Callus Induction and Cellular Suspensions from Murtilla (Ugni molinae Turcz.) for trans-resveratrol Production

The present study reports for the first time the quantification of resveratrol and the use of biotechnological techniques applied to stilbene productions in species from genus Ugni and calli production from adult explants (fruits). Resveratrol is synthesized by a series of families of higher plants, which has generated much interest in recent years for its antioxidant, anticancer and antitumor properties, which would allow longevity of cells to be prolonged. In this study, leaves and mature fruits were collected from three sites in Southern Chile and correspond to three ecotypes of the Chilean endemic species Ugni molinae Turz. (murtilla). These were established in vitro to prepare the callus and subsequent development of cellular suspensions for trans-resveratrol production. Our results showed that these stilbenes are present in murtilla and that their concentrations vary between ecotypes and tissues, reaching up to 553.5 µg g-1 of t-resveratrol produced in ecotype 3 callus. These values are relatively higher than those found in other plant species. Under optimum culture conditions, extraction of resveratrol from Ugni molinae is scalable to industrial levels, which makes it a viable alternative for obtaining stilbenes.This research was supported by a project of the Innova BíoBío, Corfo-Chile No.12.247

Obesidad, inflamación e insulino-resistencia: papel de los ligandos del receptor gp 130

Obesity can be considered as a low grade inflammatory disease, characterized by increased plasma levels of proinflammatory cytokines such as tumoral necrosis factor-a (TNF-a), and acute phase reactant proteins like C-reactive protein. In this context, some cytokines of the interleukin-6 (IL-6) family have been involved in the inflammatory processes associated to obesity. In addition to IL-6, the IL-6 cytokine family includes IL-11, ciliary neurotrophic factor (cntf), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF) y Oncostatin M (OsM). These proteins are also known as gp130 cytokines because all of them exert their action via the glycoprotein 130 (gp130) as a common transducer protein within their functional receptor complexes. However, their role in obesity and related disorders is controversial; thus, whereas some studies have described the involvement of gp130 cytokines in the development of obesity and its related cluster of pathophysiologic conditions like insulin-resistance, fatty liver and cardiovascular diseases, other trials have proposed the gp130 receptor ligands as therapeutic targets in the treatment of obesity and its related disorders. In fact, CNTF treatment has demonstrated to be effective in the reduction of body weight, by promoting the inhibition of food intake and the activation of the energy expenditure, together with an improvement of insulin sensitivity. This review analyzes the potential therapeutic role of some of the gp130 ligands in obesity and related diseases

Eicosapentaenoic acid stimulates AMP-activated protein kinase and increases visfatin secretion in cultured murine adipocytes

Visfatin is an adipokine highly expressed in visceral AT (adipose tissue) of humans and rodents, the production of which seems to be dysregulated in excessive fat accumulation and conditions of insulin resistance. EPA (eicosapentaenoic acid), an n−3 PUFA (polyunsaturated fatty acid), has been demonstrated to exert beneficial effects in obesity and insulin resistance conditions, which have been further linked to its reported ability to modulate adipokine production by adipocytes. TNF-α (tumour necrosis factor-α) is a pro-inflammatory cytokine whose production is increased in obesity and is involved in the development of insulin resistance. Control of adipokine production by some insulin-sensitizing compounds has been associated with the stimulation of AMPK (AMP-activated protein kinase). The aim of the present study was to examine in vitro the effects of EPA on visfatin production and the potential involvement of AMPK both in the absence or presence of TNF-α. Treatment with the pro-inflammatory cytokine TNF-α (1 ng/ml) did not modify visfatin gene expression and protein secretion in primary cultured rat adipocytes. However, treatment of these primary adipocytes with EPA (200 μmol/l) for 24 h significantly increased visfatin secretion (P<0.001) and mRNA gene expression (P<0.05). Moreover, the stimulatory effect of EPA on visfatin secretion was prevented by treatment with the AMPK inhibitor Compound C, but not with the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Similar results were observed in 3T3-L1 adipocytes. Moreover, EPA strongly stimulated AMPK phosphorylation alone or in combination with TNF-α in 3T3-L1 adipocytes and pre-adipocytes. The results of the present study suggest that the stimulatory action of EPA on visfatin production involves AMPK activation in adipocytes

Los programas de empleo en Argentina: el ascenso del neoliberalismo y la estrategia de workfare

Dicha ponencia se propone versar exclusivamente sobre la noción de Workfare, que emerge como un nuevo paradigma de la política social y laboral a nivel global, en consecuencia, con las transformaciones socioeconómicas sucedidas a partir de 1970 y con la consolidación del modelo neoliberal. Como señala Brenda Brown (2015) el workfare se ha asociado a una nueva forma de gestionar los riesgos sociales que nace junto a la adopción del neoliberalismo como nueva racionalidad de gobierno en los países centrales. Las primeras definiciones sobre paradigma de activación o workfare datan de mediado de los años 70 y responden a un cambio en la concepción de los riesgos sociales y en la forma de gestionar a las poblaciones desempleadas y pobres. En la región latinoamericana, la implementación de programas y dispositivos que responden a la lógica del workfare comienza a evidenciarse a partir de los años 90, década en la que se expanden diversos programas de transferencias monetarias condicionadas (PTMC) de la mano de los organismos internacionales de crédito como el Banco Mundial y el Banco Interamericano de Desarrollo.Fil: Bustos, Matilde. Universidad Nacional de Villa María; Argentina

Role of Succinate Dehydrogenase D (SDHD) in Hepatocytic Cells

Motivation: Mitochondria are the principal site of important cellular functions including ATP production via oxidative phosphorylation (OXPHOS) but also play a central role in cell physiology.OXPHOS comprises five respiratory complexes (RCs). Complex II or succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex composed of four subunits (SDHA, SDHB, SDHC, SDHD). It´s responsible for transferring electrons into mitochondrial respiratory chain for ATP production but also for the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle.Changes in TCA cycle have been considered as potential mechanisms of aerobic glycolysis that contributes to the alteration of cellular metabolism with implications in tumorigenesis. It has been reported that polymorphisms in SDHD are associated with early recurrence of hepatocellular carcinoma. Our goal was to study the role of SDHD in tumor and no tumor liver cells.Methods: Murine hepatocytic cells, AML12 and Hepa1-6 were obtained from the ATCC. In order to analyze the function of the SDHD we used murine-SDHD-specific siRNA and control siRNA. SDHD overexpression was performed using a plasmid vectors with murine SDHD constructed using standard molecular cloning techniques. In both cases cells were transfected with lipofectamine.To analyze metabolic changes induced by SDHD we performed real-time PCR. Proteins were examined by Western blots. Cell viability assay, colony formation assay and wound healing assay have been performed to determinate cell survival, proliferation and migration.Results: Inhibition of SDHD in hepatocytic cells increased glycolytic genes at 48 h and 72 h after transfection compared to controls, with higher rate in the Hepa1-6 than AML12 cells. Overexpression of SDHD, did not induce significant changes in metabolic genes as compared to control cells. Furthermore, we observed that the inhibition of SDHD in hepatocytic cells produced an increase in cell death. In addition, clonogenic growth decreased greatly, resulting in reduced colony size and numbers in SDHD-inhibiting cells. In a wound healing migration assay, cell confluence was reached significantly later in the SDHD-inhibiting cells than the si-control transfected cells. Finally, the cell cycle analyses showed that SDHD-knockdown cells showed less percentage of cells in G1 than control cells.Conclusions: Our findings suggest that SDHD plays an important role in the survival, growth and cellular proliferation

Cardiotrophin-1 defends the liver against ischemia-reperfusion injury and mediates the protective effect of ischemic preconditioning

Ischemia-reperfusion (I/R) liver injury occurs when blood flow is restored after prolonged ischemia. A short interruption of blood flow (ischemic preconditioning [IP]) induces tolerance to subsequent prolonged ischemia through ill-defined mechanisms. Cardiotrophin (CT)-1, a cytokine of the interleukin-6 family, exerts hepatoprotective effects and activates key survival pathways like JAK/STAT3. Here we show that administration of CT-1 to rats or mice protects against I/R liver injury and that CT-1-deficient mice are exceedingly sensitive to this type of damage. IP markedly reduced transaminase levels and abrogated caspase-3 and c-Jun-NH2-terminal kinase activation after I/R in normal mice but not in CT-1-null mice. Moreover, the protective effect afforded by IP was reduced by previous administration of neutralizing anti-CT-1 antibody. Prominent STAT3 phosphorylation in liver tissue was observed after IP plus I/R in normal mice but not in CT-1-null mice. Oxidative stress, a process involved in IP-induced hepatoprotection, was found to stimulate CT-1 release from isolated hepatocytes. Interestingly, brief ischemia followed by short reperfusion caused mild serum transaminase elevation and strong STAT3 activation in normal and IL-6-deficient mice, but failed to activate STAT3 and provoked marked hypertransaminasemia in CT-1-null animals. In conclusion, CT-1 is an essential endogenous defense of the liver against I/R and is a key mediator of the protective effect induced by IP

Role of the hepatoprotective citoquine cardiotrophin-1 (CT-1) in macrophages polarization

Motivation: Cardiotrophin-1 (CT-1) is a member of the interleukine-6 (IL-6) family, which signals through gp130/leukemia inhibitory factor receptor (LIFR). Signal transduction via gp130 involves three major downstream pathways: the STAT-3 (signal transducer and activator of transcription-3), the ERK and the AKT pathways, all of them described as antiapoptotics. CT-1 has been described as hepatoprotective.However, the role of cardiotrophine in inflammation is a non-well studied process. Whenever an inflammation occurs, macrophages are the first cells of the inmune system to respond. These, in respond to various enviromental cues or under different pathophysiologic conditions, can aquire distinct functional phenotypes via by undergoing different phenotypic polarization. Macrophages M1 (proinflammatory) and M2 (antiinflammatory) are two well- separated groups, which describe opposing activities of killing and repairing, respectively. The aim of our study was to evaluate the role of CT-1 in macrophages polarization.Methods: To analyze the macrophages polarization into M1 or M2, different proinflammatories and antiinflammatories genes expression were measured, as well as the pathways involved, using molecular biology techniques, such as quantitative PCR (qPCR) and Western Blots.Results: 1. In vivo model: a septic shock was induced to mice through a LPS injection. By analysis of the expression of proinflammatories genes in hepatic samples, we observed that pretreated mice with CT-1, showed a decrease on the levels of genes expression involved in proinflammation or M1, such as TNFα, IL1β and IL12p40.2. In vitro model: macrophages polarization into M1 or M2 was induced by using LPS or IL4 respectively. The analysis of pro and antiinflammatories genes expression, showed us that pretreated macrophages with CT-1 had some modifications in the level of genes expression when compared with those non-treated. M1 genes expression diminished in relation to non-treated. On the other hand, M2 genes expression augmented with respect to non-treated. Interestingly, CT-1 can't modify these expression genes by itself.Conclusions:1. CT-1 modulates inflammatory response against LPS, by reducing proinflammatories cytokines within an in vivo model.2. CT-1 modifies macrophage polarization into M1 or M2 induced by LPS or IL4, reducing proinflammatories cytokines and promoting antiinflammatories, but, interestingly, is not able to polarize macrophages by itself

Role of the cardiotrophin-1 in the physiological adaptation to fasting

Motivation: Cardiotrophin-1 (CT-1) is a member of the interleukin-6 (IL-6) family of cytokines. CT-1 is expressed in several metabolic tissues and it is a nutritionally regulated metabolic gene with a key role in glucose and lipid metabolism (1). CT-1 is expressed in white adipose tissue (WAT) and it has been reported to be upregulated in the metabolic syndrome. Our goal was to investigate the role of CT-1 in metabolic adaptations. We analysed the role of CT-1 in fasting, a physiological stress that elicits well-known metabolic adaptations (2, 3).Methods: A differential study was carried out with wild type (WT) and CT-1 deficient animals. Mice were fed or fasted for 24 or 48 hours. Mice were males of 10-16 weeks of age. Glucose, free fatty acid (FFA) and ketone bodies were determined. WAT, liver and skeletal muscle were examined. Analysis of proteins was studied by Western-blot, mRNA levels were quantified by real-time PCR and histological studies were performed with H&amp;E staining. We performed immunohistochemistry in WAT with tyrosine hydroxylase (TH) to determine sympathetic innervation. Adipocyte size was quantified using the software adiposoft.Results: CT-1 mRNA expression in WAT increased markedly when mice were subjected to 48 hours fast. We did not observe any differences between WT and CT-1 null mice in fed state. However, by nutrient deprivation (24 and 48 hours) CT-1 knock-out mice exhibited less weight loss and a higher visceral adiposity compared to WT mice. The increase of FFA and ketone bodies in serum was reduced in CT-1 null mice as compared to WT animals. Analysis of WAT showed lower levels of phosphorylation (Ser-563 and 660) of the main lipase HSL (hormone sensitive lipase) in CT-1-/- mice at 48 hours as well as the levels of lysosomal acid lipase (Lipa), genes involved in lipophagy such as lysosome-associated membrane protein 1 (LAMP1), autophagy-related genes (ATG) and the LC3II/LC3I ratio. We analysed activation of AMPK (AMP-activated protein kinase) and the forkhead homeobox type O1 (FoxO1) transcription factor in WAT. In fasted animals, WAT from CT-1-/- mice showed higher number of increased adipocyte size with less expression of TH immunostaining as compared to WT animals. Analysis of the liver showed that fasting-induced lipid droplet formation was impaired in CT-1 null mice together with a higher LC3II/LC3I ratio.Conclusions: Our findings suggest that CT-1 plays a relevant role in fasting adaptation

Induction of TIMP-1 expression in rat hepatic stellate cells and hepatocytes: a new role for homocysteine in liver fibrosis

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies