Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.

miR-381 Attenuates Peripheral Neuropathic Phenotype Caused by Overexpression of PMP22

Charcot-Marie Tooth disease type 1A (CMT1A), the major type of CMT, is caused by duplication of peripheral myelin protein 22 (PMP22) gene whose overexpression causes structural and functional abnormalities in myelination. We investigated whether miRNA-mediated regulation of PMP22 expression could reduce the expression level of PMP22, thereby alleviating the demyelinating neuropathic phenotype of CMT1A. We found that several miRNAs were down-regulated in C22 mouse, a CMT1A mouse model. Among them, miR-381 could target 3′ untranslated region (3′UTR) of PMP22 in vitro based on Western botting and quantitative Real Time-PCR (qRT-PCR) results. In vivo efficacy of miR-381 was assessed by administration of LV-miR-381, an miR-381 expressing lentiviral vector, into the sciatic nerve of C22 mice by a single injection at postnatal day 6 (p6). Administration of LV-miR-381 reduced expression level of PMP22 along with elevated level of miR-381 in the sciatic nerve. Rotarod performance analysis revealed that locomotor coordination of LV-miR-381 administered C22 mice was significantly enhanced from 8 weeks post administration. Electrophysiologically, increased motor nerve conduction velocity was observed in treated mice. Histologically, toluidine blue staining and electron microscopy revealed that structural abnormalities of myelination were improved in sciatic nerves of LV-miR-381 treated mice. Therefore, delivery of miR-381 ameliorated the phenotype of peripheral neuropathy in CMT1A mouse model by down-regulating PMP22 expression. These data suggest that miRNA can be used as a potent therapeutic strategy to control diseases with copy number variations such as CMT1A.

Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing / Caracterización de las regiones no traducidas ARN [UTR] de las isoformas de Trypanosoma cruzi LYT1 derivadas de un trans-empalme alternativo / Caracterização das regiões não-traduzidas do RNA [UTR] das isoformas de Trypanosoma cruzi LYT1 derivadas de uma junção trans-alternativa

Abstract In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of the mRNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYTl and kLYTl, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5' and 3' UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5' UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T. cruzi DTU. In addition, for the first time, was unscovered the existence of two types of LYT1 mRNAs transcripts, differing in length by 116 nts, that are generated by alternative polyadenylation. Furthermore, an in-silico analysis of the experimentally obtained UTRs, and ten additional LYT1 sequences retrieved from TritrypDB and GenBank databases, together with a thoroughly search of structural motifs, showed a remarkable conservation of relevant structural motifs previously associated with RNA metabolism in the different UTRs; these elements might be involved in the differential stage-specific expression of each LYT1 isoform.

Resumen En los trypanosomátidos, la expresión génica se regula principalmente en el nivel post-transcripcional mediante mecanismos basados en la interacción entre las proteínas de unión del ARN [RBP] y las figuras presentes en las regiones no traducidas [UTR] de las ARN, que en conjunto forman complejos ribonucleoproteicos [RNP] que definen el destino de la ARN. El pre-ARN derivado del gen LYT1 del Trypanosoma cruzi es procesado por trans-empalme alternativo, dando como resultado diferentes ARN que codifican las isoformas mLYTl y kLYTl, proteínas con expresión diferencial, localización celular y función. El objetivo de este estudio fue caracterizar los 5' y 3' UTR de las ARN LYT1 como el paso inicial hacia la identificación de los RPB responsables de la expresión diferencial. Se confirmó la presencia de los dos tipos de 5' UTR en dos aislantes del T. cruzi pertenecientes al DTU I; de esta forma también se comprobó la ocurrencia del trans-empalme alternativo en el gen LYT1 de este T. cruzi DTU. Además, por primera vez, se pudo demostrar la existencia de dos tipos de transcripciones de ARN LYT1, que difieren en longitud por 116 nts, y son generadas por poliadenilación alternativa. Adicionalmente, se realizó un análisis in-silico de la UTR obtenida experimentalmente, y otras diez secuencias LYT1 recuperadas de las bases de datos TritrypDB y GenBank, junto con una búsqueda exhaustiva de figuras estructuradas, mostrando una notable conservación de los figuras estructurales asociadas con el metabolismo del ARN en los diferentes UTR; estos elementos podrían estar implicados en la expresión diferenciada de la etapa específica de cada isoforma LYT1.

Resumo Nos tripanossomatídeos, a expressão génica é regulada principalmente a nível pós-transcricional mediante mecanismos baseados na interação entre as proteínas de união do RNA [RBPs] e as fugiras presentes nas regiões não-traduzidas [UTRs] do RNA. O pré-RNA derivado do gene LYT1 do Trypanosoma cruzí é processado por uma junção trans-alternativa, resultando em diferentes RNA que codificam as isoformas mLYTl e kLYTl, proteínas com expressão, localização celular e função diferenciadas. O objetivo de este estudo foi caracterizar as 5' e 3' UTRs dos RNAs LYT1 como sendo o passo inicial na identificação das RBPs responsáveis pela expressão diferenciada. A presença dos dois tipos de 5' UTRs foi confirmada em dois isolados de T. cruzí pertencentes ao DTU I; corroborando assim com a ocorrência da junção trans-alternativa no gene LYT1 de este T. crují DTU. Adicionalmente, se demonstrou pela primeira vez a existência de dois tipos de transcrições de RNA LYT1, que se diferenciam em comprimento por 116 nts, e são geradas por poliadenização alternativa. Além disso, realizou-se uma análise in-sílico da UTR obtida experimentalmente e outras dez sequencias LYT1 recuperadas das bases de dados TritrypDB e GenBank, junto com uma busca exaustiva de figuras estruturadas, mostrando uma notável conservação das figuras estruturais associadas com o metabolismo do RNA nas diferentes UTRs. Estes elementos poderiam estar envolvidos na expressão estágio-específica diferenciada de cada isoforma LYT1.

miR-195/miR-497 Regulate CD274 Expression of Immune Regulatory Ligands in Triple-Negative Breast Cancer / 한국유방암학회지

PURPOSE: Immune suppression is common in patients with advanced breast cancer but the mechanisms underlying this phenomenon have not been sufficiently studied. In this study, we aimed to identify B7 family members that were able to predict the immune status of patients, and which may serve as potential targets for the treatment of breast cancer. We also aimed to identify microRNAs that may regulate the expression of B7 family members. METHODS: The Cancer Genome Atlas data from 1,092 patients with breast cancer, including gene expression, microRNA expression and survival data, were used for statistical and survival analyses. Polymerase chain reaction and Western blot were used to measure messenger RNA and protein expression, respectively. Luciferase assay was used to investigate direct microRNA target. RESULTS: Bioinformatic analysis predicted that microRNA (miR)-93, miR-195, miR-497, and miR-340 are potential regulators of the immune evasion of breast cancer cells, and that they exert this function by targeting CD274, PDCD1LG2, and NCR3LG1. We chose CD274 for further investigations. We found that miR-195, miR-497, and CD274 expression levels were inversely correlated in MDA-MB-231 cells, and miR-195 and miR-497 expressions mimic inhibited CD274 expression in vitro. Mechanistic investigations demonstrated that miR-195 and miR-497 directly target CD274 3′ untranslated region. CONCLUSION: Our data indicated that the level of B7 family members can predict the prognosis of breast cancer patients, and miR-195/miR-497 regulate CD274 expression in triple negative breast cancer. This regulation may further influence tumor progression and the immune tolerance mechanism in breast cancer and may be able to predict the effect of immunotherapy on patients.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

miR-483-5p promotes prostate cancer cell proliferation and invasion by targeting RBM5

ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.

Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication

BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.

Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder

MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3’ untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5’-CAGUCG-3’) in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3’ UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study.

miR-30c regulates proliferation, apoptosis and differentiation via the Shh signaling pathway in P19 cells

MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that suppress protein expression by binding to the 3′ untranslated regions of their target genes. Many studies have shown that miRNAs have important roles in congenital heart diseases (CHDs) by regulating gene expression and signaling pathways. We previously found that miR-30c was highly expressed in the heart tissues of aborted embryos with ventricular septal defects. Therefore, this study aimed to explore the effects of miR-30c in CHDs. miR-30c was overexpressed or knocked down in P19 cells, a myocardial cell model that is widely used to study cardiogenesis. We found that miR-30c overexpression not only increased cell proliferation by promoting cell entry into S phase but also suppressed cell apoptosis. In addition, we found that miR-30c inhibited dimethyl sulfoxide-induced differentiation of P19 cells. miR-30c knockdown, in contrast, inhibited cell proliferation and increased apoptosis and differentiation. The Sonic hedgehog (Shh) signaling pathway is essential for normal embryonic development. Western blotting and luciferase assays revealed that Gli2, a transcriptional factor that has essential roles in the Shh signaling pathway, was a potential target gene of miR-30c. Ptch1, another important player in the Shh signaling pathway and a transcriptional target of Gli2, was downregulated by miR-30c overexpression and upregulated by miR-30c knockdown. Collectively, our study revealed that miR-30c suppressed P19 cell differentiation by inhibiting the Shh signaling pathway and altered the balance between cell proliferation and apoptosis, which may result in embryonic cardiac malfunctions.

Sequence Analysis of the UGT1A1 Gene Untranslated Region in Chinese Han Population / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the 5' and 3'-untranslated region sequences of the UGT1A1 gene in Chinese Han population and to find polymorphic variants within the untranslated region.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral leukocytes in 220 healthy Han individuals. The 5' and 3'-untranslated region sequences of the UGT1A1 gene were amplified by polymerase chain reaction, and followed by DNA sequencing.</p><p><b>RESULTS</b>Two polymorphic loci were identified in the 5'-untranslated region of the UGT1A1 gene with -64(G/C) and A(TA)6TAA/A(TA)7TAA in TATAA box region among Chinese Han population. Genotype frequencies were 98.4% (G) and 1.6% (C) in -64 locus of the UGT1A1 gene among the 220 individuals. The allele frequency of A(TA)6TAA and A(TA)7TAA within the promoter region was found to be 93.4% and 6.6%, respectively. Two polymorphic loci of 1813(C/T) and 1941(C/G) were detected in the 3'-untranslated region of the UGT1A1 gene, they showed a homozygous state at two loci with cosegregation pattern at 1813 and 1941 locus. The haplotype frequencies were 73.6% (CC/1813+CC/1941) and 26.4% (TT/1813+GG/1941) for 1813 and 1941 loci in the UGT1A1 gene.</p><p><b>CONCLUTION</b>Cosegregation pattern, at 1813 and 1941 locus with homozygous state in the 3'-untranslated region of the UGT1A1 gene may be selected from the human genome among Chinese Han population. More studies should be focused on the mechanism of homozygous cosegregation.</p>

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

Genetic Analysis of SCN5A in Korean Patients Associated with Atrioventricular Conduction Block

Recent several studies have shown that the genetic variation of SCN5A is related with atrioventricular conduction block (AVB); no study has yet been published in Koreans. Therefore, to determine the AVB-associated genetic variation in Korean patients, we investigated the genetic variation of SCN5A in Korean patients with AVB and compared with normal control subjects. We enrolled 113 patients with AVB and 80 normal controls with no cardiac symptoms. DNA was isolated from the peripheral blood, and all exons (exon 2-exon 28) except the untranslated region and exon-intron boundaries of the SCN5A gene were amplified by multiplex PCR and directly sequenced using an ABI PRISM 3100 Genetic Analyzer. When a variation was discovered in genomic DNA from AVB patients, we confirmed whether the same variation existed in the control genomic DNA. In the present study, a total of 7 genetic variations were detected in 113 AVB patients. Of the 7 variations, 5 (G87A-A29A, intervening sequence 9-3C>A, A1673G-H558R, G3578A-R1193Q, and T5457C-D1819D) have been reported in previous studies, and 2 (C48G-F16L and G3048A-T1016T) were novel variations that have not been reported. The 2 newly discovered variations were not found in the 80 normal controls. In addition, G298S, G514C, P1008S, G1406R, and D1595N, identified in other ethnic populations, were not detected in this study. We found 2 novel genetic variations in the SCN5A gene in Korean patients with AVB. However, further functional study might be needed.

Genetic Analysis of SCN5A in Korean Patients Associated with Atrioventricular Conduction Block

Recent several studies have shown that the genetic variation of SCN5A is related with atrioventricular conduction block (AVB); no study has yet been published in Koreans. Therefore, to determine the AVB-associated genetic variation in Korean patients, we investigated the genetic variation of SCN5A in Korean patients with AVB and compared with normal control subjects. We enrolled 113 patients with AVB and 80 normal controls with no cardiac symptoms. DNA was isolated from the peripheral blood, and all exons (exon 2-exon 28) except the untranslated region and exon-intron boundaries of the SCN5A gene were amplified by multiplex PCR and directly sequenced using an ABI PRISM 3100 Genetic Analyzer. When a variation was discovered in genomic DNA from AVB patients, we confirmed whether the same variation existed in the control genomic DNA. In the present study, a total of 7 genetic variations were detected in 113 AVB patients. Of the 7 variations, 5 (G87A-A29A, intervening sequence 9-3C>A, A1673G-H558R, G3578A-R1193Q, and T5457C-D1819D) have been reported in previous studies, and 2 (C48G-F16L and G3048A-T1016T) were novel variations that have not been reported. The 2 newly discovered variations were not found in the 80 normal controls. In addition, G298S, G514C, P1008S, G1406R, and D1595N, identified in other ethnic populations, were not detected in this study. We found 2 novel genetic variations in the SCN5A gene in Korean patients with AVB. However, further functional study might be needed.

Expression of YB-1 enhances production of murine leukemia virus vectors by stabilizing genomic viral RNA

Murine leukemia virus (MLV)-based retroviral vectors is widely used for gene transfer and basic research, and production of high-titer retroviral vectors is very important. Here we report that expression of the Y-box binding protein 1 (YB-1) enhanced the production of infectious MLV vectors. YB-1 specifically increased the stability of viral genomic RNA in virus-producing cells, and thus increasing viral RNA levels in both producer cells and virion particles. The viral element responsive to YB-1 was mapped to the repeat sequence (R region) in MLV genomic RNA. These results identified YB-1 as a MLV mRNA stabilizer, which can be used for improving production of MLV vectors.

Estudios de la región 5'UTR del gen FMR-1 en pacientes con falla ovárica prematura / Studies of the 5' - UTRTR region in the FMR-1 gene in patients withe Premature Ovarian Failure

La falla ovárica prematura (FOP) es un síndrome de patogénesis multicausal que afecta aproximadamente al 1% de las mujeres en edad reproductiva. Numerosos estudios asocian el estado de premutación (amplificación del número de tripletes CGG entre 50/55 y 200 repeticiones) en el gen FMR-1 y FOP. Alrededor de un 4% de las pacientes FOP presentan alelos con premutación. La amplificación del número de tripletes por encima de 200 repeticiones causa el Síndrome de Fragilidad del X (SFX). El objetivo del presente trabajo fue estudiar la región 5´ no codificante del gen en un grupo de pacientes FOP de Argentina. La región de interés se amplificó por PCR a partir de muestras de ADN de 100 pacientes FOP y 145 mujeres controles. Los alelos de las pacientes y controles fueron agrupados en 7 categorías de acuerdo al número de tripletes obtenidos. Se observó que el número de repeticiones más frecuente se encuentra en el rango de 26 a 30 tripletes, tanto en pacientes como en controles. En el grupo de pacientes FOP, 5/197 (2.6%) alelos no relacionados estudiados presentaron un número de tripletes CGG mayor a 50, mientras que sólo 1 de 290 (0.34%) para el grupo control. Todas las pacientes FOP con valores de tripletes CGG mayor a 50 presentaron amenorrea secundaria. Estos resultados están en concordancia con lo comunicado para otras poblaciones acerca de la existencia de una asociación entre la premutación del gen FMR-1 y el desarrollo de FOP. Asimismo, los resultados obtenidos refuerzan la importancia de la genotipificación del gen FMR-1 en las pacientes FOP, a los efectos de estimar el riesgo de su descendencia para el SFX.

Premature ovarian failure (POF) is a syndrome of multicausal pathogenesis that affects 1% of women before the age of 40. Several studies associate the premutation state (CGG repeats increased in number between 50/55 and 200) in the FMR-1 gene and POF. About 4% of POF women have alleles in the FMR-1 gene in the permutation range. An increase above 200 in the number of triplets in this gene causes the Fragile X Syndrome (FXS). The purpose of the present study was to analyze the 5´untranslated region of the FMR-1 gene in a group of patients from Argentina. The region of interest was amplified by PCR from DNA samples of 100 POF patients and 145 control women. Alleles from controls and patients were grouped in 7 categories according to the number of triplets obtained. We observed that the most frequent number of repeats ranged from 26 to 30 triplets, in both patient and control groups. In the POF group, 5 out of 197 (2.6%) not related alleles presented a number of CGG triplets higher than 50, while only 1 out of 290 (0.34%) was present in controls. All POF patients with a number of CGG repeats higher than 50 presented secondary amenorrhea. These results are in accordance with previous reports from other populations showing an association between the premutation state in the FMR-1 gene and POF development. In addition, these results reinforce the importance of genotyping POF patients to estimate the risk of their offspring for Fragile X Syndrome.

Functional genomics of Salvia militiorrhiza IV--analysis of ethylene responsive element binding protein gene / 中国中药杂志

<p><b>OBJECTIVE</b>To study the ethylene responsive element binding protein genes of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression in regenerated plantlets.</p><p><b>METHOD</b>The ethylene responsive element binding protein genes were obtained by cDNA microarray analyze. BLAST was used for alignment, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT- PCR method was used to detect the gene expression level.</p><p><b>RESULT</b>One ethylene responsive element binding protein was obtained, named as SmERF. SmERF had an open reading frame of 699 bp with 5'-URT 87 bp and 3'-URT 166 bp. The putative protein SmERF contains a highly conserved ERF/AP2 domain. Semiquantitative RT- PCR illustrated that SmERF was expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in root than in stem and leaf.</p><p><b>CONCLUSION</b>It was the first time to obtain ERF gene in S. miltiorrhiza and set a good foundation for its further functional study.</p>

Torque teno virus (TTV) is prevalent in Brazilian non-human primates and chickens (Gallus gallus domesticus) / Torque teno virus (TTV) es prevalente en primates no humanos brasileños y pollos (Gallus gallus domesticus)

Torque Teno virus (TTV) is an infectious agent of worldwide distribution isolated by the first time as the agent of an acute post-transfusion hepatitis in a patient in Japan. It has been classified into a new floating genus called Anellovirus. Recent studies showed that TTV can also be identified in serum specimens obtained from domesticated farm animals and from non-human primates. To better understand the relationship between TTV and their hosts, a study to detect virus in the serum and whole blood of Brazilian non-human primates and in the plasm of chickens was performed by applying the PCR-UTR-A technique, followed by a genomic sequence and phylogenetic analysis. By nested-PCR-UTR, the DNA of TTV was detected in sera from 4 (5.3 percent) of 75 Cebus apella, 2 (40 percent) of 5 Alouatafusca, 1 (20 percent) of 5 Alouata caraya, 1 (5.2 percent) of 19 Callithrixpenicilata, 1 (4 percent) of 25 Callithrixjacchus, 1 (20 percent) of 5 Saimiri sciureus and 1 (25 percent) of 4 Leontopithecus chrysomelas. Phylogenetic analysis revealed that sequences detected in 8 samples clustered with TTV sequences So-TTV2 (Sagüínus oedipus) and At-TTV3 (Aotes Trivirgatus). Three sequences showed similarity with a human Torque Teno Minivirus (TLMV). TTV ORF2 DNA was detected in one sera sample and one whole blood sample of non-human primates and in one plasm sample of chicken. Phylogenetic analysis revealed that the sequences amplified by the ORF2 region show no difference between human, non-human primates and chicken. This is the first report of TTV in Brazilian new world non-human primates and chicken.

Torque Teno virus (TTV) es una agente infeccioso de distribución mundial, aislado por primera vez como el agente de una hepatitis aguda posterior a la transfusión de un paciente en Japón. Se ha clasificado en un nuevo género flotante llamado Anellovirus. Recientes estudios han demostrado que TTV también puede ser identificado en el suero de especímenes obtenidos desde granjas de animales domésticos y desde primates no humanos. Para entender mejor la relación entre la TTV y sus huéspedes, fue realizado un estudio para detectar el virus en el suero y la sangre de primates no humanos brasileños y en el plasma de pollos mediante la aplicación de la técnica PCR-UTR-A, seguida de una secuencia genómica y análisis filogenético. Por medio de PCR-UTR-anidado, el ADN de TTV fue detectado en sueros de 4 de 75 (5,3 por ciento)Cebus apella, 2 de 5 (40 por ciento) Alouata fusca, 1 de 5 (20 por ciento) de Alouata caraya, 1 de 19 (5,2 por ciento) de Callithrixpenicilata, 1 de 25 (4 por ciento) Callithrixjacchus, 1 de 5 (20 por ciento) de Saimiri sciureus y 1 de 4 (25 por ciento) de Leontopithecus chrysomelas. El análisis filogenético reveló secuencias detectadas en 8 muestras agrupadas con TTV secuencias So-TTV2 (Sagüínus oedipus) y At-TTV3 (Aotes Trivirgatus). Tres secuencias mostraron similitud con el Torque Teno Minivirus humano (TLMV). Fue detectado TTV ORF2 ADN en una muestra de suero y una muestra de sangre de primates no-humanos y en una muestra de plasma de pollo. El análisis filogenético reveló que las secuencias amplificadas por la región ORF2 no muestran ninguna diferencia entre humanos, primates no humanos y pollos. Este es el primer informe de nuevos TTV en primates-no humanos brasileños y en pollos.

Overexpression of protein tyrosine phosphatase IB in HepG2 cells ameliorates insulin-mediated suppression of apolipoprotein B mRNA translation via its untranslated regions

The hepatic secretion of apolipoprotein B [apoB], containing lipoproteins, is known to be regulated by insulin, and the overproduction of these atherogenic lipoproteins occurs in insulin-resistant states. Protein tyrosine phosphatase 1B [PTP-1B] is a key regulator of hepatic insulin signaling and is also upregulated in insulin resistance. We aimed to investigate the role of PTP-1B in regulating apoB mRNA translational efficiency mediated by 5'/3' untranslated regions [UTRs] under conditions of insulin stimulation. Human hepatoma HepG2 cells were transfected with a vector carrying the firefly luciferase reporter gene and either a chimeric apoB mRNA encoding the 5'/3' untranslated region [5' LUC3' -pGL3] or a null sequence of length equivalent to apoB 5' UTR [LUC-pGL3]. The transfected cells were then infected with adenovirus carrying the mouse PTP-1B gene [AdPTP1B] in the absence or presence of insulin, and the cellular luciferase activity was determined. The RNA extracts from cells were transfected with constructs carrying 5'/3' apoB UTR, or a null sequence was also translated in vitro in a rabbit reticulocyte translation system. The luciferase activity of the cells transfected with constructs containing the apoB UTR sequences [5' LUC3'] was significantly higher than that of the control constructs carrying a null sequence [p<0.01, n=12]. Similar results were observed following in vitro translation studies showing a significantly higher expression of the 5'/3' UTR constructs [p<0.001, n=6]. Treatment with 100 nM insulin led to a significant reduction in the luciferase activity of the constructs carrying apoB 5'/3' UTR [p<0.0001, n=12]. The down regulation of the apoB mRNA translation mediated by insulin was mediated by the apoB 5'/3' UTR sequences since insulin did not affect the control constructs containing a null sequence. The infection of HepG2 cells expressing 5' LUC3' or control constructs with AdPTP-1B attenuated the inhibitory effect of insulin and led to higher levels of luciferase activity compared to the Ad beta-gal infected control cells [p<0.05, n=12]. However, the activity was lower than that in the control cells infected with 5' LUC3' -pGL3 but not treated with insulin [p<0.05, n=12]. Our data suggest that PTP-1B can potentially modulate apoB synthesis by blocking insulin-mediated inhibition of the apoB mRNA translation via its 5'/3' UTR sequences. We hypothesize that the PTP-1B-mediated attenuation of the insulin action can lead to the upregulation of the apoB mRNA translation and contribute to a lipoprotein overproduction in conditions such as insulin resistance

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.