Rebeca Martínez

Mendelian transmission, transgene dosage and growth phenotype in transgenic tilapia (Oreochromis hornorum) showing ectopic expression of homologous growth hormone

Abstract Gene transfer has offered a new tool for the development of improved fish strains for aquaculture. However, characterization is required before these strains can be introduced into national aquaculture programs. Transgenic tilapia ( O. hornorum urolepis ) were produced by the microinjection into early embryos of a transgene containing the tilapia growth hormone (tiGH) cDNA under the regulatory sequences derived from the human cytomegolovirus (CMV). A male containing 1 copy/cell of the transgene was selected to establish a transgenic tilapia line. The transgene was transmitted to F 1 –F 4 generations in a Mendelian fashion. Previous studies showed ectopic, low level expression of tiGH in brain, heart, gonad, liver and muscle cells of transgenic tilapia. Biochemical analyses indicated lower levels of cholesterol, free alanine and aspartic acid in the muscle of transgenic animals. Four month old transgenic homozygous (F 2 +/+ ) and heterozygous (F 2 −/+ ) tilapia and non-transgenic siblings ( N TRANSGENICS =14; N CONTROLS =11; N F2 −/+ =8; N F2 +/+ =6) were studied for 3 months grown communally in the same pond. Transgenic (F 2 −/+ +F 2 +/+ ), F 2 −/+ , and F 2 +/+ progeny were larger than non-transgenic siblings at P =0.009, P =0.005 and P =0.07 (Student t -test), respectively, suggesting a transgene-dosage effect. These results indicate stable germ line transformation in this fast-growing transgenic tilapia line.

Growth regulation and enhancement in tilapia: basic research findings and their applications

Growth manipulation in fish is one of the targets of gene transfer experiments. The aim is to produce strains with improved growth performance. The transfer of growth hormone transgenes has been successful in many fish species. Now detailed knowledge of the molecular events that control growth in fish is necessary in order to efficiently manipulate this process. We have selected tilapia for our studies because these species are suitable for basic research as well as for the development of improved strains for aquaculture. Here we review the results of basic and applied research in the field of growth control and manipulation in tilapia. Our experiments produced new scientific results on growth control in tilapia. These results were used to develop a new aquacultured line with improved growth performance. Many of these results are probably applicable to other teleosts.

A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish.

In teleosts fish, secretion of GH is regulated by several hypothalamic factors that are influenced by the physiological state of the animal. There is an interaction between immune and endocrine systems through hormones and cytokines. GH in fish is involved in many physiological processes that are not overtly growth related, such as saltwater osmoregulation, antifreeze synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide compound A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [d-Lys(3)]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments propose a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfish and tilapia larvae (P<0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 designed by molecular modeling is able to function as a GHS in teleosts and enhance parameters of the innate immune system in the fish larvae.

Factores pronósticos de la bacteriemia: estudio prospectivo

BACKGROUND: To identify the epidemiology and risk factors with influence in the outcome and mortality of a series of bacteriemic patients. MATERIAL AND METHODS: A prospective study of bloodstream infections with clinical significance detected in a secondary hospital of 650 beds over period from May 1998 to May 1999. The true bacteriemia was defined in basis to the criteria both the physician and microbiologist. A total of 16 variables were defined and categorized such as clinical-epidemiologic, intrinsic risk factor, extrinsic risk factor, outcome and survival. We used SPSS statistical package: For cuantitative variables we carried out with the mean with confidence interval of 95%, for cualitative variables: number and %. Univariate analysis of the results was carried out with the X2 test and t Student, the survival was expressed with Kaplan Meyer graphics and the logistic regression model. RESULTS: A total of 320 positive blood cultures were studied but only 272 blood cultures were considered true bacteriemia in 259 patients. The calculated incidence of significant episodes of bacteriemia per 1000 admissions/year was 13. The overall mortality was 22% whereas death attributable to bacteriemia was 16%. The mean age was 66.9 years (IC 95% 65-69), 59% episodes occurred in men. The 78% episodes occurred in patients hospitalized in medical services. 52% episodes were of nosocomial infection and 48% of community acquired infection. According to the severity of the underlying disease, 15% had fatal diseases and 35% episodes occurred in patients without underlying disease. According to the univariant analysis, the variables which where significantly associated with greater risk death were: etiology (fungus), septic shock, the inadequate antibiotic therapy, presence of extrinsic factors (central intravenous catheter, performance of invasive procedures, previous antimicrobial therapy) and the hospital stay of less than 10 days. According to the multivariable analysis showed that the factors remaining independent predictors of mortality were: septic shock (p < 0.0001, OR: 8), inadequate antimicrobial therapy (p < 0.005, OR: 6.7), existence of two or more extrinsic risk factors (p < 0.04). CONCLUSIONS: The presence of septic shock was the most important variable which influenced in the mortality in our serie, together with inappropriate antimicrobian therapy and the association of various extrinsic risk factors. These variables could be modified partly, for this reason the aggressive hemodynamic control and the early and appropriate antibiotic therapy would be the support of the successful bacteriemia management.

Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response

Background Growth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood. Methods The effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation. Results The functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection. Conclusions The growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model. General Significance The increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.

Growth Hormone Secretagogue (A233) Improves Growth and Changes the Tissue Fatty Acid Profile in Juvenile Tilapia (Oreochromis niloticus)

Growth hormone (GH) release is a process that is well regulated by several factors, including GH secretagogues. GH can mediate the regulation of the fatty acid level and composition. The aim of this study was to determine the effect of a synthetic GH secretagogue peptide (A233) on the growth and fatty acid composition in tilapia (Oreochromis niloticus). To address this objective, we administrated a diet supplemented with A233 to juvenile tilapia for 60 days. The group fed with a diet supplemented with 600 μg of A233 per kg of feed increased in weight (4.81 ± 0.09 g) and specific growth rate (2.49 ± 0.03%/day) compared to the control diet group (3.63 ± 0.08 g, 2.07 ± 0.04%/day; respectively) (p < 0.001). In the muscle, the total lipids for the control diet group were higher than that in the group fed with 600 μg of A233 per kg feed; however, no differences were detected in the liver. In both tissues, the patterns of fatty acid composition and content were generally similar, with some exceptions. Tilapia fed with 600 μg of A233 per kg of feed showed, in liver and muscle, a significantly higher composition and content of n-3 polyunsaturated fatty acids (such as 20:5n-3, 22:5n-3, 22:6n-3) and n-3/n-6 PUFA than animals fed with the control diet. To our knowledge, this is the first report on the the effects of natural or synthetic GH secretagogues (GHS) on fatty acid composition, implying an increase in the nutritional quality of the tilapia.

A233 Peptide: A Growth Hormone Secretagogue that Promotes an Antiviral Signaling Pathway

Rebeca Martinez1, Lazaro Gil2, Yassel Ramos3, Luis J Gonzalez3, Mario P Estrada1* and Vladimir Besada3* 1Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, Cuba 2Vaccines Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba 3Department of Systems Biology, Center for Genetic Engineering and Biotechnology; Havana, Cuba *Corresponding authors: Vladimir Besada, Department of Systems Biology, Center for Genetic Engineering and Biotechnology; Havana, Cuba, Tel: +53-7250-41-50; Fax: +53-7271-8070; E-mail: vladimir.besada@cigb.edu.cu