The expression of the genes involved in redox metabolism and hydrogen peroxide balance is associated with the resistance of cowpea [Vigna unguiculata (L.) Walp.] to the hemibiotrophic fungus Colletotrichum gloeosporioides.

Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid.

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata.

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.