Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses

<p>Abstract</p> <p>Background</p> <p>The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, <it>SOD </it>gene, was isolated from a <it>Deschampsia antarctica </it>Desv. by cDNA library screening. The expression of SOD gene in the leaves of <it>D. antarctica </it>was determined by RT-PCR and its differential expression of gene transcripts in conditions of cold and UV radiation stresses was revealed by northern blot.</p> <p>Findings</p> <p>The molecular characterization shows that <it>SOD </it>cDNA is 709 bp in length, which translates an ORF of 152 amino acids that correspond to a protein of predicted molecular mass of 15 kDa. The assay shows that the expression of <it>SOD </it>gene increases when <it>D. antarctica </it>is acclimatised to 4°C and exposed to UV radiation. These results indicate that the <it>SOD </it>gene of <it>D. antarctica </it>is involved in the antioxidative process triggered by oxidative stress induced by the conditions of environmental change in which they live.</p> <p>Conclusion</p> <p>The present results allow us to know the characteristics of Cu/ZnSOD gene from <it>D. antarctica </it>and understand that its expression is regulated by cold and UV radiation.</p

Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p><it>Deschampsia antarctica </it>shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the <it>D. antarctica </it>Cu/ZnSOD gene into <it>Pichia pastoris </it>and to characterize the heterologous protein.</p> <p>Findings</p> <p>The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, <it>SOD </it>gene, was isolated from a <it>D. antarctica </it>by cDNA library screening. This <it>SOD </it>gene was cloned in the expression vector pGAPZαA and successfully integrated into the genome of the yeast <it>P. pastoris </it>SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg.</p> <p>Conclusion</p> <p><it>D. antarctica </it>Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40°C for 30 minutes and it is stable (80% of activity) between -20°C and 20°C. The recombinant SOD described in this study can be used in various biotechnological applications.</p

Immunotherapy for liver tumors: present status and future prospects

Increasing evidence suggests that immune responses are involved in the control of cancer and that the immune system can be manipulated in different ways to recognize and attack tumors. Progress in immune-based strategies has opened new therapeutic avenues using a number of techniques destined to eliminate malignant cells. In the present review, we overview current knowledge on the importance, successes and difficulties of immunotherapy in liver tumors, including preclinical data available in animal models and information from clinical trials carried out during the lasts years. This review shows that new options for the treatment of advanced liver tumors are urgently needed and that there is a ground for future advances in the field

A tumor-stroma targeted oncolytic adenovirus replicated in human ovary cancer samples and inhibited growth of disseminated solid tumors in mice

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 10(10) v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15-40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression.Fil: Lopez, Maria Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Rivera, Angel A.. University Of Alabama At Birmingahm; Estados UnidosFil: Viale, Diego Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Benedetti, Lorena Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Cuneo, Nicasio. Hospital Municipal de Oncología Marie Curie; ArgentinaFil: Kimball, Kristopher J.. University Of Alabama At Birmingahm; Estados UnidosFil: Wang, Minghui. University Of Alabama At Birmingahm. School Of Medicine. Division Of Human Gene Therapy; Estados UnidosFil: Douglas, Joanne T.. University Of Alabama At Birmingahm. School Of Medicine. Division Of Human Gene Therapy; Estados UnidosFil: Zhu, Zeng B.. University Of Alabama At Birmingahm. School Of Medicine. Division Of Human Gene Therapy; Estados UnidosFil: Bravo, Alicia I.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "eva Peron"; ArgentinaFil: Gidekel, Manuel. Universidad de la Frontera; ChileFil: Alvarez, Ronald D.. University Of Alabama At Birmingahm; Estados UnidosFil: Curiel, David T.. University Of Alabama At Birmingahm. School Of Medicine. Division Of Human Gene Therapy; Estados Unidos. University of Washington; Estados UnidosFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP- 1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profle of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fbroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCCFil: Bayo Fina, Juan Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Real, Alejandrina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Fiore, Esteban Juan. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Malvicini, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Sganga, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Adriani, Oscar. Hospital Universitario Austral; ArgentinaFil: Bizama, Carolina. Universidad Católica de Chile; ChileFil: Fresno Rodríguez, Cristóbal. Universidad Católica de Córdoba; ArgentinaFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Fernandez, Elmer Andres. Universidad Católica de Córdoba; ArgentinaFil: Gidekel, Manuel. Universidad de la Frontera. Núcleo Científico y Tecnológico en Recursos Naturales; Chile. Universidad Autónoma de Chile; ChileFil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentina. Hospital Universitario Austral; ChileFil: García, Mariana Gabriela. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentin

Isolation and characterisation of pectic substances from murta (Ugni molinae Turcz) fruits

Cell walls polysaccharides from murta fruit (Ugni molinae Turcz), an endemic Chilean species with relevant food uses, were fractionated by water, ammonium oxalate, hot diluted HCl and cold diluted NaOH extractions. The polysaccharide fractions were analysed for monosaccharide composition and physicochemical properties. Pectic substances were found in all extracts, but mainly in the oxalate and acid soluble fractions, in which they appear as homogalacturonan polymers. Murta pectin was further extracted by hot diluted acid treatment using industrial conditions, yielding 30% by weight of dry fruit. The polymer showed similar composition and physicochemical properties to those of commercial citrus pectin, presenting a galacturonic acid content of 70.9% (w/w), a molecular weight of 597. kDa, and a methoxylation degree of 57%. The FT-IR spectrum of murta pectin suggests the presence of ferulic acid residues on its structure and the NMR analysis confirmed the structure of this polysaccharide. It is concluded that murta fruit can be considered as a valuable source of high quality pectin. © 2010 Elsevier Ltd

Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclophosphamide and gene transfer of IL-12

Immunotherapy-based strategies for gastrointestinal carcinomas (GIC) have been exploited so far, but these approaches have to face strong mechanisms of immune escape induced by tumours. We previously demonstrated that sub-therapeutic doses of an adenovirus expressing IL-12 genes (AdIL-12) mediated a potent antitumour effect against subcutaneous (s.c.) colorectal carcinomas (CRC) in mice pre-treated with low doses of cyclophosphamide (Cy). In our study we used this combination to assess its impact on the immunosuppressive microenvironment. In s.c. CRC model we demonstrated that non-responder mice failed to decrease Tregs in tumour, spleen and peripheral blood. Reconstitution of Tregs into tumour-bearing mice treated with combined therapy abolished the antitumoural effect. In addition, Cy + AdIL-12 modified Tregs functionality by inhibiting the in vitro secretion of IL-10 and TGF-β and their ability to inhibit dendritic cells activation. Combined treatment decreased the number of myeloid-derived suppressor cells (MDSCs) in comparison to non-treated mice and, interestingly, administration of Tregs restored splenic MDSCs population. Furthermore, combined therapy potently generated specific cytotoxic IFN-γ-secreting CD4+ T cells able to eradicate established CRC tumours after adoptive transfer. Finally, we evaluated the combination on disseminated CRC and pancreatic carcinoma (PC). Cy + AdIL-12 were able to eradicate liver metastatic CRC (47%) and PC tumour nodules (40%) and to prolong animal survival. The results of this study support the hypothesis that Cy + AdIL-12 might be a valid immunotherapeutic strategy for advanced GIC.Fil: Malvicini, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Ingolotti, Mariana. Universidad Austral; ArgentinaFil: Piccioni, Flavia Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: García, Mariana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Bayo Fina, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Atorrasagasti, María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Alaniz, Laura Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Aquino, Jorge Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; ArgentinaFil: Espinoza, Jaime A.. Universidad Adolfo Ibañez; Chile. Universidad de La Frontera; ChileFil: Gidekel, Manuel. Universidad Adolfo Ibañez; ChileFil: Scharovsky, Olga Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Matar, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Mazzolini Rizzo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Austral; Argentin

Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed