ABSTRACT
The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudo-aspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it's non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it's possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries.
Subject(s)
Arthropod Proteins/metabolism , Aspartic Acid Proteases/metabolism , Digestive System/enzymology , Rhipicephalus/enzymology , Amino Acid Sequence , Animals , Arthropod Proteins/genetics , Arthropod Proteins/isolation & purification , Aspartic Acid Proteases/genetics , Aspartic Acid Proteases/isolation & purification , Cattle/parasitology , Cloning, Molecular , Female , Gene Expression Regulation, Enzymologic , Pseudogenes/genetics , RNA Interference , Rhipicephalus/genetics , Rhipicephalus/physiology , Sequence Homology, Amino Acid , Tick Infestations/parasitologyABSTRACT
The major goal in restorative dentistry is to develop a true regenerative approach that fully recovers hydroxyapatite crystals within the caries lesion. Recently, a rationally designed self-assembling peptide P11-4 (Ace-QQRFEWEFEQQ-NH2) has been developed to enhance remineralization on initial caries lesions, yet its applicability on dentin tissues remains unclear. Thus, the present study investigated the interaction of P11-4 with the organic dentin components as well as the effect of P11-4 on the proteolytic activity, mechanical properties of the bonding interface, and nanoleakage evaluation to artificial caries-affected dentin. Surface plasmon resonance and atomic force microscopy indicated that P11-4 binds to collagen type I fibers, increasing their width from 214 ± 4 nm to 308 ± 5 nm ( P < 0.0001). P11-4 also increased the resistance of collagen type I fibers against the proteolytic activity of collagenases. The immediate treatment of artificial caries-affected dentin with P11-4 enhanced the microtensile bonding strength of the bonding interface ( P < 0.0001), reaching values close to sound dentin and decreasing the proteolytic activity at the hybrid layer; however, such effects decreased after 6 mo of water storage ( P < 0.05). In conclusion, P11-4 interacts with collagen type I, increasing the resistance of collagen fibers to proteolysis, and improves stability of the hybrid layer formed by artificial caries-affected dentin.
Subject(s)
Dental Bonding , Dental Caries , Dentin/metabolism , Collagen , Dentin-Bonding Agents , Glycosyltransferases , Humans , Materials Testing , Proteolysis , Resin Cements , Tensile StrengthABSTRACT
Blood coagulation is an important process in haemostasis, and disorders of blood coagulation can lead to an increased risk of haemorrhage and thrombosis. Coagulation is highly conserved in mammals and has been comprehensively studied in humans in the investigation of bleeding or thrombotic diseases. Some substances can act as inhibitors of blood coagulation and may affect one or multiple enzymes throughout the process. A specific thrombin inhibitor called infestin has been isolated from the midgut of the haematophagous insect Triatoma infestans. Infestin is a member of the nonclassical Kazal-type serine protease inhibitors and is composed of four domains, all of which have a short central α-helix and a small antiparallel ß-sheet. Domains 1 and 4 of infestin (infestins 1 and 4) possess specific inhibitory activities. Infestin 1 inhibits thrombin, while infestin 4 is an inhibitor of factor XIIa, plasmin and factor Xa. Here, the structure determination and structural analysis of infestin 1 complexed with trypsin and of infestin 4 alone are reported. Through molecular modelling and docking, it is suggested that the protein-protein binding site is conserved in the infestin 1-thrombin complex compared with other Kazal-type inhibitors. Infestin 4 is able to bind factor XIIa, and the F9N and N11R mutants selected by phage display were shown to be more selective for factor XIIa in comparison to the wild type.
Subject(s)
Insect Proteins/chemistry , Triatoma/chemistry , Animals , Insect Proteins/metabolism , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Structural Homology, Protein , Thrombin/chemistry , Thrombin/metabolismABSTRACT
Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor X(a). Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.
