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The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58) is a molecular chaperone member of the endoplasmic reticulum (ER) stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency). We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a recently discovered adipocytokine mainly secreted from visceral adipose tissue, which plays a main role in insulin sensitivity. In this study, we have investigated the regulation of vaspin gene expression in rat white adipose tissue (WAT) in different physiological (nutritional status, pregnancy, age and gender) and pathophysiological (gonadectomy, thyroid status and growth hormone deficiency) settings known to be associated with energy homeostasis and alterations in insulin sensitivity. We have determined vaspin gene expression by real-time PCR. Vaspin was decreased after fasting and its levels were partially recovered after leptin treatment. Chronic treatment with metformin increased vaspin gene expression. Vaspin mRNA expression reached the highest peak at 45 days in both sexes after birth and its expression was higher in females than males, but its levels did not change throughout pregnancy. Finally, decreased levels of growth hormone and thyroid hormones suppressed vaspin expression. These findings suggest that WAT vaspin mRNA expression is regulated by nutritional status, and leptin seems to be the nutrient signal responsible for those changes. Vaspin is influenced by age and gender, and its expression is increased after treatment with insulin sensitizers. Finally, alterations in pituitary functions modify vaspin levels. Understanding the molecular mechanisms regulating vaspin will provide new insights into the pathogenesis of the metabolic syndrome.

Objective : The purpose of the presera study was to devélop a molecular genotyping method test by using a real time PCR hybridization probé and applying it to the analysis of C1843T mutations of the Sus scrofa RYR1 gene. Animáis population Three PSS-susceptible and PSS non-susceptible crossbred swine races were used for the experiments: Pietrain X Landrace Belga, Pietrain X Large White and Pietrain X Duroc. Methods: We have devéloped a genotyping method by using a hybridization probé and applied it to the analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results obtained by hybridization probé strategies were confirmed by restriction analysis and sequencing. In addi-tion, phenotype/genotype correlation analyses were devéloped by using the in vitro contracture test and confirmed the in vivo hálothane-succinylcholine challenge. Results: The real-time PCR with fluorescent hybridization probé methodology was designed to identify ho-mozygous PSS-resistant, PSS-susceptible animáis as well as heterozygous carriers. All cases genotyped by fluorescent hybridization probes were in agreement with PCR restriction enzyme digestión and sequencing and showed a 100% concordance between the in vivo and in vitro porcine stress syndrome (PSS) susceptibility results. Conclusions and clinical relevance: The real-time PCR with fluorescent hybridization probé method described here provides a rapid, easily interpretable and réliáble tool for genotyping the C1843T (Arg615-Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of PSS-susceptibility and genetic selection.

Increased efforts have been made during the last few decades to develop new technologies capable of identifying and quantifying the expression proteome in different cellular systems in physiological and physiopathological conditions for determining illness biomarkers, pharmaceutical targets and/or posttranslational modifications (PTM) by means of proteomic techniques. 2D gel electrophoresis, with immobilized pH gradients, associated with mass spectrometry, is one of the fundamentals steps in studying proteomics. The 2D technique can be used in studying the quantitative expression of protein profiles according to iso-electric point (Ip), molecular weight (Mr), protein solubility and the relative abundance of the above. This methodology provides a protein profile reflecting changes in protein expression levels, isoforms and PTM. Proteins can be classified into those known by their structure and function, those recognized by determined domains and about which there is some knowledge, and those whose function is still not known. Proteomics is defined as the large-scale study of proteins expressed for a specific tissue from a genome, (global proteomics) or differentially expressed proteins (differential proteomics). Determining differentially expressed proteins, or proteins suffering a change in physiological circumstances, is the clue to understanding such pathology’s cellular mechanisms. Although an expressed gene in specific tissues (as an answer to biologic alterations) could be analyzed by a mRNA expression study (transcriptomics), these results do not always coincide with the expected expression profiles since the number and activity of proteins associated with the same regulation in different stages could be modified. Genomic data integration is required, as well as transcritomics

Amylin (islet amyloid polypeptide) and vaspin (visceral adipose tissue specific serpin) are gut and adipocyte hormones involved in the regulation of body weight homeostasis. The aim of this study was to examine whether amylin and vaspin are expressed in human and rat placenta, as well as their regulation by nutritional status. Our results demonstrate that amylin and vaspin are localized in both human and rat placenta. In the rat term placenta vaspin was demonstrated in the trophoblast of the fetal villi, the labyrinth. Vaspin immunostaining in human placenta was localized in cytotrophoblast and syncytiotrophoblast in the first trimester placentas while in the third trimester vaspin was localized in the syncytiotrophoblast. Regarding amylin, rat placenta of 16 days of gestational age showed an intense immunostaining, mainly localized in the labyrinth. On the other hand, in the human third trimester placenta amylin immunoreactivity was intense in the syncytiotrophoblast of the chorionic villi and in decidual cells. Furthermore, placental amylin and vaspin showed an opposite pattern of expression during pregnancy, with vaspin showing the highest expression level at the end and amylin at the beginning of pregnancy. Finally, food restriction also has contrary effects on their expression, increasing vaspin but decreasing amylin placental mRNA and protein levels. Taken together, our results demonstrate that vaspin and amylin are modulated by energy status in the placenta, which suggests that these proteins may be involved in the regulation of placental metabolic functions.

Pulmonary arterial hypertension (PAH) is the major complication of systemic sclerosis (SSc) and the main cause of morbi-mortality. It is important to find predictors for this vascular problem. The objective of this study was to determine the serum levels of different biomarkers in patients with SSc and secondary PAH and to compare them with those of healthy control subjects to define their potential role as predictors of PAH. Cross-section study in which 20 patients with SSc were included. PAH was diagnosed by echocardiogram. The optical densities of endoglin (Eng), endothelin-1 (ET-1), platelet-derived growth factor (PDGF), tumoral necrosis factor alpha (TNF-alpha), Transforming growth factor beta 2 (TGF-beta2) and Interleukin 8 (IL-8) were measured in 20 patients with SSc and 20 healthy controls matched by sex. The differences found between the group of patients with PAH and the control group were (mean or median and range): ET-1 (0.20; 0.10-0.35 vs. 0.16; 0.10-0.24; P = 0.0276), IL-8 (195.7; 45.5-504 vs. 118.9; 23-299.5; P = 0.0364), TNF-alpha (0.70; 0.50-0.96 vs. 0.48; 0.38-0.65; P = 1 x 10(-8)) and Eng (0.95; 0.57-1.72 vs. 0.75; 0.57-0.89; P = 0.0028). A correlation was found between the progression of the disease and the development of Raynaud's phenomenon (Rho: 0.67 and P = 0.0011), ET-1 and Eng (Rho: 0.53 and P = 0.0196), and between IL-8 and Eng (Rho: 0.68 and P = 0.0019). In conclusions, the elevation of the serum levels of Eng and ET-1 could represent a useful tool as PAH biomarkers. Nevertheless, the diagnostic value of these markers needs to be determined by prospective studies.

Background Neuropeptide Y (NPY), agouti related peptide (AgRP), cocaine and amphetamine-regulated transcript (CART) and melanocortins, the products of the proopiomelanocortin (POMC), are hypothalamic peptides involved in feeding regulation and energy homeostasis. Recent evidence has demonstrated their expression in rat and human placenta. Methods In the current study, we have investigated the expression of those neuropeptides in the rat placenta by real-time PCR using a model of maternal food restriction. Results Our results showed that placental-derived neuropeptides were regulated through pregnancy and following food restriction. Conclusion These data could indicate that placental-derived neuropeptides represent a local regulatory circuit that may fine-tune control of energy balance during pregnancy.

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.

Among the four parasite species causing malaria in humans, Plasmodium vivax prevails on both the Asian and the American continents. Several antigens from this parasite’s erythrocytic stages have been characterised and some of them are considered to be good vaccine candidates. The P. vivax merozoite surface protein-1 (PvMSP-1) is a 200 kDa antigen, thought to mediate the initial contact between the merozoite and the erythrocyte. An effective blockage of this interaction could be important in anti-malarial vaccine design. This study analyses the genetic polymorphism, binding to both reticulocytes and erythrocytes, antigenicity and immunogenicity of two recombinant proteins belonging to the 33 kDa PvMSP-1 proteolytic fragment. Both regions showed very low genetic variation, bound reticulocytes with higher affinity than erythrocytes, were recognised by naturally P. vivax -infected patient sera and were immunogenic when used to immunise rabbits, making them good vaccine candidates against P. vivax , to be further preclinically tested in the Aotus monkey model.