Clonación y caracterización de fragmentos RAPD y loci microsatélite, asociados a Psidium guajava L. cultivada en 4 estados de la República Mexicana

Introduction. People around the world use medicinal plants to attend their health problems so government and international institutions are now taking attention in quality control methods to guarantee the security and efficiency of plant drug material, avoiding the incorporation of related species that lack the active compound or intrinsically have inhibitory compounds. Official guidelines recognize taxonomic identification and chemical fingerprinting with authenticity tests; however RAPD, RFLP, AFLP, specific-sequence and microsatellital DNA markers are used on species identification and adulterant discrimination in basic preparative research projects. Guava (Psidium guajava L.) leaf is an extensive crop in México and it is used in abdominal disorders, and recently in the treatment of irritable bowel syndrome. Justification. Due to limited genetic information of P. guajava, the purpose of this work was to identify genetic markers related to quercetin accumulation and that allow the authentication of this species to be used in the development of phytodrugs, as a tool of quality control. Hypothesis. Some genetic variations between individuals of P. guajava cultivated in different states from Mexico are associated with a variation in the accumulation of flavonoids while conserved regions are associated with the identification of the species. Objectives. a) To characterize varieties or homologies in RAPD fragments sequences from P. guajava DNA; b) To obtain a dinucleotide microsatellite genomic library from P. guajava L. Material and methods. Young clean leaves of 40 trees where collected from four Mexican states and processed for DNA genomic and quercetin extraction. The raw material was used to obtain a methanolic hydrolyzed extract for quercetin quantification by the HPLC technique using RP-18 columns and a PDA detector. On the other hand, it was used for DNA extraction, amplification of random DNA regions by the RAPD technique using 10-mer primers, and to obtain DNA fragments with restriction enzymes. RAPD analysis was correlated with high quercetin accumulation. RFLP fragments were hybridized with biotilinated tandem sequences. RAPD and microsatellites library were cloned, sequenced, analyzed and specific primers were designed. DNA markers were used in authentication and discrimination from other morphologically alike species. Results. Selected states account for 84% of the guava fruit production in Mexico. Spring samples collected from Aguascalientes showed a high quercetin accumulation, but one individual sample from the state of Mexico had the highest. Six decamer commercial RAPD primers generated a total of 91 fragments, sharing 74 ± 100% of bands among collected trees. The oligonucleotide 1 amplified two polymorphic segments of 560 and 610 bp to recognize the 1a, 1b and 1c banding patterns, oligonucleotide 2 amplified one polymorphic segment of 370 bp to recognize the 2a, and 2b banding patterns, oligonucleotide 3 amplified one polymorphic segment of 690 bp to recognize the 3a and 3b banding patterns, oligonucleotide 4 amplified two polymorphic segments of 460 and 480 bp to recognize the 4a, 4b and 4c banding patterns, oligonucleotides 5 and 6 amplified a pattern band each. Quercetin accumulation increase was correlated between 1a and 2a with 4c pattern bands. Thirty-six sequences were obtained from genomic libraries, five of them with tandem regions, one with a similarity of 91% with Arabidopsis thaliana, and only 22 sequences were viable for specific primer design. Amplified conditions were developed for 11 sequences using 10 ng of genomic DNA and 1 mM MgCl2 or 12 sequences using 5 ng of genomic DNA and 1.5 mM MgCl2. Five fragments were specific for P. guajava. Conclusions. The chemical marker quercetin allowed the identification of individuals from Aguascalientes state in spring with the highest accumulation of quercetin among selected regions. The banding patterns 1a and 2a with 4c are correlated with an increase of quercetin with a LoD value of 9.498 (p<0.05). In the authentication of species markers 1.A, 4.1, 4.3, 5.8 and 5.5 amplified for P. guajava, allowing to discriminate against guava from others individuals morphologically similar.Introducción. El gran número de habitantes en el mundo que utilizan a las plantas medicinales como recurso para solucionar sus problemas de salud, atrae la atención de instancias gubernamentales e internacionales que establecen métodos de control de calidad para garantizar su seguridad y eficacia. Los documentos oficiales reconocen como pruebas de autenticidad, a las de identificación taxonómica y de compuestos químicos que son característicos de la especie; sin embargo, en años recientes se están empleando marcadores de ADN para la identificación de la especie y discriminación de adulterantes. Entre los marcadores de ADN se encuentran los del tipo RAPD, RFLP, AFLP, secuencia específica y microsatelital. La hoja de guayaba (Psidium guajava L.) ampliamente cultivada en México, se utiliza popularmente en el tratamiento de trastornos estomacales y estudios clínicos recientes demuestran su eficacia en el tratamiento del síndrome de colon irritable. Planteamiento del problema. Debido a la escasa información genética existente de P. guajava, el presente trabajo se enfocó en la obtención de marcadores genéticos que se relacionen con la acumulación de quercetina y permitan la autentificación de la especie, como una herramienta en el control de calidad en el desarrollo de fitofármacos. Hipótesis. Algunas variaciones en regiones del ADN entre individuos de P. guajava cultivados en diferentes estados de la República Mexicana están asociadas a la acumulación de flavonoides mientras que las regiones conservadas lo están con la identificación de la especie. Objetivos. a) Caracterizar las variaciones u homologías de secuencia en segmentos tipo RAPD provenientes del ADN genómico de P. guajava L.; b) Obtener la distribución de la frecuencia alélica de diferentes muestras de P. guajava L. a partir de la construcción de una biblioteca genómica de loci microsatélite de la clase dinucleótida. Materiales y Métodos. Retoños limpios se colectaron en 40 árboles en cuatro estados de la República Mexicana. El material vegetal se procesó para obtener por un lado un extracto metanólico hidrolizado para la cuantificación de quercetina por HPLC usando columnas RP-18 y un detector PDA, y por otro lado se obtuvo ADN genómico. En éste se amplificaron regiones aleatorias tipo RAPD con seis oligonucleótidos de 10-mer (Amersham Bioscience, UK), y por otra parte se fraccionó con enzimas de restricción de acuerdo a lo informado previamente. Los amplificados aleatorios RAPD se analizaron y correlacionaron con la acumulación de quercetina total, mientras que los fragmentos RFLP se hibridaron con sondas (GT)15 biotiniladas, para capturar fragmentos con secuencias dinucleótidas repetidas. Las bibliotecas tipo RAPD y microsatelitales, se clonaron, secuenciaron, analizaron y de acuerdo con esto se diseñaron oligonucleótidos específicos. Los marcadores de ADN seleccionados se utilizaron en la autentificación de la especie y discriminación con otras plantas morfológicamente semejantes. Resultados. Los estados seleccionados producen el 84% de la guayaba que se cultiva en México. Las muestras de Aguascalientes mostraron la mayor acumulación de quercetina total en primavera, pero en el estado de México se encontró un árbol con la mayor acumulación de quercetina total. Las otras muestras de los estados de Querétaro y Michoacán tuvieron valores inferiores. En el amplificado aleatorio RAPD se usaron seis oligonucleótidos para generar 91 amplificados con una homología entre 74 y 100% entre los individuos. El oligonucleótido 1 generó dos amplificados polimórficos de 560 y 610pb diferenciando los patrones de bandeo 1a, 1b y 1c; el 2 mostró un amplificado polimórfico de 370pb con lo que se diferenciaron los patrones 2a y 2b; el 3 dió un amplificado polimórfico de 690pb que distingue los patrones 3a y 3b; el 4 generó dos amplificados polimórficos de 460 y 480pb diferenciando los patrones de bandeo 4a, 4b y 4c, y los oligonucleótidos 5 y 6 mostraron un solo patrón de bandeo cada uno. El aumento en la acumulación de quercetina se correlaciona con la presencia de los patrones de bandeo 1a y 2a con 4c. De las bibliotecas se obtuvieron 36 secuencias; 5 de ellas con microsatélites dinucleótidos; una similar en un 91% a Arabidopsis thaliana y se logró diseñar oligonucleótidos específicos para 22 secuencias de P. guajava. Se establecieron las condiciones de amplificación para 11 secuencias usando 10 ng de ADN genómico y 1 mM MgCl2 o para 12 secuencias usando 5 ng de ADN genómico y 1.5 mM MgCl2. Cinco de las secuencias seleccionadas fueron exclusivas para P. guajava. Conclusiones. El marcador químico de quercetina, permitió identificar a los individuos del estado de Aguascalientes en primavera como los de mayor acumulación de quercetina. Los marcadores genéticos tipo RAPD 1a y 2a con 4c se correlacionan con un aumento de quercetina con un valor LoD de 9.498 (p<0.05). En la autentificación de la especie los marcadores 1.2A, 4.1, 4.3, 5.8 y 5.5 fueron específicos para P. guajava, permitiendo discriminar la guayaba de otros individuos taxonómicamente similares de la familia Mirtacea

Revisiting Pharmacokinetics and Pharmacogenetics of Methadone in Healthy Volunteers

Methadone acts as a μ opioid agonist, a serotonin and norepinephrine reuptake inhibitor, and a noncompetitive N-methyl-D-aspartate receptor antagonist. These actions altogether are responsible for its efficacy in the management of chronic pain. It is available as a racemic mixture of (R)- and (S)-methadone, both being stereoisomers responsible for its analgesic effect. Methadone elimination occurs mainly through metabolism in the liver by CYP3A4, CYP2B6, and CY2C19 and to a lesser extent by CYP2D6 and in the intestine by CYP3A4. The relative intestinal content of CYP2B6 and CY2C19 is unknown but it seems that CYP2B6 is not present at the intestine. CYP3A4, CYP2B6, and CYP2C19 convert methadone mainly into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine(EDDP). CYP2B6 and CYP2C19 are stereoselective to S- and R-enantiomer, respectively. The pharmacokinetic study carried out in healthy volunteers by our research group confirmed that MTD undergoes recirculation via gastric secretion and intestinal reabsorption and revealed that the drug is extensively metabolized in the liver but intestinal metabolism is not only relevant but also stereoselective. Polymorphisms of the CYP2B6 and CYP2C19 isoenzymes and their relationship with the pharmacokinetics of MTD were also assessed

Autoimmune Epilepsy: New Development and Future Directions

In recent years, there has been accumulating evidence to support an autoimmune etiology for some patients with drug-resistant seizures, typically in the context of an antibody-mediated encephalopathy; any seizure disorder that may be caused by pathogenic autoantibodies, are an example of autoimmune epilepsy. Autoimmunity is characterized by loss of immune tolerance that causes the destruction of cells and tissues. The largest complex histocompatibility system has had a strong association with autoimmune disease, although certain genes encoding cytokines and co-stimulatory molecules increase genetic susceptibility. In spite of having scientific advances in this research area, the conditions underlying mechanisms are unknown.Goal: this chapter aims to present in synthesized form, the genetic, immunological, and environmental factors role in the autoimmunity to epilepsy, as well as the therapeutic approach that has been used to control seizures, mainly where there is a suspected anti-neuronal-antibodies circulation. Methods: a review of the work achieved during the last years in patients with this condition provides information and experience in the diagnosis and treatment of this epilepsy type. For this, a systematic search of PUBMED is conducted using the search terms “autoimmune and epilepsy, auto antibodies and epilepsy, NMDA and epilepsy, AMPA and epilepsy, and GAD and epilepsy.” The list of identified articles was complemented by additional searches for relevant articles in the reference section of the publications captured by the initial search

The preventive effects of broccoli bioactives against cancer: Evidence from a validated rat glioma model

The aggressive and incurable diffuse gliomas constitute 80% of malignant brain tumors, and patients succumb to recurrent surgeries and drug resistance. Epidemiological research indicates that substantial consumption of fruits and vegetables diminishes the risk of developing this tumor type. Broccoli consumption has shown beneficial effects in both cancer and neurodegenerative diseases. These effects are partially attributed to the isothiocyanate sulforaphane (SFN), which can regulate the Keap1/Nrf2/ARE signaling pathway, stimulate detoxifying enzymes, and activate cellular antioxidant defense processes. This study employs a C6 rat glioma model to assess the chemoprotective potential of aqueous extracts from broccoli seeds, sprouts, and inflorescences, all rich in SFN, and pure SFN as positive control. The findings reveal that administering a dose of 100 mg/kg of broccoli sprout aqueous extract and 0.1 mg/kg of SFN to animals for 30 days before introducing 1 × 104 cells effectively halts tumor growth and progression. This study underscores the significance of exploring foods abundant in bioactive compounds, such as derivatives of broccoli, for potential preventive integration into daily diets. Using broccoli sprouts as a natural defense against cancer development might seem idealistic, yet this investigation establishes that administering this extract proves to be a valuable approach in designing strategies for glioma prevention. Although the findings stem from a rat glioma model, they offer promising insights for subsequent preclinical and clinical research endeavorsThis work was supported by the Health Research Coordination-IMSS through financing FIS/IMSS/PROT/G17-2/1725. NB is granted by a “Juan de la Cierva Incorporación”(IJC-2020-044496-I) post-doctoral contract funded by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033). This research was partially supported by Fundación Seneca - Murcia Regional Agency for Science and Technology (CARM), Project Reference N# 20855/PI/18Peer reviewe

Electroacupuncture Reduces Seizure Activity and Enhances GAD 67 and Glutamate Transporter Expression in Kainic Acid Induced Status Epilepticus in Infant Rats

Status epilepticus (SE) is one of the most significant complications in pediatric neurology. Clinical studies have shown positive effects of electroacupuncture (EA) as a therapeutic alternative in the control of partial seizures and secondary generalized clonic seizures. EA promotes the release of neurotransmitters such as GABA and some opioids. The present study aimed to evaluate the anticonvulsive and neuromodulatory effects of Shui Gou DM26 (SG_DM26) acupuncture point electrostimulation on the expression of the glutamate decarboxylase 67 (GAD67) enzyme and the glutamate transporter EAAC1 in an early SE model. At ten postnatal days (10-PD), male rats weighing 22&ndash;26 g were divided into 16 groups, including control and treatment groups: Simple stimulation, electrostimulation, anticonvulsant drug treatment, and combined treatment&mdash;electrostimulation and pentobarbital (PB). SE was induced with kainic acid (KA), and the following parameters were measured: Motor behavior, and expression of GAD67 and EAAC1. The results suggest an antiepileptic effect derived from SG DM26 point EA. The possible mechanism is most likely the increased production of the inhibitory neurotransmitter GABA, which is observed as an increase in the expression of both GAD67 and EAAC1, as well as the potential synergy between the neuromodulator effects of EA and PB

GABAERGIC ALTERATIONS IN NEOCORTEX OF PATIENTS WITH PHARMACORESISTANT TEMPORAL LOBE EPILEPSY CAN EXPLAIN THE COMORBIDITY OF ANXIETY AND DEPRESSION: THE POTENTIAL IMPACT OF CLINICAL FACTORS

Temporal lobe epilepsy (TLE) is a chronic neurodegenerative disease with a high prevalence of psychiatric disorders. Temporal neocortex contributes to either seizure propagation or generation in TLE, a situation that has been associated with alterations of the γ-aminobutyric acid (GABA) system. On the other hand, an impaired neurotransmission mediated by GABA in temporal neocortex has also been involved with the pathophysiology of psychiatric disorders. In spite of these situations, the role of the necortical GABA system in the comorbidity of TLE and mood disorders has not been investigated. The present study was designed to identify alterations in the GABA system such as: binding to GABAA and GABAB receptors and benzodiazepine site, the tissue content of GABA and the expression of the mRNA encoding the α1-6, β1-3 and γ GABAA subunits, in the temporal neocortex of surgically treated patients with TLE with and without anxiety and/or depression. Neocortex of patients with TLE and comorbid anxiety and/or depression showed increased expression of the mRNA encoding the γ2-subunit, reduced GABAB-induced G protein activation in spite of elevated GABAB binding, and lower tissue content of GABA when compared to autopsy controls. Some of these changes significantly correlated with seizure frequency and duration of epilepsy. The results obtained suggest a dysfunction of the GABAergic neurotransmission in temporal neocortex of patients with TLE and comorbid anxiety and/or depression that could be also influenced by clinical factors such as seizure frequency and duration of illness