RESUMEN

BACKGROUND: The kinetic parameters of peach rootstock are a major factor contributing to its nitrogen (N) uptake efficiency, which directly affects the yield and quality parameters. However, the impact of grafting on N kinetic parameters is not sufficiently known as rootstocks are grafted with other scion cultivars before being transplanted to the field. The objective of this study was therefore to evaluate whether the grafting of 'Okinawa' rootstock with the scion cultivar 'Chimarrita' can alter the kinetic parameters related to N uptake. RESULTS: The plants were acclimatized in Hoagland solution for 21 days and, sequentially, were tested to assess the depletion of the internal reserves at 15 and 30 days in CaSO4 solution. Morphological and physiological parameters were evaluated, and kinetic parameters were calculated. The depletion period for to reach the minimum concentration (Cmin ) of NO3 - and NH4 + in the plant was 62 h and 56 h, respectively. The optimal time for internal reserve depletion for 'Okinawa' rootstock with and without the 'Chimarrita' scion was 30 days. CONCLUSION: 'Okinawa' rootstock grafted with 'Chimarrita' scion cultivar was more efficient in NO3 - and NH4 + uptake. © 2022 Society of Chemical Industry.

Asunto(s)

Prunus persica , Nitrógeno , Raíces de Plantas/fisiología

RESUMEN

Maud Leonora Menten nació en Canadá, tuvo cuatro títulos universitarios: Bachiller en Artes, Master en Fisiología, médica y Doctora en Bioquímica. Trabajó en Estados Unidos, Alemania y Canadá. Trabajó en diferentes áreas: en la distribución de los iones cloruro en el sistema nervioso central, en tumores experimentales y su tratamiento con bromuro de radio, en el equilibrio ácido-base durante la anestesia, en el mecanismo hiperglucemiante de toxinas bacterianas, en el descubrimiento de un mecanismo de acoplamiento en química orgánica y hasta en la electroforesis de las hemoglobinas humanas. Sin embargo, el aporte por el cual es más conocida es su trabajo en el estudio de la cinética enzimática junto a Leonor Michaelis en 1913. El propósito de este trabajo es exponer la vida personal y académica de una científica conocida por la gran mayoría de los profesionales de la salud. La mujer que a principios del siglo XX trabajó con grandes investigadores de Canadá, Estados Unidos y Alemania, cuyos aportes científicos fueron reconocidos muchas décadas después. (AU)

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Maud Leonora Menten was born in Canada; she had four university degrees, Bachelor of Arts, Master of Physiology, Physician and Doctor of Biochemistry. She worked in the United States, Germany, and Canada. Maud worked in different areas: the distribution of chloride ions in the central nervous system, experimental tumors and their treatment with radium bromide, the acid-base balance during anesthesia, the hyperglycemic mechanism of bacterial toxins, the discovery of a coupling mechanism in organic chemistry and even the electrophoresis of human hemoglobins. However, the contribution for which she is best known is for her work in the study of enzymatic kinetics with Leonor Michaelis in 1913. The aim of this paper is to expose the personal and academic life of a scientist known to the vast majority of Health professionals. The woman who, at the beginning of the 20th century, worked with great researchers from Canada, the United States and Germany, whose scientific contributions were recognized many decades later. (AU)

Asunto(s)

Humanos , Femenino , Médicos Mujeres/historia , Historia de la Medicina , Mujeres Trabajadoras/historia , Historia del Siglo XX

RESUMEN

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16-2.6 µM; Vmax,app = 224-2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 µM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.

Asunto(s)

3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Proteínas de la Membrana/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/química , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Análisis Mutacional de ADN , Células HEK293 , Humanos , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

Monocarboxylate transporter 4 (MCT4) is an H+-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mm, the reported Km of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective Km values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a Km for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous Km estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in high-lactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate.

Asunto(s)

Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Transporte Biológico/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Diclofenaco/farmacología , Fluoresceínas/química , Edición Génica , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Cinética , Macrófagos/citología , Macrófagos/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ácido Pirúvico/metabolismo

RESUMEN

In this article, the occurrence of dead core in catalytic particles containing immobilized enzymes is analyzed for the Michaelis-Menten kinetics. An assessment of numerical methods is performed to solve the boundary value problem generated by the mathematical modeling of diffusion and reaction processes under steady state and isothermal conditions. Two classes of numerical methods were employed: shooting and collocation. The shooting method used the ode function from Scilab software. The collocation methods included: that implemented by the bvode function of Scilab, the orthogonal collocation, and the orthogonal collocation on finite elements. The methods were validated for simplified forms of the Michaelis-Menten equation (zero-order and first-order kinetics), for which analytical solutions are available. Among the methods covered in this article, the orthogonal collocation on finite elements proved to be the most robust and efficient method to solve the boundary value problem concerning Michaelis-Menten kinetics. For this enzyme kinetics, it was found that the dead core can occur when verified certain conditions of diffusion-reaction within the catalytic particle. The application of the concepts and methods presented in this study will allow for a more generalized analysis and more accurate designs of heterogeneous enzymatic reactors.

Asunto(s)

Enzimas Inmovilizadas/química , Modelos Químicos , Catálisis , Cinética

RESUMEN

In this article, Perturbation Method (PM) is employed to obtain a handy approximate solution to the steady state nonlinear reaction diffusion equation containing a nonlinear term related to Michaelis-Menten of the enzymatic reaction. Comparing graphics between the approximate and exact solutions, it will be shown that the PM method is quite efficient.

RESUMEN

Linear (linear-plateau, quadratic, quadraticplateau, square root) and non-linear (exponential and Michaelis-Menten) models to describe the ruminal microbial growth as a function of sucrose levels were evaluated. Is was used a database of 76 observations of microbial growth in roll tubes (mix of bacterial) containing final concentrations of 0.0; 0.375; 0.75; 1.50; 3.0; 6.0; 12 and 24g/L of sucrose. The microbial growth was evaluated by measuring optical density (OD - 600nm) at 0; 6; 12; 18 and 24 hours of fermentation. The evaluation of linear regression parameters of predicted and observed values indicated adequacy of all models in the prediction of microbial growth. The partition of the mean square prediction error did not allow identifying differences in relation to prediction quality of the models. However, the marginal growth as a function of sucrose supply was inadequate to models linear-plateau and quadratic (in low sucrose levels). The square root, Mitscherlich and Michaelis-Menten models can be used to describe biological responses to nutrients. However, differences are observed in estimates of substrates efficiencies, which implies differences in the recommendations of optimum levels of nutrients. In this sense, the choice of these models can be modulated by the level of nutrient supply desired.

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Avaliaram-se diferentes modelos lineares (quadrático, linear-platô, quadrático-platô e raiz quadrada) e não lineares (exponencial-Mitscherlich e Michaelis-Menten) para descrever o crescimento microbiano ruminal em função do suprimento de nutrientes. Utilizou-se banco de dados compostos por 76 observações de meios de cultivo ruminal in vitro (mix de bactéria) contendo 0,0; 0,375; 0,75; 1,50; 3,0; 6,0; 12 e 24g de sacarose/L. O crescimento microbiano foi avaliado por meio de mensuração da densidade óptica (OD - 600nm) após 0; 6; 12; 18 e 24 horas de fermentação. A avaliação conjunta dos parâmetros da regressão linear dos valores preditos e observados indicou adequação de todos os modelos na predição do crescimento microbiano. A partição do quadrado médio do erro de predição não permitiu identificar diferenças na qualidade da predição do crescimento microbiano entre os modelos. Porém, ao avaliar o crescimento marginal em função do suprimento de glicose verificou-se inadequação do modelo linear-platô e do modelo quadrático (em baixos níveis substratos). Os modelos quadrático-platô, raiz quadrada, Mitscherlich ou Michaelis-Menten podem ser utilizados para descrever o comportamento do crescimento microbiano ruminal ao suprimento de nutrientes. Entretanto, diferenças são observadas quanto às estimativas das eficiências de uso nos diferentes níveis de substratos, o que implica em diferenças nas recomendações dos níveis ótimos de nutrientes. Neste sentido, a escolha desses modelos poderá ser modulada pelo nível de suprimento de nutrientes desejado.