Expresión del locus GH en el ojo de primates superiores

Introducción: La GH es una hormona que regula el crecimiento y el metabolismo en vertebrados. En humanos el locus de la hormona de crecimiento (GH) se encuentra en el cromosoma 17, y está conformado por cinco genes: dos de tipo GH (GH-1 y GH-2) y tres de tipo hormona somatomamotropina corónica (CSH-L, CSH-1 y CSH-2). El gen GH-1 se expresa principalmente en las células somatotrofas de la pituitaria anterior, mientras que el resto de los genes del locus se expresan en el sincitiotrofoblasto de la placenta. Recientemente se ha reportado la expresión de GH en numerosos tejidos extrapituitarios, siendo uno de ellos el ojo, lo que apunta a un rol más complejo de la hormona y a posibles nuevas funciones. Por lo que comprender la biología molecular de esta hormona en el ojo ayudará a dilucidar su función. Objetivo: Investigar la biología molecular del locus de la GH en el ojo humano y del babuino (Papio hamadryas). Materiales: Se recolectaron muestras de tejidos oculares humanos (152 tejidos frescos y 209 tejidos embebidos en parafina) de pacientes que por indicación médica y/o traumatismos se les retiró el tejido, con su respectiva firma de carta de consentimiento informado. También se recolectaron muestras de tejidos oculares de babuinos (6 ojos completos para extracción de ARN y de proteínas; y 3 ojos completos embebidos en parafina). Metodología: Los tejidos oculares, tanto de babuinos como de humanos que se almacenaron en RNA later®, se utilizaron para ensayos de tiempo real. Los tejidos almacenados en parafina se usaron para los ensayos de inmunofluorescencia, en donde se analizaron los siguientes genes y proteínas respectivamente: GH-1, GH-2, CSH-1, CSH-2, GHR, Pit-1, SST, SSTR-1, SSTR-2, SSTR-3, SSTR-4, SSTR-5, GHRH y GHRHR. También se realizaron ensayos de proteómica para analizar en muestras de vítreo de babuino las distintas isoformas o cambios postraduccionales de la GH. Resultados y Discusión: Del locus GH sólo se detectó la expresión del gen GH-1, tanto en muestras oculares de humanos como de babuinos. La falta de expresión ocular del resto de los genes del locus en primates es un hallazgo inédito. Este resultado no es una sorpresa, ya que se ha reportado que la mayoría de los tejidos extrapituitarios sólo expresan el gen GH-1. El ARNm del gen GH-1 en el humano no sólo se encontró en retina, como previamente ya se había reportado, sino también se encontró en coroides y trabéculo. En el babuino se encontró en retina-coroides. Por medio de ensayos de inmunofluorescencia la GH-1, se encontró en células ganglionares de retina y en las demás estirpes celulares de este tejido tanto en humanos como en babuinos. Estos hallazgos apuntan a que la GH pudiera tener otras funciones además de la función neuroprotectora propuesta para las células RGC. Tanto el ARNm como la proteína de GHR se encontraron en córnea, coroides y retina de humanos y babuinos. Un descubrimiento importante es la identificación de GHR en las células basales del epitelio corneal. Por lo que nosotros proponemos que probablemente la GH participe como un factor de proliferación celular en córnea. Al encontrarse co-expresión de GHR y de GH-1 se postula acción autocrina, y al encontrarse en ciertos tejidos oculares sólo la expresión de GHR también se postula acción paracrina. Ninguno de los tejidos oculares expresó Pit-1, por lo que la regulación de la expresión de la GH ocular es independiente de Pit-1. Tanto el ARNm y las proteínas de SST, sus receptores (SSTR1-SSTR5), GHRH y su receptor GHRHR, se localizaron principalmente en retina y coroides de humanos y babuinos. En base a lo anterior se propone que estos dos neurotransmisores podrían estar involucrados en la regulación de la GH ocular en primates.Por medio de ensayos de proteómica LC/FT-MS/MS en muestras de vítreos de babuinos se encontró solo la presencia de la GH-1 de 22 kD. Por lo que se descarta en este primate la presencia de isoformas y/o otros miembros del locus GH. Conclusión: De los cinco genes que componen el locus de la GH humana, sólo se detectó la expresión de GH-1 en los distintos tejidos oculares. La GH no sólo se encontró en retina en células ganglionares sino también en las demás estirpes celulares de este tejido, tanto en humanos como en babuinos. Lo anterior apunta a que la GH puede tener otras funciones además de la función neuroprotectora en células ganglionares. En primates, la GH probablemente actúa de manera autocrina y paracrina, al estar presente su receptor en varios de los tejidos de este órgano. La expresión del gen de la GH-1 en el ojo parece ser independiente del factor transcripcional hipofisiario Pit-1. De entre los posibles candidatos a regularle están los neurotransmisores GHRH y SST. Aunque aún falta mucho por entender sobre el papel de la GH en el ojo, dados los hallazgos, se postula para ésta una función neuroprotectora en retina, mientras que en el caso de córnea posiblemente se trate de regeneración celular

Biobanks: Experience of the School of Medicine and the “Dr. José Eleuterio González” University Hospital of the Universidad Autónoma de Nuevo León

Medical research has greatly beneited from molecular biology and increasingly relies on tools from the “omics” disciplines (mainly genomics, transcriptomics, proteomics and metabolomics). The availability of biological samples preserved with high quality standards is a sine qua non condition for such studies and their repositories are referred to as biobanks. Biobanks support the transportation, storage, preservation, and initial pathological and analytical examinations of biospecimens, as well as the protection of relevant information and the comparison of clinical and laboratory findings. A biobank facility is one of the most valuable tools the academic medicine organizations can offer to their researchers to improve the competitiveness of their current and future medical research. it acts as an essential bridge and an effective catalyst for research synergies between basic and clinical sciences, and it can be potentiated with efforts to raise funds for acquiring and maintaining cutting-edge analytical infrastructure to better serve its clinical, pharmaceutical and biotech clients

DPYD pathogenic variants associated with fluoropyrimidines toxicity

Background: Genetic variants in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism. DPYD contributes to the development of severe FPs-related toxicity, and pathogenic DPYD variants detection reduces side effects and complications associated with FP-toxicity. The allelic frequency of these variants in the Mexican population is currently unknown. Methods: The study was carried out at the Centro Universitario Contra el Cáncer (CUCC) of the Universidad Autónoma de Nuevo León (UANL) in Monterrey México. Genomic DNA was isolated from 154 subjects using the QIAamp DNA Blood Midi kit (QIAGEN) following the manufacturer\u27s recommendations. We analyze the variants c.1156G-\u3eT, c.2846A-\u3eT, and c.1129-5923C-\u3eG by qPCR using predesigned probes. For the remaining genomic variants (c.1905+1G-\u3eA, c.1679T-\u3eG, c.1898delC and c.299_302delTCAT), we design sequencing oligos using the software Oligo Primer v.7®. The allele frequency was calculated for each variant. Results: We analyzed a total of 154 samples to detect the seven variants analyzed. So far, only 2 samples have been found that presented the variant c.1129-5923C\u3eG in a state of heterozygosis, representing 1.2987% of the total of our population. Conclusions: The allele frequency for the variant c.1129-5923C-\u3eG was higher than reported in other populations. So this allele is more common in our population, which could attribute to the large percentage of side effects in our patients. However, more studies and increasing the number of samples are needed to establish DPYD the allele frequency more precisely

Risk Association of TOX3 and MMP7 Gene Polymorphisms with Sporadic Breast Cancer in Mexican Women

Breast cancer (BC) has one of the highest incidences and mortality worldwide. Single nucleotide polymorphisms (SNPs) in TOX3 rs3803662 and MMP7 rs1943779 have been associated with susceptibility to BC. In this case-control study, we evaluated the association of rs3803662 (TOX3)/rs1943779 (MMP7) SNPs with clinical features, immunohistochemical reactivity, and risk association with BC in women from northeastern Mexico. We compared 212 BC cases and 212 controls. DNA was isolated from peripheral blood to perform the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. We calculated genotype frequencies, odds ratios, and 95% confidence intervals. We found that CT (Cytocine-Thymine) and TT (Thymine -Thymine) genotypes, and T alleles of TOX3 rs3803662, were associated with BC risk (p = 0.034, p = 0.011, respectively). SNP TOX3 rs3803662 was associated with positive progesterone receptors (PR) and triple-negative BC (TNBC) but not with estrogen receptor (ER) or HER2 reactivity. CT and TT genotypes (p = 0.006) and T alleles (p = 0.002) of SNP MMP7 rs1943779 were associated with risk of BC. We found that T alleles of TOX3 rs3803662 and MMP7 rs1943779 SNPs are associated with BC risk. These findings contribute to personalized medicine in Mexican women

Olfactomedin‑like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon)

Background: The olfactomedin‑like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. Objective: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. Methods: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT‑PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. Results: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human’s orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Conclusions: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high simi‑ larity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons

Abstract. The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney‑specific member of the aldo‑keto reductase family. MIOX catalyzes the first reaction involved in the myo‑inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo‑Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo‑Inositol is achieved by utilizing the Fe2+/Fe3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription‑polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces. Key words: animal models, gene expression, kidney, myo-inositol oxygenase, Old World monke

Prolactin Expression in the Baboon (Papio hamadryas) Eye

Prolactin (PRL) is a hormone expressed in lactotrophs cells of the pituitary gland in primates. Extra pituitary expression of PRL has been reported, including the eye; however, expression in the developing eye of primates is limited. The aim of the study was determining the expression of PRL and PRL receptor (PRLR) (mRNAs and proteins) in adult and fetal baboon (Papio hamadryas) ocular tissues. Methods: We analyzed PRL and PRLR in baboon eyes tissues by immunofluorescence. The mRNAs of PRL and PRLR were detected by RT-PCR, cDNA was cloned, and sequenced. Furthermore, we performed a phylogenetic analysis to identify the evolutionary forces that underlie the divergence of PRL and PRLR primate genes. Results: We observed the expression of PRL and PRLR (mRNAs and proteins) in all retinal cell lineages of fetal and adult baboon. PRL and PRLR fit the hypothesis of evolutionary purifying gene selection. Conclusions: mRNA and protein of PRL and PRLR are expressed in fetal and adult baboon retinal tissue. PRL may trigger autocrine and paracrine-specific actions in retinal cell lines