Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates.

Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. ß-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the ß-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (+1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to ß-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral ß-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with ß-defensins from vertebrates, it is conceivable to further support the finding of a new family of ß-defensins in invertebrate.

Molecular and conventional epidemiology of tuberculosis in an inner city district.

SETTING: Molecular epidemiology has underlined the importance of recent tuberculosis (TB) transmission and has uncovered notable discrepancies compared with conventional epidemiology. OBJECTIVES: 1) To determine, by RFLP analysis, the percentage of clustered cases in an inner city district with a high incidence of TB (163/100,000) and the groups at risk of being clustered; and 2) to compare the role of conventional contact tracing (CCT) with that of RFLP. DESIGN: RFLP was carried out using the IS6110 and pTBN12 (PGRS) sequences of 165 cultures positive for Mycobacterium tuberculosis in the Ciutat Vella district of Barcelona during 1997-1998. Contact tracing was carried out in 171 of 251 declared cases (68.1%). Associations were assessed by calculating odds ratios (OR) with 95% confidence intervals (CI). Logistic regression was used for multivariate analysis. RESULTS: Using RFLP, 76 (46.0%) strains were found to be clustered by IS6110 and PGRS. From CCT, 30 new patients were found among 858 contacts (3.5%) and 57 patients were linked. In terms of RFLP and CCT, the main risk factor was intravenous drug use (IVDU). In 44 cases who lived alone and were not involved in CCT, 50% were in RFLP clusters. The concordance rate between RFLP and CCT was 8/13 (61.5%); the disagreement corresponded to a cluster of five recent immigrants from Africa. Subsequent to RFLP, an epidemiological connection was found in 15/55 cases (27.2%). CONCLUSIONS: The percentage of clustered cases is very high. CCT was useful for identifying new cases, but it was insufficient for detecting the pathways of transmission. CCT coverage needs to be improved in marginalized individuals, and the results correlated with those of RFLP.

Growth efficiency in transgenic tilapia (Oreochromis sp.) carrying a single copy of an homologous cDNA growth hormone.

Growth hormone (GH) has been shown to have a profound impact on fish physiology and metabolism. However, detailed studies in transgenic fish have not been conducted. We have characterized the food conversion efficiency, protein profile, and biochemical correlates of growth rate in transgenic tilapia expressing the tilapia GH cDNA under the control of human cytomegalovirus regulatory sequences. Transgenic tilapia exhibited about 3.6-fold less food consumption than nontransgenic controls (P < 0.001). The food conversion efficiency was significantly (P < 0.05) higher (290%) in transgenic tilapia (2.3 +/- 0.4) than in the control group (0.8 +/- 0.2). Efficiency of growth, synthesis retention, anabolic stimulation, and average protein synthesis were higher in transgenic than in nontransgenic tilapia. Distinctive metabolic differences were found in transgenic juvenile tilapia. We had found differences in hepatic glucose, and in agreement with previous results we observed differences in the level of enzymatic activities in target organs. We conclude that GH-transgenic juvenile tilapia show altered physiological and metabolic conditions and are biologically more efficient.

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.

Characterization of transgenic tilapia lines with different ectopic expression of tilapia growth hormone.

The transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. However, the ectopic GH levels to optimize growth acceleration in fish, and specially in tilapia, are not known and must be determined experimentally. The tilapia GH (tiGH) cDNA was used to construct chimeric genes expressing different levels of tiGH in vitro and in vivo. These constructs were used to generate four lines of transgenic tilapia by microinjection into one-cell embryos. Different patterns and levels of ectopic expression of tiGH and IGF were detected in organs of transgenic tilapia by RNA or protein analysis. The two lines with lower ectopic tiGH mRNA levels were the only ones showing growth acceleration, suggesting that the expression of ectopic tiGH promoted growth only at low expression levels. The effect of higher ectopic tiGH levels resembled the physiologic situation of low condition factor and permitted us to postulate a model for growth acceleration in transgenic tilapia expressing ectopic tiGH.

Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.

The generation of transgenic fish with the transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. The tilapia growth hormone (tiGH) cDNA was linked to the human cytomegalovirus (CMV) enhancer-promoter and used to generate transgenic tilapia by microinjection into one-cell embryos. Five transgenic tilapia were obtained from 40 injected embryos. A transgenic animal containing one copy of the transgene per cell was selected to establish a transgenic line. The transgene was stably transmitted to F1 and F2 generations in a Mendelian fashion. Ectopic, low-level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by immunohystochemical analysis of tissue sections. Nine-month-old transgenic F1 progeny were 82% larger than nontransgenic fish at p = .001. These results showed that low-level ectopic expression of tiGH resulted in a growth acceleration in transgenic tilapia. Tilapia GH gene transfer is an alternative for growth acceleration in tilapia.

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.

High level expression of streptokinase in Escherichia coli.

Streptokinase (SK), which activates human plasminogen by promoting its conversion to plasmin, is normally obtained from beta-hemolytic streptococci. Treatment with SK is an effective therapy for improving survival and preserving left ventricular function after coronary thrombosis. We report the cloning, expression in E. coli to levels of 25% of the total cell protein, and characterization of a novel SK (SKC-2) gene, the product of which is functionally equivalent to the naturally-derived protein. The availability of a recombinant streptokinase (rSK) in high yield and purity offers a potentially attractive alternative source of this important therapeutic agent.

Clonación y expresión del ADNc para el activador tisular del plasminógeno en cèlulas de animales / Cloning and expresion of human tissue-type plasminogen activator cDNA in eukararyotic cells

Una alta expresión de tPA humana recombinante en células de hamster chino (CHO), fue obtenida usando el sistema de coamplificación del gen de la dehidrofolato reductasa (DHFR). La proteina fue secretada activa al medio de cultivo. En varios clones se mantuvo la producción estable, aun en ausencia de presión selectiva. No obstante, cuando el mismo ADN complementario fue clonado en un vector de expresión de baculovirus, sólo fue posible detectar niveles muy bajos de actividad tPA

Cloning of coliphage-T4 gene pseT and high-level synthesis of polynucleotide kinase in Escherichia coli.

The gene, pseT, of coliphage T4 which encodes polynucleotide kinase (PNK) was cloned directly into an expression plasmid using the polymerase chain reaction. When placed under the control of the trp promoter, the pse T gene can be maintained stably in Escherichia coli and yields high levels of the enzyme upon induction. The system described facilitates purification and provides very high yields of PNK.

Determinación de actividad estreptoquinasa utilizando un ensayo cuantitativo / Determination of streptokinase activity by quantitative assay

Para determinar la actividad estreptoquinasa (SK) presente en muestras de cultivo y purificación, se desarrolló un método cuantitativo a partir de la formación del complejo estequiométrico estreptoquinasa-plasminógeno (sk-plg). Las determinaciones fueron realizadas utilizando el método del sustrato cromogénico S-2251. La sk se obtuvo del cultivo de Streptococcus equisimilis beta hemolítico (grupo C). El plasminógeno empleado en el ensayo fue purificado a partir de plasma humano en nuestro laboratorio. El método permite la detección de hasta 7 UI de sk/ml. En el trabajo se describen los resultados obtenidos a diferentes concentraciones de sales presentes en las muestras, así como el efecto estimulador del fibrinógeno en la reacción

Expresión directa de la hormona de crecimiento humana madura en Escherichia coli / Direct expression of the mature human growth hormone in Escherichia coli

En el presente trabajo se describe una forma novedosa para la expresión directa de proteinas maduras de mamíferos en E.coli. Las regiones 3' y 5' no taducidas, y la secuencia señal de la hormona de crecimiento humana (hGH) fueron removidas, y un codón de iniciación se situó adyacente al primer codón de la secuencia correspondiente a la proteína madura. Los sitios de restricción Ncol Y BamHI fueron introducidos en las regiones 5' y 3' respectivamente, y la secuencia correspondiente a la proteína madura se amplificó por el método de la reacción en cadena de la polimerasa (PCR), clonándose directamente en un vector de expresión. Se alcanzaron altos niveles de expresión en E. coli