Carlos Bloch

Functional and structural features of γ-zeathionins, a new class of sodium channel blockers

γ1‐ and γ2‐zeathionins (γ1‐Z and γ2‐Z) are members of a family of small and basic peptides involved in plant protection. These plant defensins exhibit remarkable structural similarity to scorpion neurotoxins and insect defensins. In the present report, we used the whole‐cell patch clamp technique to investigate the inhibition of the sodium current (I Na) by γ1‐Z and γ2‐Z in the GH3 cell line. Both γ1‐Z and γ2‐Z rapidly and reversibly inhibited I Na without changing the kinetics or voltage dependence of activation or inactivation. To our knowledge, this is the first example of a plant protein that inhibits the sodium channel. From structural comparisons with the μ‐conotoxins, a family of peptides that block the sodium channel, we detected some similar features that could provide the basis of inhibition of sodium channels by γ‐zeathionins.

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree‐frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier‐transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti‐parallel β‐sheet. A conformational variation was observed under experimental conditions mimicking a membrane‐like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.

Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta1

Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 microM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.

Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalus ephippium (Anura: Brachycephalidae).

Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.

Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes.

Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human alpha2-macroglobulin (alpha2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.

Zinc Binding to Lambda Phage DNA Studied by Voltammetric Techniques

Binding of zinc to lambda phage DNA was investigated by differential pulse voltammetry and cyclic voltammetry. These methods rely on the direct monitoring of reduction and oxidation current of zinc in the absence and presence of this virion DNA. Titration graphs of Zn2+ with DNA were obtained in the concentration ranges from 3.57 x 10-12 to 3.92 x 10-11 molxa0L-1 and 6.97 x 10-12 to 5.56 x 10-11 molxa0L-1. These data were used to calculate the dissociation constant of the complex and the stoichiometry. The mechanism of this reaction was studied by cyclic voltammetry through curves I (oxidation current of Zn2+) versus v1/2 (square root of scan rate). These curves showed that the oxidation-reduction process of Zn2+ was not controlled by diffusion in the presence of lambda phage DNA.

Characterization of a methionine-rich protein from the seeds of Cereus jamacaru Mill. (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Cereus jamacaru (Cactaceae), to which we gave the trivial name of cactin. This protein has a molecular mass of 11.3 kDa and is formed by a light chain (3.67 kDa) and a heavy chain (7.63 kDa). This protein was isolated using a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of cactin was determined and found to resemble that of the 2S seed reserve protein from the Brazil nut, a protein remarkable for its high methionine content. The usefulness of cactin as a molecular marker in the taxonomy of the Cactaceae is discussed.

Isolation and Characterisation of a Reserve Protein from the Seeds of Cereus jamacaru (Cactaceae)

We describe here the isolation and characterisation of a major reserve protein from the seeds of Cereus jamacaru. (Cactaceae). This protein has a molecular mass of 5319 kDa and was isolated by a combination of gel filtration chromatography and reverse phase HPLC. The amino acid composition of the protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The usefulness of this protein as a molecular marker in the Cactaceae is also discussed.