Proteínas Involucradas en los Mecanismos de Defensa de Plantas Proteínas Involucradas en los Mecanismos de Defensa de Plantas

The plants lacking a defense system based on similar antibodies to which exists in animals, bases their protection on physical characteristics and in a series of components that the own plant synthesizes. Inside those compounds, the proteins constitute one of the main defense sources, not only for their high specificity and efficiency, but because some of them are also highly regulated, their synthesis responding to the attack of the insects orby pathogens. These proteins represent an interesting alternative to produce plants with better resistance characteristics, since through breeding mechanisms or introducing the gene a susceptible plant, by means of the genetic engineering. The knowledge of its action mechanisms represents an important form to learn how to combat plagues and illnesses of the plants, without having to use compound highly polluting, like the commercial insecticides. Las plantas careciendo de un sistema de defensa basado en anticuerpos similar al que existe en animales, basan su protección en características físicas y en una serie de componentes que la propia planta sintetiza. Dentro de esos compuestos, las proteínas constituyen una de las principales fuentes de defensa, no sólo por su elevada especificidad y eficiencia, sino porque además algunas de ellas son altamente reguladas, respondiendo su síntesis al ataque de los depredadores (insectos) o de los patógenos. Estas proteínas representan una interesante alternativa para producir plantas con mejores características de resistencia, ya que a través de mecanismos de fitomejoramiento o bien introduciendo el gen a plantas sensibles, por medio de la ingeniería genética. El conocimiento de sus mecanismos de acción representa una forma importante para aprender a combatir plagas y enfermedades de las plantas, sin tener que utilizar compuestos altamente contaminantes, como son los insecticidas comerciales. </span

Potencial y Algunos de los Mecanismos de Acción de los Hongos Entomopatógenos para el Control de Insectos Plaga Potencial y Algunos de los Mecanismos de Acción de los Hongos Entomopatógenos para el Control de Insectos Plaga

<span style="font-family: Times New Roman; font-size: small;"> </span><p style="margin: 0cm 0cm 0pt; line-height: normal; mso-layout-grid-align: none;" class="MsoNormal"><span lang="EN-US" style="color: #231f20; font-family: BookmanOldStyle; font-size: 7.5pt; mso-bidi-font-family: BookmanOldStyle; mso-hansi-font-family: Calibri; mso-hansi-theme-font: minor-latin; mso-ansi-language: EN-US;">This paper describes the use of an ecological alternative to control insect pests. One viable strategy, developed to answer the growing concern about the negative effects that chemical pesticides have on human health and/or the environment, consists in exploiting the use of natural insect pathogens. Among several options, the use of entomopathogenic fungi (EMF) as possible microbial control agents has been considered worldwide. EMF are widely recognized for their potential use as biological control agents. Due to their distinctive mode of action, EMF could have a unique and/or complementary role in insect control. Unlike bacterial, viral or protozoan entomopathogens, EMF do not require to be ingested by their host; instead, germinating fungal spores can penetrate directly through the insect’s cuticle. This mode of infection is possible thanks to the synergistic effect of cuticle-degrading enzymes, plus the mechanic pressure produced upon their contact. The enzymes participating in this process are a determinant factor for an effective infection.</span></p><span style="font-family: Times New Roman; font-size: small;"> </span><br><span style="font-family: Times New Roman; font-size: small;"> </span><p style="margin: 0cm 0cm 0pt; line-height: normal; mso-layout-grid-align: none;" class="MsoNormal"><span style="color: #231f20; font-family: "BookmanOldStyle","serif"; font-size: 7.5pt; mso-bidi-font-family: BookmanOldStyle;">El presente artículo enfatiza el uso de una alternativa ecológica para el control efi caz de plagas. Considerando que existe una preocupación general sobre los efectos negativos de los pesticidas químicos en la salud pública y/o en el medio ambiente, una alternativa consiste en el aprovechamiento de patógenos naturales de insectos. El uso de hongos entomopatógenos (HEP) como agentes de control biológico ha sido considerado a nivel mundial; estos hongos son ampliamente reconocidos por su potencial como agentes de control biológico. Debido a su modo de infección distintivo, los hongos pueden tener un papel único o complementario como agentes de control de insectos plaga. A diferencia de otros agentes entomopatógenos, como bacterias, virus o protozoarios, los HEP no requieren ser ingeridos por su hospedero para causar la infección; en su lugar, las esporas pueden penetrar directamente a través de la cutícula. Este modo de infección es posible gracias a la acción coordinada de enzimas hidrolíticas, además de la presión mecánica ejercida en el punto de contacto. Las enzimas participantes son factores determinantes en el éxito de la infección.</span></p><span style="font-family: Times New Roman; font-size: small;"> </span

Expression of Lectins in Heterologous Systems

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages

A novel alpha-amylase inhibitor from amaranth (Amaranthus hypocondriacus) seeds.

The major alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypocondriacus, a variety of the Mexican crop plant amaranth, is a 32-residue-long polypeptide with three disulfide bridges. Purified AAI strongly inhibits the alpha-amylase activity of insect larvae (Tribolium castaneum and Prostephanus truncatus) and does not inhibit proteases and mammalian alpha-amylases. AAI was sequenced with the automated Edman method, and the disulfide bridges were localized using enzymatic and chemical fragmentation methods combined with N-terminal sequencing. AAI is the shortest alpha-amylase inhibitor described so far which has no known close homologs in the sequence data bases. Its residue conservation patterns and disulfide connectivity are related to the squash family of proteinase inhibitors, to the cellulose binding domain of cellobiohydrolase, and to omega-conotoxin, i.e. a group of small proteins termed "knottins" by Nguyen, D. L., Heitz, A., Chiche, L., Castro, B., Boigegrain, R., Favel, A., and Coletti-Previero, M. ((1990) (Biochimie 72, 431-435) The three-dimensional model of AAI was built according to the common structural features of this group of proteins using side-chain replacement and molecular dynamics refinement techniques

Respuestas del insecto Max (Scyphophorus acupunctatus Gyllenhal [Coleoptera: Curculionidae] hacia algunos compuestos atrayentes del henequ\ue9n

Scyphophorus acupunctatus Gyllenhal 1838 (Coleoptera: Curculionidae) is one of the most important pests of cultivated agave; causing severe damage to the leaves of the plant; Yield losses up to 40% have been reported due to this insect; Previous studies have shown the attractive effect of henequen leaves towards the insect S; acupunctatus and the effective control of this pest using henequen leaves treated with common insecticides; Therefore; this study was undertaken to identify the compounds responsible for the strong attraction towards the insect S; acupunctatus; Henequen leaves were steam distilled and subsequently extracted with different organic solvents; Free choice bioassays were conducted to determine the attractive activity of each extract towards this insect; using groups of insects separeted on the basis of their sex; or else; taken at random; Statistical analysis showed significant differences among the different extracts tested; The five main compounds from the most active extracts determined by GC-MS were: 4-ethyl cumene; p-metoxy-ethyl benzene; 1(methyl 4-(1-methyl ethyl) cyclohexanol; p-mentha-1;5- dien-8-ol and butirofenone; Since these compounds are commercially available; it raises the possibility of using them in the control of this pest;El max Scyphophorus acupunctatus Gyllenhal 1838 (Coleoptera: Curculionidae) es una de las plagas más importante en agaves cultivados; produciendo daños severos a las hojas de las plantas y pérdidas de rendimiento hasta de 40% en el Estado de Yucatán; En estudios previos se demostró el efecto atrayente que ejercen las hojas de henequén sobre el insecto adulto de S; acupunctatus y el control efectivo que se logra de esta plaga usando hojas de henequén tratadas con insecticidas comunes; Por lo tanto; en el presente trabajo se llevó a cabo la identificación de los compuestos responsables de la actividad atrayente del henequén hacia el insecto S; Acupunctatus; Fragmentos de hojas de henequén se sometieron a una destilación por arrastre con vapor y el destilado se extrajo con diferentes solventes orgánicos; Se realizaron bioensayos de elección libre para determinar la actividad atrayente de cada uno de los extractos obtenidos; hacia el insecto S; acupunctatus; utilizando grupos de insectos separados por sexo; o bien tomados al azar; El análisis estadístico de estos tratamientos mostró que existían diferencias significativas entre los diferentes extractos probados; Los extractos con mayor actividad atrayente se analizaron por CG-EM pudiéndose identificar 5 compuestos en común: 4-etil cumeno; p-metoxi-etil-benceno; 1- metil-4-(1-metil etil)-ciclohexanol; p-menta-1;5-dien-8-ol y butirofenona; Considerando que estos compuestos están disponibles en forma comercial; se plantea la posibilidad de utilizarlos para lograr un control efectivo de esta plaga en la zona henequenera

Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N-glycans’ characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins

Snake Venom Hemotoxic Enzymes: Biochemical Comparison between <i>Crotalus</i> Species from Central Mexico

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A2 (PLA2), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA2 activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA2 activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms

Optimization of a Recombinant Lectin Production in Pichia pastoris Using Crude Glycerol in a Fed-Batch System

The production of heterologous proteins for medical use is an important area of interest. The optimization of the bioprocesses includes the improvement of time, costs, and unit operations. Our study shows that a lectin fraction from Tepary bean (Phaseolus acutifolius) (TBLF) has cytotoxic effects on colon cancer cells and in vivo antitumorigenic activity. However, the low-yield, time-consuming, and expensive process made us focus on the development of a strategy to obtain a recombinant lectin using engineered Pichia pastoris yeast. Pure glycerol is one of the most expensive supplies; therefore, we worked on process optimization using crude glycerol from biodiesel production. Recombinant lectin (rTBL-1) production and purification were evaluated for the first time by an experimental design where crude glycerol (G65) was used and compared against pure glycerol (G99) in a controlled stirred-tank bioreactor with a fed-batch system. The recombinant lectin was detected and identified by SDS-PAGE, Western blot, and UHPLC–ESI–QTOF/MS analysis. The results show that the recombinant lectin can be produced from G65 with no significant differences with respect to G99: the reaction rates were 2.04 and 1.43 mg L−1 h−1, and the yields were 264.95 and 274.67 mgL−1, respectively. The current low cost of crude glycerol and our results show the possibility of producing heterologous proteins using this substrate with high productivity