Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

• MeSH
• antifibrinolytika chemie   imunologie   MeSH
• antikoagulancia chemie   imunologie   MeSH
• faktor Xa imunologie   MeSH
• hemostáza * MeSH
• infekce červy třídy Trematoda krev   imunologie   parazitologie   veterinární   MeSH
• inhibitory enzymů chemie   imunologie   MeSH
• inhibitory faktoru Xa chemie   imunologie   MeSH
• interakce hostitele a parazita MeSH
• kapři krev   imunologie   parazitologie   MeSH
• komplement imunologie   MeSH
• nemoci ryb krev   imunologie   parazitologie   MeSH
• plasmin imunologie   MeSH
• plazmatický kalikrein antagonisté a inhibitory   imunologie   MeSH
• proteiny červů chemie   genetika   imunologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Trematoda chemie   genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.

• MeSH
• 2D gelová elektroforéza MeSH
• alergeny imunologie   MeSH
• alergie na pšenici diagnóza   imunologie   MeSH
• alfa-amylasy antagonisté a inhibitory   MeSH
• dítě MeSH
• dospělí MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• financování organizované MeSH
• imunoblotting MeSH
• imunoglobulin E krev   MeSH
• inhibitory enzymů imunologie   metabolismus   MeSH
• lidé MeSH
• mladiství MeSH
• peptidové fragmenty analýza   metabolismus   MeSH
• průtoková cytometrie MeSH
• pšenice imunologie   metabolismus   škodlivé účinky   MeSH
• rostlinné proteiny imunologie   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

• MeSH
• cyklin-dependentní kinasy analýza   antagonisté a inhibitory   metabolismus   MeSH
• cykliny * imunologie   MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• inhibitory enzymů * imunologie   MeSH
• lidé MeSH
• molekulární sondy biosyntéza   chemie   metabolismus   MeSH
• monoklonální protilátky biosyntéza   genetika   metabolismus   MeSH
• multigenová rodina imunologie   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádory prsu enzymologie   chemie   MeSH
• nádory tračníku enzymologie   chemie   MeSH
• orgánová specificita imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH