RESUMEN
High-resolution mass spectrometry-based peptidomics has been used to characterize several components in electro-stimulated skin secretions of the endemic Mexican frog Pachymedusa dacnicolor. Peptide mass screening performed in an Orbitrap-XL mass spectrometer showed that P. dacnicolor skin secretions possess 194 different components with molecular masses ranging mainly from 500 to 6,000 Da. Dozens of molecules were partially sequenced including two novel protease inhibitors. Additionally, one posttranslationally modified bradykinin and two novel dermaseptin-like antimicrobial peptides were fully sequenced. The novel peptide named here DMS-DA5 was fully characterized and showed potent antibacterial activity against various bacteria such as Escherichia coli, Bacillus subtilis, Salmonella enterica serovar typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations from 3.10 to 25.0 microM.
Asunto(s)
Antibacterianos/química , Anuros , Piel/química , Secuencia de Aminoácidos , Animales , Antibacterianos/metabolismo , Anuros/metabolismo , Bacterias/efectos de los fármacos , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Alineación de Secuencia , Piel/metabolismoRESUMEN
In this work, we describe the original characterization of peptides and proteins present in the skin secretions of the Mexican amphibian Hyla eximia. To this purpose, a novel water/dark extraction method, as well as the classic electrical stimulation procedure, was applied in order to extract the skin secretion. Two novel antimicrobial peptides He-1 and He-2 were sequenced. In addition, a molecular mass fingerprint revealed more than one hundred different molecules. Eight peptides in homogeneous form were assayed against five species of bacteria. Thereafter, the peptide He-2 demonstrated high antiparasitic activity against ookinete forms of malaria parasites at low concentration.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Anuros , Secreciones Corporales/química , Piel/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias , Proliferación Celular/efectos de los fármacos , Fraccionamiento Químico/métodos , México , Pruebas de Sensibilidad Microbiana , Plasmodium berghei , Análisis de Secuencia de ProteínaRESUMEN
The protein composition of the soluble venom from the South American fish-eating coral snake Micrurus surinamensis surinamensis, here abbreviated M. surinamensis, was separated by RP-HPLC and 2-DE, and their components were analyzed by automatic Edman degradation, MALDI-TOF and ESI-MS/MS. Approximately 100 different molecules were identified. Sixty-two components possess molecular masses between 6 and 8 kDa, are basically charged molecules, among which are cytotoxins and neurotoxins lethal to fish (Brachidanios rerio). Six new toxins (abbreviated Ms1-Ms5 and Ms11) were fully sequenced. Amino acid sequences similar to the enzymes phospholipase A2 and amino acid oxidase were identified. Over 20 additional peptides were identified by sequencing minor components of the HPLC separation and from 2-DE gels. A functional assessment of the physiological activity of the six toxins was also performed by patch clamp using muscular nicotinic acetylcholine receptor assays. Variable degrees of blockade were observed, most of them reversible. The structural and functional data obtained were used for phylogenetic analysis, providing information on some evolutionary aspects of the venom components of this snake. This contribution increases by a factor of two the total number of alpha-neurotoxins sequenced from the Micrurus genus in currently available literature.
Asunto(s)
Proteómica/métodos , Venenos de Serpiente/análisis , Aminoácido Oxidorreductasas/metabolismo , Animales , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Peces , Humanos , Técnicas de Placa-Clamp , Fosfolipasas A2/metabolismo , Filogenia , Receptores Colinérgicos/metabolismo , Venenos de Serpiente/química , Serpientes , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales.
Asunto(s)
Conjugación Genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genes Reguladores , Plásmidos/genética , Rhizobium etli/genética , Sinorhizobium meliloti/genética , Simbiosis/genética , Elementos Transponibles de ADN , Silenciador del Gen , Mutagénesis Insercional , Operón , Homología de Secuencia de Aminoácido , SinteníaRESUMEN
The structure of rotaviruses and many steps of their replication cycle depend on the concentration of calcium in the microenvironment. In this work, to learn about the role of calcium during the early steps of the infection, we characterized the effect of increasing the calcium concentration in the medium on the infectivity of rotaviruses. We found that a fivefold increase in the calcium concentration of the cell culture medium results in an increased viral titer in all rotavirus strains tested. The effect of this divalent ion seems to be mainly on the viral particle and not on the surface of the cell. Analysis of the intrinsic fluorescence spectra of purified triple-layered particles revealed that changes in the environment of tryptophan residues occurred as calcium concentration increased, suggesting that conformational changes in the viral particle might be responsible for the effect of this ion on the viral infectivity.