Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

ppiGReMLIN: a graph mining based detection of conserved structural arrangements in protein-protein interfaces.

BACKGROUND: Protein-protein interactions (PPIs) are fundamental in many biological processes and understanding these interactions is key for a myriad of applications including drug development, peptide design and identification of drug targets. The biological data deluge demands efficient and scalable methods to characterize and understand protein-protein interfaces. In this paper, we present ppiGReMLIN, a graph based strategy to infer interaction patterns in a set of protein-protein complexes. Our method combines an unsupervised learning strategy with frequent subgraph mining in order to detect conserved structural arrangements (patterns) based on the physicochemical properties of atoms on protein interfaces. To assess the ability of ppiGReMLIN to point out relevant conserved substructures on protein-protein interfaces, we compared our results to experimentally determined patterns that are key for protein-protein interactions in 2 datasets of complexes, Serine-protease and BCL-2. RESULTS: ppiGReMLIN was able to detect, in an automatic fashion, conserved structural arrangements that represent highly conserved interactions at the specificity binding pocket of trypsin and trypsin-like proteins from Serine-protease dataset. Also, for the BCL-2 dataset, our method pointed out conserved arrangements that include critical residue interactions within the conserved motif LXXXXD, pivotal to the binding specificity of BH3 domains of pro-apoptotic BCL-2 proteins towards apoptotic suppressors. Quantitatively, ppiGReMLIN was able to find all of the most relevant residues described in literature for our datasets, showing precision of at least 69% up to 100% and recall of 100%. CONCLUSIONS: ppiGReMLIN was able to find highly conserved structures on the interfaces of protein-protein complexes, with minimum support value of 60%, in datasets of similar proteins. We showed that the patterns automatically detected on protein interfaces by our method are in agreement with interaction patterns described in the literature.

Kinetic Characterization of Anticarsia gemmatalis Digestive Serine- Proteases and the Inhibitory Effect of Synthetic Peptides.

BACKGROUND: Enzyme kinetics contributes to understanding the structure and function of insect digestive serine proteases. Kinetic parameters allow to understanding active sites and mechanisms of enzymes efficacy, identifying the inhibition of the insects digestive protease system by inhibitors produced by plants, or via the application of synthetic inhibitors Objectives: The aim of this study was to purify digestive serine proteases of A. gemmatalis, determining their kinetic properties using the chromogenic substrates tripeptidyl and characterizing the effects of synthetic inhibitors on their activity. In order to provide new opportunities for sustainable pest management through the development of protease inhibitors. METHODS: The enzymes were purified on p-aminobenzamidine agarose affinity column in an FPLC system using electrophoresis with 12.5% polyacrylamide gel. Michaelis-Menten constants and the inhibition model were determined according to the Dixon methodology and Lineweaver-Burk's double reciprocal. RESULTS: The KM values and catalytic constants of peptide substrates show that A. gemmatalis trypsin- like has a higher affinity for substrates with arginine in the P1 position. Inhibition by Gor 3, Gor 4, and Gor 5, in the presence of L-BApNA, was linear competitive. The inhibition constant for the Gor 5 peptide was higher due to its strong interaction with hydrophobic residues in the secondary site region of A. gemmatalis trypsin-like. CONCLUSION: It is observed that among the three peptides analyzed, the Gor 5 presented lower inhibition constant and therefore, the most potent among the tested ones. The predominance of hydrophobic residues in the region of the secondary site of the enzymes favored the interaction of the peptide. After characterization by three different types of graphs profiles, it is possible to verify that the inhibition model of the trypsin-like enzymes for the tested peptides is of the linear competitive type, in the concentration range of inhibitors and substrates analyzed. However, by the graphing profiles it is observed that the inhibition occurred due to the interaction of the peptides at the secondary site S2' in the hydrophobic cavity of the enzymes analyzed.

Factores relacionados con hábitos y conductas de adultos con sobrepeso. Caldas, Colombia / Factors Related to Habits and Behaviors of Overweight Adults in Caldas, Colombia

Resumen Antecedentes: la obesidad es una condición de salud que ha aumentado su prevalencia en Colombia y en Caldas. Objetivo: identificar factores relacionados con hábitos y conductas de adultos con sobrepeso, residentes en tres zonas urbanas de Manizales, La Dorada y Chinchiná durante 2015. Materiales y métodos: estudio cualitativo en 23 adultos seleccionados por conveniencia. El procedimiento para la recolección de datos se centró en entrevistas a profundidad. Se desarrolló una guía de entrevista y se realizó una prueba piloto para optimizar la secuencia temática en la interacción con los participantes. El análisis de datos se realizó con el software Dedosse. En la codificación de fragmentos y categorización se utilizó codificación abierta nombrando códigos de frases en vivo y se debatieron procedimientos entre los investigadores para lograr consenso. Resultados: se identificaron cuatro categorías de factores relacionados con hábitos y conductas: 1) hábitos y conductas no saludables en ambiente obesogénico; 2) información nutricional; 3) conductas racionales y no racionales/actitudes e imaginarios; y 4) la atención de salud. Conclusiones: un estilo de vida sedentario, ambiente obesogénico, ausencia de políticas públicas para el control del sobrepeso y poca oferta y disponibilidad de programas, de servicios y de nutricionistas se identifican como factores relacionados con hábitos y conductas de adultos con sobrepeso en Caldas.

Abstract Background: Obesity is a health condition that has increased in prevalence in Colombia and the city of Caldas. Objective: To identify factors related to habits and behaviors of overweight adults living in three urban areas of Manizales, La Dorada and Chinchiná during 2015. Materials and Methods: This was a qualitative study with a convenience sample of 23 adults. The procedure for data collection focused on in-depth interviews. An interview guide was developed and a pilot test was conducted to optimize the thematic sequence in the interaction with the participants. Data analysis was done using the software Dedosse. In the coding of fragments and categorization, open coding was used, naming live codes of sentences and procedures were discussed among researchers to achieve consensus. Results: Four categories of factors related to habits and behaviors were identified: unhealthy habits and behaviors in an obesogenic environment, nutritional information, rational and irrational behavior / attitudes, and imaginary and health care services. Conclusions: Sedentary lifestyle, obesogenic environment, absence of public policies for the control of overweight, and low supply and availability of programs, services, and nutritionists are identified as factors related to habits and behaviors of overweight adults in Caldas.