RESUMEN
Polyphenols are plant secondary metabolites that function mostly as a general stress-induced protective mechanism. Polyphenols have also gained interest due to their beneficial properties for human health. Strawberry leaves represent an agro-industrial waste material with relevant bioactive polyphenol content, which could be incorporated into circular economy strategies. However, due to the low quantities of polyphenols in plants, their production needs to be improved for cost-effective applications. The objective of this research was to compare polyphenol production in strawberry (Fragaria × ananassa cv. Festival) leaves in plants grown in greenhouse conditions and plants grown in vitro, using three possible elicitor treatments (UV irradiation, cold exposure, and cysteine). General vegetative effects were morphologically evaluated, and specific polyphenolic compounds were quantified by UHPLC-DAD-MS/MS. Gallic acid was the most abundant polyphenol found in the leaves, both in vivo and in vitro. The results showed higher amounts and faster accumulation of polyphenols in the in vitro regenerated plants, highlighting the relevance of in vitro tissue culture strategies for producing compounds such as polyphenols in this species and cultivar.
Asunto(s)
Fragaria , Hojas de la Planta , Polifenoles , Fragaria/química , Fragaria/metabolismo , Polifenoles/química , Hojas de la Planta/química , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Ácido Gálico/químicaRESUMEN
Alphaproteobacteria include organisms living in close association with plants or animals. This interaction relies partly on orthologous two-component regulatory systems (TCS), with sensor and regulator proteins modulating the expression of conserved genes related to symbiosis/virulence. We assessed the ability of the exoS+Sm gene, encoding a sensor protein from the plant endosymbiont Sinorhizobium meliloti to substitute its orthologous bvrS in the related animal/human pathogen Brucella abortus. ExoS phosphorylated the B. abortus regulator BvrR in vitro and in cultured bacteria, showing conserved biological function. Production of ExoS in a B. abortus bvrS mutant reestablished replication in host cells and the capacity to infect mice. Bacterial outer membrane properties, the production of the type IV secretion system VirB, and its transcriptional regulators VjbR and BvrR were restored as compared to parental B. abortus. These results indicate that conserved traits of orthologous TCS from bacteria living in and sensing different environments are sufficient to achieve phenotypic plasticity and support bacterial survival. The knowledge of bacterial genetic networks regulating host interactions allows for an understanding of the subtle differences between symbiosis and parasitism. Rewiring these networks could provide new alternatives to control and prevent bacterial infection.
Asunto(s)
Brucella abortus , Genes Bacterianos , Animales , Ratones , Humanos , Virulencia/genética , Histidina Quinasa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and ß-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales.
RESUMEN
Berry fruits are an important dietary source of health-promoting antioxidant polyphenols. Interestingly, berry leaves of diverse species, including strawberries, have shown higher bioactive phytochemical content in the leaves than in the fruit. Moreover, the vegetative part of the plants is usually discarded, representing a presumably large source of underutilized bioactive biomass. In this investigation, the polyphenol profiles of tropical highland strawberry (Fragaria x ananassa cv. Festival) leaves and fruits were compared by high-performance liquid chromatography coupled with a diode array detector (UHPLC-DAD) and mass spectrometry (HPLC-MS). The total polyphenol strawberry leaf extracts exhibited a 122-fold-higher total polyphenol content and 13-fold higher antioxidant activity (ORAC) than strawberry fruits, and they showed evidence of possible photoprotective effects against UV damage in human melanoma cells (SK-MEL-28) and in murine embryo fibroblasts (NIH/3T3), together with promising anti-proliferative activities against the same melanoma cells. Seven polyphenols were confirmed by HPLC-DAD in the leaf extracts, with differences depending on fraction solubility. Moreover, three substituted quercetin derivatives, three substituted kaempferol derivatives, two anthocyanins, and catechin were confirmed in the soluble fraction by HPLC-MS. Given their higher total polyphenol content and bioactive activities, underutilized strawberry Festival leaves are a potential source of apparently abundant biomass with prospective bioactive applications.
Asunto(s)
Fragaria , Polifenoles , Animales , Humanos , Ratones , Polifenoles/análisis , Fragaria/química , Frutas/química , Antocianinas/química , Vacaciones y Feriados , Estudios Prospectivos , Antioxidantes/química , Fitoquímicos/análisisRESUMEN
Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.
Asunto(s)
Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/fisiología , Fosfolipasas de Tipo C/metabolismo , Fosfolipasas de Tipo C/fisiología , Animales , Clostridium perfringens/enzimología , Clostridium perfringens/patogenicidad , Humanos , Listeria monocytogenes/enzimología , Listeria monocytogenes/patogenicidad , Fosfolipasas , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Fosfolipasas de Tipo C/genéticaRESUMEN
Around 5.5 million people suffer from snakebites per year, with about 400,000 cases with some type of sequelae, such as amputation, and 20,000 to 125,000 cases with the fatal end. Usually, the victim outcome depends on correct, agile and many times in situ intervention based on the proper identification of the snake venom type and its potential effects, among other factors. Therefore, knowledge on the snake venom composition and a research on inhibitors of snake venom target components might ameliorate envenoming dangerous outcome. Herein, two thrombin-like serine proteases from the Crotalus simus snake venom - SVSP1 and SVSP2 - were isolated in two chromatographic steps, using gel filtration and then RP-HPLC. They showed molecular masses of around 31.3 and 24.6â¯kDa, respectively, and mostly ß-sheet secondary structure features. The SVSP1 and SVSP2 were sequenced using tandem mass spectrometry (Q-TOF). Using the known serine protease structure (PDB entry: 4e7n), which was evaluated as homologous to the two target proteins, in silico docking results showed that hesperetin is its excellent inhibitor. Using in vitro tests with the commercial hesperetin, kinetic parameters were obtained for SVSPs against the synthetic substrate BApNA. Obtained results pointed that hesperetin might act as an uncompetitive (SVSP1) or mixed (SVSP2) inhibitor. Also, the fluorescence quenching upon inhibition was observed, as well as, red shift in maximums of around 20â¯nm, which indicate that the tryptophan residues in the target enzymes suffered conformational changes caused by hesperetin binding. Thus, a naturally occurring flavone that can easily be extracted from oranges might serve as low-cost inhibitor of the investigated snake venom proteases.
Asunto(s)
Venenos de Crotálidos/antagonistas & inhibidores , Crotalus , Hesperidina/farmacología , Serina Proteasas/efectos de los fármacos , Animales , Venenos de Crotálidos/enzimología , Fibrinólisis/efectos de los fármacos , Humanos , Cinética , Simulación del Acoplamiento Molecular , Conformación Proteica , Análisis de Secuencia de Proteína , Serina Proteasas/química , Espectrometría de Masas en TándemRESUMEN
Snakebites represent an important public health problem, with a great number of victims with permanent sequelae or fatal outcomes, particularly in rural, agriculturally active areas. The snake venom metalloproteases (SVMPs) are the principal proteins responsible for some clinically-relevant effects, such as local and systemic hemorrhage, dermonecrosis, and myonecrosis. Because of the difficulties in neutralizing them rapidly and locally by antivenoms, the search and design of small molecules as inhibitors of SVMPs are proposed. The Bothrops asper metalloprotease P1 (BaP1) is hereby used as a target protein and by High Throughput Virtual Screening (HTVS) approach, the free access virtual libraries: ZINC, PubChem and ChEMBL, were searched for potent small molecule inhibitors. Results from the aforementioned approaches provided strong evidences on the structural requirements for the efficient BaP1 inhibition such as the presence of the pyrimidine-2,4,6-trione moiety. The two proposed compounds have also shown excellent results in performed in vitro interaction studies against BaP1.
Asunto(s)
Antídotos/química , Antídotos/farmacología , Bothrops/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Pirimidinonas/química , Pirimidinonas/farmacología , Venenos de Serpiente/antagonistas & inhibidores , Animales , Simulación por Computador , Descubrimiento de Drogas , Metaloendopeptidasas/metabolismo , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The term "agrochemicals" is used in its generic form to represent a spectrum of pesticides, such as insecticides, fungicides or bactericides. They contain active components designed for optimized pest management and control, therefore allowing for economically sound and labor efficient agricultural production. A "drug" on the other side is a term that is used for compounds designed for controlling human diseases. Although drugs are subjected to much more severe testing and regulation procedures before reaching the market, they might contain exactly the same active ingredient as certain agrochemicals, what is the case described in present work, showing how a small chemical compound might be used to control pathogenicity of Gram negative bacteria Xylella fastidiosa which devastates citrus plantations, as well as for control of, for example, meningitis in humans. It is also clear that so far the production of new agrochemicals is not benefiting as much from the in silico new chemical compound identification/discovery as pharmaceutical production. Rational drug design crucially depends on detailed knowledge of structural information about the receptor (target protein) and the ligand (drug/agrochemical). The interaction between the two molecules is the subject of analysis that aims to understand relationship between structure and function, mainly deciphering some fundamental elements of the nanoenvironment where the interaction occurs. In this work we will emphasize the role of understanding nanoenvironmental factors that guide recognition and interaction of target protein and its function modifier, an agrochemical or a drug. The repertoire of nanoenvironment descriptors is used for two selected and specific cases we have approached in order to offer a technological solution for some very important problems that needs special attention in agriculture: elimination of pathogenicity of a bacterium which is attacking citrus plants and formulation of a new fungicide. Finally, we also briefly describe a workflow which might be useful when research requires that model structures of target proteins are firstly generated (starting from genome sequences), followed by identification of ligand-target sites at the surface of those modeled structures, then application of procedures that adequately prepare both protein and ligand structures (the latter also involving filtration that satisfies acceptable adsorption/desorption/metabolism/excretion/toxicity [ADMET] parameters) for virtual high throughput screening (involving docking of ligands to indicated sites) and terminating by ranking of best pairs: target protein with selected ligand.
Asunto(s)
Agroquímicos/metabolismo , Aminoácidos/metabolismo , Biología Computacional/métodos , Diseño de Fármacos , Secuencia de Aminoácidos , Sitios de Unión , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Poligalacturonasa/química , Alineación de SecuenciaRESUMEN
The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP.