were removed from seminal plasma by antibody to protein C inhibitor, demonstrating that protein C inhibitor is the only significant inhibitor of hK2 in semen. hK2 reacted more rapidly with α2-macroglobulin than with any other inhibitor in plasma or serum. Divalent metal ions and heparin did not appreciably affect the rate of formation of any of the hK2 complexes in serum or plasma or with purified α2-macroglobulin or C1-inactivator. Measurement of one or more of the hK2 forms identified here may have diagnostic or prognostic potential for prostate cancer.

Bradykinin release from high molecular weight kininogen and increase in plasma kallikrein-like activity following sensory stimulation by food in the rat.

Cephalic stimulation by food elicits, among other responses, dilatation of mesenteric blood vessels preparatory for digestion. The possible participation of bradykinin (BK), a powerful endogenous vasodilator, in this response was studied in fasted rats prior and following stimulation by sight and scent of food (sensory stimulation, SS), actual ingestion being denied to the animals. BK content of plasma high (HK) and low molecular weight kininogen (LK) was determined by bioassay on the atropinized, antihistamine-treated isolated guinea-pig ileum following release by trypsin from heat/acid denatured plasma. BK corresponding to LK was estimated in plasma which prior to denaturation had been incubated with kaolin, a process which leads to quantitative release and inactivation of BK from HK, but does not affect LK. BK corresponding to (HK + LK) was determined in plasma not exposed to kaolin. BK contained in HK was the difference between BK of (HK + LK) and of BK of LK. Plasma and glandular kallikreins were estimated by fluorimetry, using specific synthetic substrates. A 40.6+/-4.0% decrease (P<0.001) of BK in HK occurred in rats after 90 s of SS; LK remained unaffected. Ten minutes of SS did not result in further change. Atropine inhibited the effect of SS. Return of HK to pretreatment levels occurred when, following 90s of SS, rats were allowed to rest for 60 min in the absence of food. Renewed capacity to respond to SS was then observed. Plasma kallikrein, but not glandular kallikrein, increased in plasma of rats after SS. Increased free BK was detected in the circulation of Enalapril-protected rats after SS. Electrical stimulation of the distal sector of the sectioned left abdominal vagus nerve of Nembutal-anesthetized fasted rats reproduced the effect of SS on HK. It is concluded that visuo-olfactory stimulation by food generates nerve impulses, possibly carried by the vagus nerve, which by activating plasma kallikrein lead to cleavage of circulatory HK and release of BK in the rat.

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.

In prostatism patients the ratio of human glandular kallikrein to free PSA improves the discrimination between prostate cancer and benign hyperplasia within the diagnostic “gray zone” of total PSA 4 to 10 ng/mL

Objectives. Human glandular kallikrein (hK2) possesses approximately 80% structure identity with prostate-specific antigen (PSA). Moreover, messenger ribonucleic acid for hK2 and for PSA is expressed in both benign and malignant prostatic tissue. We investigated whether the hK2 serum measurement may improve the detection of prostate cancer (PCa) in patients with total PSA of 4 to 10 ng/mL (diagnostic “gray zone”). Methods. Blood samples were obtained from 90 consecutive male patients with lower urinary tract symptoms and total PSA values of 4 to 10 ng/mL. Eighty-one patients underwent transurethral resection of the prostate and 6 radical prostatectomy. The patients were divided into two groups: I, patients with PCa (n = 20) and II, patients with benign prostatic hyperplasia (BPH) (n = 70). An “in-house” immunofluorometric assay with analytical sensitivity of 0.01 ng/mL and the functional sensitivity of 0.05 ng/mL (at this level the mean coefficient of variation, calculated from the precision profile based on the assays of serum samples, was less than 20%) was used to determine serum hK2 concentrations. Total PSA, free PSA (ProStatus), and PSA complexed to alpha 1-antichymotrypsin (PSA-ACT) were also measured. Free/total PSA, hK2/total PSA, and hK2/free PSA ratios were calculated. Results. The serum hK2 could be detected in all samples and in 76 (84.4%) of 90 samples (PCa, n = 18; BPH, n = 58) at given functional sensitivity level. For these cases the median concentration of hK2 was 0.135 ng/mL in PCa and 0.09 ng/mL in BPH ( P <0.1). The median hK2/total PSA ratio was 2% for PCa and 1.6% for BPH ( P <0.2). The median free/total PSA ratio was 0.122 for PCa and 0.215 for BPH ( P <0.0008) and the hK2/free PSA ratio was 0.139 for PCa and 0.075 for BPH ( P <0.000003). At a 7.2% cutoff, the specificity of hK2/free PSA ratio was 48.2% at 100% sensitivity and increased to 60.3% at 94.4% sensitivity level (the area under the receiver operating characteristic curve was 0.86). In comparison, the free/total PSA ratio at a 25.2% cutoff had a sensitivity of 94.4% and a specificity of 27.6% (area under the curve = 0.76). Conclusions. hK2 was detected in all sera with total PSA values of 4 to 10 ng/mL. Of particular clinical interest is the finding that the hK2/free PSA ratio had a better specificity without loss of sensitivity for PCa than total PSA or the PSA free/total ratio within the range of 4 to 10 ng/mL total PSA. hK2 in combination with free PSA may offer a new diagnostic means for PCa detection.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Kallikrein concentration in mixed saliva after contraceptive administration or postmenopausal hormone replacement

The aim of our study was to evaluate the premise (Jenzano 1992) that estrogen influences the female mixed saliva kallikrein level (MSK). We estimated the MSK level from woman between the ages of 17 and 39 with (n = 20) and without (20) contraceptive medication (at least an one year treatment) and postmenopausal women between the ages of 47 and 65 with (n = 20) and without (n = 22) hormonal substitution. Kallikrein was estimated by means of the cleavage of the chromogenic tripeptide Val-Leu-Arg-p-nitranilide (SIGMA) and absorbance measuring of p-nitraniline at 405 nm. A Wilcoxon test was conducted for statistical analysis. The MSK level decreases significantly (p = 0.000) under contraceptive medication (estrogen component: synthetic ethinyl-estrogen). However the MSK level of postmenopausal women showed no significant alteration (p = 0.158) after hormone substitution (estrogen component: natural estrogen). The finding may be due to the weaker effect of the estrogen used in post-menopausal hormone therapy. Our results demonstrate that estrogenic substances affect salivary kallikrein levels.

Detection of disseminated prostate cells by reverse transcription-polymerase chain reaction (RT-PCR): technical and clinical aspects.

Metastasis of prostate cancer (CaP) to bone is responsible for much of the morbidity and mortality associated with this disease. While the primary goal of current methods of treatment is to prevent, slow down or reverse metastatic spread, the sensitivity of today’s conventional staging techniques is insufficient to detect micrometastatic disease. Radical prostatectomy is intended to be a complete cure, but 15–30% of these cases recur, because CaP cells that have escaped the prostate prior to surgery give rise to metastases. Although macroscopic techniques such as the bone scan cannot detect micrometastases, new molecular approaches that are under development may prove to be capable of filling this need. There is now hope that detection of prostate cells in circulation may provide a new and highly sensitive staging tool. Because prostate-specific antigen (PSA) is produced almost exclusively by prostate epithelial cells (Clements and Mukhtar, 1994; Levesque et al., 1995a,b; Oesterling, 1991; Zarghami and Diamandis, 1996; Zarghami et al., 1997), the presence of PSA-positive cells in circulation clearly indicates an abnormal condition, and a strong correlation between the appearance of prostate cells in circulation and the advent of metastasis might be expected. Current models of metastasis hold that cells ‘‘shed’’ by primary tumors are borne by the circulation (blood and lymphatics) to other organs, where they are deposited and form secondary tumors. It is not known whether these circulating cells have metastatic potential, or whether they must undergo further changes to cause metastases. Protocols based on the reverse transcription-polymerase chain reaction (RT-PCR) have been developed in response to the need for identification and widespread adoption of superior methods for detecting these extra-prostatic cells, so that more systematic studies with long-term follow-up may be undertaken. Currently, the mRNAs encoding PSA, prostate-specific membrane antigen (PSMA), and human glandular kallikrein (hK2) are the 3 messages most frequently used for detection of prostate cells, but these messages, while prostate-specific, are not cancer-specific, and therefore yield little or no information about the metastatic potential of the cells detected. Markers related to cancer progression are desired from the viewpoints of both prognostic methods and basic understanding. The limit of detection (LoD) of RT-PCR is better than that of other methods in current use, such as flow cytometry and immunohistochemistry (Pelkey et al., 1996). There is therefore considerable interest in the use of RT-PCR for detection and possible quantification of prostate cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples from CaP patients. However, despite the promise of this method, it is not yet clear whether RT-PCR will prove to be of clinical value in prostatecancer staging. In this review we will summarize published reports on RT-PCR detection of circulating prostate cells. Because the field is developing rapidly, there have been several significant advances since the last major reviews were published (Gomella et al., 1997; Olsson et al., 1997), including the use of longer primers with 2-temperature PCR for specificity, development of internal controls and targeting of the hK2 message. There have also been a number of additional reports describing RT-PCR protocols and clinical results in CaP. With a very few exceptions, we have restricted the scope of this review to published articles. The review is divided into 2 parts: a technical evaluation of RT-PCR assays used for detection of prostate cells and a discussion of the results of clinical studies using these procedures.

Peptide aldehyde inhibitors of the kallikreins: an investigation of subsite interactions with tripeptides containing structural variations at the amino terminus.

A series of tripeptide aldehyde derivatives containing variations at the P3 subsite and the amino terminus has been prepared and evaluated for trypsin-like serine protease inhibition. These compounds exhibit strong in vitro inhibition of human plasma kallikrein (HPK), porcine pancreatic kallikrein (PPK) and human plasmin (HP). As suspected from an examination of a related crystal structure, the presence of a hydrophobic residue (adamantyl) at the amino terminus dramatically improves the binding to PPK. The adamantyl group, however, represents a peak in binding; larger residues cause the binding to be reduced, and thus are less well accommodated in this subsite. Although both HP and HPK also can accept large molecular volume at the amino terminus, they do not exhibit the same preference for large residues at this subsite that is demonstrated by PPK. Selectivity differences also are observed with P3 subsite substitution; with PPK preferring a bulky, but compact side-chain (t-butyl) and HP and HPK preferring a more extended (e.g. benzyl) group.

ALTERATIONS IN CONSTITUENT URINARY PROTEINS IN RESPONSE TO BLADDER OUTLET OBSTRUCTION IN RATS

Purpose Benign prostatic hyperplasia, resulting in bladder outflow obstruction, induces well recognized clinical symptoms and morphologic bladder changes. Despite these phenomenon, relatively little is known with regard to the precise molecular events occurring in the bladder as a consequence of obstruction. In an effort to screen for alterations in bladder gene expression induced by obstruction, and/or alterations in uroepithelial integrity, this study compared pre-and post-obstructive constituent urinary proteins in an animal model. Materials and Methods Outlet obstruction was created using a previously established model system. Experimental animals were surgically obstructed for either 2 or 7 days, at which time the urine was aspirated and the bladders removed and weighed. Urinary proteins were separated using 2-D PAGE. Following comparison of sham versus experimental animals, microsequencing was performed on proteins that were down regulated. Results Duplicate experiments confirmed the presence of outflow obstruction. Statistically significant increases (p <0.01) in bladder weights were seen at 2 and 7 days in the obstructed groups as compared with both sham and control groups. 2-D PAGE demonstrated a down regulation of three urinary proteins post-obstruction. Microsequencing identified these proteins as prostatic steroid-binding protein C3 precursor (pI = 5.5, MW = 15000), glandular kallikrein 9 (S3) precursor (pI = 6.2, MW = 19000), and glandular kallikrein 8 (P1) precursor (pI = 6.2, MW = 33000). Conclusions Bladder outflow obstruction alters constituent urinary protein composition in an animal model system. The precise etiology of these alterations remains to be defined.