Protein-Rich Fraction of Cnidoscolus urens (L.) Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bouhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH(2) (RGE) and IVYYPDRGETGL-NH(2) (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein. (C) 2009 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAP/FADA (UNIFESP)Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Fed Mato Grosso, Dept Nat Sci, BR-79603011 Tres Lagoas, MS, BrazilUniv Estadual Fluminense Darcy Ribeiro, Lab Prot & Peptide Biochem, CBB, BR-28015620 Campos Dos Goytacazes, RJ, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation

A systematic review and meta-analysis of structural and functional brain alterations in individuals with genetic and clinical high-risk for psychosis and bipolar disorder

Neuroimaging findings in people at either genetic risk or at clinical high-risk for psychosis (CHR-P) or bipolar disorder (CHR-B) remain unclear. A meta-analytic review of whole-brain voxel-based morphometry (VBM) and functional magnetic resonance imaging (fMRI) studies in individuals with genetic risk or CHR-P or CHR-B and controls identified 94 datasets (N = 7942). Notwithstanding no significant findings were observed following adjustment for multiple comparisons, several findings were noted at a more liberal threshold. Subjects at genetic risk for schizophrenia or bipolar disorder or at CHR-P exhibited lower gray matter (GM) volumes in the gyrus rectus (Hedges' g = −0.19). Genetic risk for psychosis was associated with GM reductions in the right cerebellum and left amygdala. CHR-P was associated with decreased GM volumes in the frontal superior gyrus and hypoactivation in the right precuneus, the superior frontal gyrus and the right inferior frontal gyrus. Genetic and CHR-P were associated with small structural and functional alterations involving regions implicated in psychosis. Further neuroimaging studies in individuals with genetic or CHR-B are warranted