2,3,7,8-Tetrachlorodibenzo-p-dioxin modifies alternative splicing in mouse liver.

Alternative splicing is a co-transcriptional mechanism that generates protein diversity by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigated whether a single po. dose of 5 µg/kg or 500 µg/kg TCDD influence hepatic alternative splicing in adult male C57BL/6Kou mouse. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that the exposure to TCDD has an impact on alternative-splicing, and suggest a new avenue for understanding TCDD-mediated toxicity and pathogenesis.

Human astroviruses: in silico analysis of the untranslated region and putative binding sites of cellular proteins.

Human astrovirus (HAstV) constitutes a major cause of acute gastroenteritis in children. The viral 5' and 3' untranslated regions (UTR) have been involved in the regulation of several molecular mechanisms. However, in astrovirues have been less characterized. Here, we analyzed the secondary structures of the 5' and 3' UTR of HAstV, as well as their putative target sites that might be recognized by cellular factors. To our knowledge, this is the first bioinformatic analysis that predicts the HAstV 5' UTR secondary structure. The analysis showed that both the UTR sequence and secondary structure are highly conserved in all HAstVs analyzed, suggesting their regulatory role of viral activities. Notably, the UTRs of HAstVs contain putative binding sites for the serine/arginine-rich factors SRSF2, SRSF5, SRSF6, SRSF3, and the multifunctional hnRNPE2 protein. More importantly, putative binding sites for PTB were localized in single-stranded RNA sequences, while hnRNPE2 sites were localized in double-stranded sequence of the HAstV 5' and 3' UTR structures. These analyses suggest that the combination of SRSF proteins, hnRNPE2 and PTB described here could be involved in the maintenance of the secondary structure of the HAstVs, possibly allowing the recruitment of the replication complex that selects and recruits viral RNA replication templates.

Life and Death of mRNA Molecules in Entamoeba histolytica.

In eukaryotic cells, the life cycle of mRNA molecules is modulated in response to environmental signals and cell-cell communication in order to support cellular homeostasis. Capping, splicing and polyadenylation in the nucleus lead to the formation of transcripts that are suitable for translation in cytoplasm, until mRNA decay occurs in P-bodies. Although pre-mRNA processing and degradation mechanisms have usually been studied separately, they occur simultaneously and in a coordinated manner through protein-protein interactions, maintaining the integrity of gene expression. In the past few years, the availability of the genome sequence of Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, coupled to the development of the so-called "omics" technologies provided new opportunities for the study of mRNA processing and turnover in this pathogen. Here, we review the current knowledge about the molecular basis for splicing, 3' end formation and mRNA degradation in amoeba, which suggest the conservation of events related to mRNA life throughout evolution. We also present the functional characterization of some key proteins and describe some interactions that indicate the relevance of cooperative regulatory events for gene expression in this human parasite.

Palmitic acid but not palmitoleic acid induces insulin resistance in a human endothelial cell line by decreasing SERCA pump expression.

Palmitic acid is a negative regulator of insulin activity. At the molecular level, palmitic acid reduces insulin stimulated Akt Ser473 phosphorylation. Interestingly, we have found that incubation with palmitic acid of human umbilical vein endothelial cells induced a biphasic effect, an initial transient elevation followed by a sustained reduction of SERCA pump protein levels. However, palmitic acid produced a sustained inhibition of SERCA pump ATPase activity. Insulin resistance state appeared before there was a significant reduction of SERCA2 expression. The mechanism by which palmitic acid impairs insulin signaling may involve endoplasmic reticulum stress, because this fatty acid induced activation of both PERK, an ER stress marker, and JNK, a kinase associated with insulin resistance. None of these effects were observed by incubating HUVEC-CS cells with palmitoleic acid. Importantly, SERCA2 overexpression decreased the palmitic acid-induced insulin resistance state. All these results suggest that SERCA pump might be the target of palmitic acid to induce the insulin resistance state in a human vascular endothelial cell line. Importantly, these data suggest that HUVEC-CS cells respond to palmitic acid-exposure with a compensatory overexpression of SERCA pump within the first hour, which eventually fades out and insulin resistance prevails.

Diagnosis and treatment of obesity among Mexican adults.

OBJECTIVES: To quantify the access to diagnosis and treatment of obesity and intentional weight loss among obese adults in Mexico and to identify the sociodemographic factors related to these events. METHODS: The 2006 Mexican National Health and Nutrition Survey - representative of the adults aged 20 to 64 years - was analyzed. Whether people had received diagnosis and treatment from health professionals and whether they had intentional weight loss were explored. The independent variables were: sex, age, socioeconomic position, locality size, and body weight perception. Analyses were carried out for obese people only (BMI ≥ 30 kg/m(2), N = 8,545). RESULTS: Among obese people, just 20.2% were diagnosed with such condition, only 8.0% undertook treatment, and barely 5.6% had lost weight intentionally. Individuals with a higher BMI, older individuals, people with higher education, those living in wealthier households, and those living in metropolitan areas were more likely to receive diagnosis and treatment for obesity. Women and people who had been diagnosed as obese were more likely to lose weight. CONCLUSION: There is an urgent need to increase access to diagnosis and treatment of obesity in Mexico, particularly for men and for lower socioeconomic groups.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.

Application of body mass index to schoolchildren of Mexico City.

BACKGROUND: The validity of body mass index (BMI)-for-age for obesity diagnosis in Latin-American children may be limited due to observed cases of overweight without obesity (i.e. body fat excess), possibly due to certain physical characteristics. In the current study, we investigated whether the usefulness of BMI-for-age in the diagnosis of obesity among Mexican schoolchildren is modified by height, trunk length, muscle mass, body frame, or waist circumference. METHODS: Our study cohort comprised 1,015 schoolchildren (aged 6-11 years) from Mexico City. Obesity diagnostics were derived from three classifications of BMI-for-age: percentiles of BMI according to the references of the Centers of Disease Control (CDC), the National Center for Health Statistics and the International Obesity Task Force. The area under the curve (AUC, through receiver-operating characteristic curves) and optimal cutoff points (by Youden index) of each classification were calculated. Body fat percentage, triceps skinfold thickness and blood pressure were used as standards. AUC and optimal cutoff point analysis were stratified according to height-for-age, sitting height, elbow breadth, arm muscle area (AMA) and waist circumference. RESULTS: For the general population, the CDC reference had the highest values of AUC (0.94 for triceps skinfold thickness and 0.96 for body fat percentage), and the optimal cutoff point was the 85th percentile. Among schoolchildren with large body frames (measured through elbow breadth) or with high muscle mass (assessed by AMA), the optimal cutoff point was the 95th percentile of the CDC reference. CONCLUSIONS: Our results suggest that the percentile cutoff to define obesity in children with high muscle mass or a large body frame should be the 95th percentile, while the 85th percentile can still be used for the other children.

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by Lactococcus lactis.

BACKGROUND: Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10. RESULTS: The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes. CONCLUSION: Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.