Aptamer-MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen.

This study reports the design and evaluation of a new synthetic receptor sensor based on the amalgamation of biomolecular recognition elements and molecular imprinting to overcome some of the challenges faced by conventional protein imprinting. A thiolated DNA aptamer with established affinity for prostate specific antigen (PSA) was complexed with PSA prior to being immobilised on the surface of a gold electrode. Controlled electropolymerisation of dopamine around the complex served to both entrap the complex, holding the aptamer in, or near to, it's binding conformation, and to localise the PSA binding sites at the sensor surface. Following removal of PSA, it was proposed that the molecularly imprinted polymer (MIP) cavity would act synergistically with the embedded aptamer to form a hybrid receptor (apta-MIP), displaying recognition properties superior to that of aptamer alone. Electrochemical impedance spectroscopy (EIS) was used to evaluate subsequent rebinding of PSA to the apta-MIP surface. The apta-MIP sensor showed high sensitivity with a linear response from 100pg/ml to 100ng/ml of PSA and a limit of detection of 1pg/ml, which was three-fold higher than aptamer alone sensor for PSA. Furthermore, the sensor demonstrated low cross-reactivity with a homologous protein (human Kallikrein 2) and low response to human serum albumin (HSA), suggesting possible resilience to the non-specific binding of serum proteins.

Tissue kallikrein promotes prostate cancer cell migration and invasion via a protease-activated receptor-1-dependent signaling pathway.

We recently demonstrated that tissue kallikrein (TK) promotes keratinocyte migration through activation of protease-activated receptor-1 (PAR(1)) and transactivation of the epi-dermal growth factor receptor (EGFR). In this study, we investigated the potential role of PAR(1) in mediating the effect of TK on cancer cell migration, invasion and proliferation. Our results show that TK promotes DU145 prostate cancer cell migration in a concentration-dependent manner, but has no effect on A549 lung cancer cells. Active TK markedly increases DU145 cell migration and invasion, which are blocked by aprotinin but minimally affected by icatibant; kinin treatment has little effect. TK-induced cell migration and invasion are abolished by inhibition of PAR(1) using a pharmacological inhibitor or RNA interference. The effect of TK on cell migration and invasion are also blocked by inhibitors of protein kinase C, c-Src, matrix metalloproteinase, EGFR and extracellular signal-regulated kinase (ERK). Moreover, TK stimulates ERK phosphorylation, which is inhibited by an EGFR antagonist. Additionally, TK but not kinin stimulates DU145 cell proliferation through activation of the kinin B2 receptor, but not PAR(1) and EGFR. These results indicate differential signaling pathways mediated by TK in promoting prostate cancer cell migration and invasion via PAR(1) activation, and proliferation via kinin B2 receptor stimulation.

Prostatic kallikrein: a new major dog allergen.

BACKGROUND: Dog dander is an important cause of respiratory allergy, but the spectrum of known dog allergens appears incomplete. Two lipocalins, Can f 1 and Can f 2, and serum albumin, Can f 3, have been characterized in detail but do not fully account for the IgE antibody-binding activity of dog dander extract. Allergen activity has previously been detected in dog urine but not further characterized. OBJECTIVE: We sought to identify, characterize, and assess the importance of allergen components in dog urine. METHODS: Dog urine was fractionated by means of size exclusion chromatography and examined for IgE antibody binding. A protein present in one fraction displaying IgE antibody-binding activity was identified by means of N-terminal sequencing and mass spectrometry. A recombinant form of the protein was produced in Pichia pastoris. IgE antibody binding to dog allergen components among sera of 37 subjects with dog allergy was determined by means of ImmunoCAP analysis. RESULTS: An IgE antibody-binding protein was isolated from dog urine and identified as prostatic kallikrein. A closely related or identical protein was detected in dog dander. The recombinant prostatic kallikrein displayed immunologic and biochemical properties similar to those of the natural protein and bound IgE antibodies from 26 (70%) of 37 sera of subjects with dog allergy, 14 of which reacted to none of Can f 1, Can f 2, or Can f 3. The dog allergen identified here was found to cross-react with human prostate-specific antigen, a key culprit in IgE-mediated vaginal reactions to semen. CONCLUSION: Prostatic kallikrein is a new major dog allergen.

Blarina toxin, a mammalian lethal venom from the short-tailed shrew Blarina brevicauda: Isolation and characterization.

Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the short-tailed shrew Blarina brevicauda. Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L(106)TFFYKTFLG(115). BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.

Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice.

The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations.