Cloning and expression of a thermostable alpha-amylase gene from Streptomyces thermoviolaceus CUB74

Streptomycetes are aerobic Gram-positive bacteria whose natural habitat is the soil. They have been of general interest because of their ability to produce a variety of secondary metabolites with antibiotic properties. Apart from antibiotic production, streptomycetes produce many extracellular enzymes including amylases, proteases, cellulases and nucleases. Amylases are widely distributed amongst Streptomyces species and have been coming under increasing scrutiny in the past few years. For example, the α-amylase genes of S. hygroscopicus, S. limosus and S. venezuelae have been cloned and sequenced and their predicted amino acid sequences have been compared. The thesis describes the cloning and expression of a thermostable α-amylase gene from S. thermoviolaceus CUB74 into Escherichia coli and S. lividans. When expressed in E. coli, the Streptomyces α-amylase enzyme was found in the periplasmic space and the supernatant. The extracellular enzyme isolated from the parent Streptomyces strain and the new hosts was further analysed by SDS-polyacry1 amide gel electrophoresis. In addition, the product of starch digestion with this enzyme was analysed by paper chromatography. The α-amylase enzyme was reactivated in the SDS protein gel and its activity was visualized using a starch-agarose gel overlay. Stability of this enzyme at high temperatures was determined in the presence and absence of calcium and substrate. Its production in the parent strain and in S. lividans was also studied in the presence of different carbon sources. The gene specifying this enzyme was sequenced and its predicted amino acid sequence was compared with α-amylases isolated from other species. mRNA produced from this gene in different plasmid constructs was detected by Northern blotting and the transcriptional start site was determined by high resolution S mapping

Redundancy and compensation in axon guidance: genetic analysis of the Drosophila Ptp10D/Ptp4E receptor tyrosine phosphatase subfamily

Background: Drosophila has six receptor protein tyrosine phosphatases (RPTPs), five of which are expressed primarily in neurons. Mutations in all five affect axon guidance, either alone or in combination. Highly penetrant CNS and motor axon guidance alterations are usually observed only when specific combinations of two or more RPTPs are removed. Here, we examine the sixth RPTP, Ptp4E, which is broadly expressed. Results: Ptp4E and Ptp10D are closely related Type III RPTPs. Non-drosophilid insect species have only one Type III RPTP, which is closest to Ptp10D. We found that Ptp4E mutants are viable and fertile. We then examined Ptp4E Ptp10D double mutants. These die before the larval stage, and have a mild CNS phenotype in which the outer longitudinal 1D4 bundle is frayed. Ptp10D Ptp69D double mutants have a strong CNS phenotype in which 1D4 axons abnormally cross the midline and the outer and middle longitudinal bundles are fused to the inner bundle. To examine if Ptp4E also exhibits synthetic phenotypes in combination with Ptp69D, we made Ptp4E Ptp69D double mutants and Ptp4E Ptp10D Ptp69D triple mutants. No phenotype was observed in the double mutant. The triple mutant phenotype differs from the Ptp10D Ptp69D phenotype in two ways. First, the longitudinal tracts appear more normal than in the double mutant; two or three bundles are observed, although they are disorganized and fused. Second, axons labelled by the SemaIIB-tMyc marker often cross in the wrong commissure. We also examined motor axon guidance, and found that no phenotypes are observed in any Ptp4E double mutant combination. However, triple mutants in which Ptp4E Ptp10D was combined with Ptp69D or Ptp52F exhibited stronger phenotypes than the corresponding Ptp10D double mutants. Conclusions: Type III RPTPs are required for viability in Drosophila, since Ptp4E Ptp10D double mutants die before the larval stage. Unlike Ptp10D, Ptp4E appears to be a relatively minor player in the control of axon guidance. Strong phenotypes are only observed in triple mutants in which both Type III RPTPs are eliminated together with Ptp69D or Ptp52F. Our results allow us to construct a complete genetic interaction matrix for all six of the RPTPs

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. Here we report the isolation of mutations in the fourth neural RPTP, DPTP10D. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. Here we show that the Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. We also show that elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals

Transmural migration of a surgical compress into the stomach after splenectomy: a case report

A surgical compress retained in the abdominal cavity following surgery is a serious problem. Here, we describe a 33-year-old female who was admitted with abdominal pain, vomiting, no passage of gas or feces, and abdominal distension for 3 days. She had a splenectomy at another medical center 4 years previously. An upright plain abdominal film revealed small bowel obstruction with marked small bowel air-fluid levels. The physical examination revealed muscular guarding and rebound tenderness in the periumbilical region. Therefore, a laparotomy was performed. A surgical compress was removed at enterotomy and the final diagnosis was gossypiboma. Because a retained surgical compress may lead to medicolegal problems, it is important to count the material used before and after a surgical procedure to reduce the risk of this problem

Fonctionnement du drainage au sein d'une oasis modernisée du sud tunisien. Cas de l'oasis de Fatnassa Nord à Kébili, Tunisie

Un dispositif pérenne de suivi de l'irrigation et du drainage a été mis en place par l'INRGREF et le Cemagref dans l'oasis de Fatnassa Nord. Ce dispositif expérimental permet un suivi temporel des volumes et des concentrations en sels des eaux d'irrigation et de drainage au niveau parcellaire et de l'ensemble du périmètre (l'oasis) et ce en vue de décrire et d'évaluer l'efficience de ces systèmes « récents » dans ce contexte oasien. Ceci nous a amené à caractériser et à quantifier les bilans hydrique et salin au niveau de l'oasis (114 ha) et d'une parcelle (0,8 ha), et de faire le lien avec les modalités de gestion de l'irrigation au sein des exploitations. Dans ce contexte, quelques résultats obtenus entre avril 2003 et septembre 2005 sont présentés dans cette communication. A l'échelle de la parcelle, l'analyse piézométrique met en évidence une très forte dynamique de la nappe superficielle suite aux irrigations, qui témoignent d'une remontée systématique de cette nappe à chaque irrigation et d'une vitesse de tarissement assez liée à la demande climatique. En termes de sels apportés, ces cumuls correspondent respectivement à 48,4 ; 30,4 et 23,3 t/ha de sels. Cela montre l'importance cruciale du drainage et de l'apport d'une fraction de lessivage pour mieux contrôler cette salinité. Les mesures au niveau de la sortie ont montré une variation saisonnière de la fraction drainée et en fonction du volume apporté à chaque tour d'eau. Par ailleurs, on a estimé l'exportation de sels due au pompage au niveau de cette parcelle vers les extensions et qui représente un ordre de grandeur quadruple de celui du drainage, soit de 60 tonnes /ha /an. Cette pratique traduit une gestion hydrosaline complexe entre l'oasis et les extensions et il est nécessaire de mieux estimer le poids de cette gestion dans l'établissement du bilan notamment à l'échelle du périmètre. A l'échelle de l'oasis, les différents termes du bilan hydrosalin (entrée - sortie) révèlent un bilan déficitaire sur toute la période de mesure. Les quantités d'eau et de sels sont estimées en entrée et en sortie du système sur deux années de mesure. Les entrées d'eau et de sels sont mesurées à l'échelle de la parcelle et extrapolées à l'échelle de l'oasis, et sont confirmées par les informations recueillies au niveau des forages. Les sorties par drainage souterrain sont mesurées indépendamment à deux exutoires. En revanche, les sorties de sel par l'écoulement naturel de la nappe vers le Chott n'ont pas été estimées. Au niveau des deux exutoires, 15 t/ha de sels ont été évacués par drainage sur 48 t/ha apportés pour l'année 1 et 5 t/ha ont été évacués sur 30 t/ha apportés pour l'année 2 avec une bonne répétabilité entre les deux exutoires. Le rapport correspondant entre volume de drainage et d'irrigation est de 11 % et 6 % respectivement aux deux années de mesure avec un coefficient de concentration quasi-constant de l'ordre de 2,8. Ces résultats traduisent un déficit respectif de 69 % et 84 % en termes de quantités de sels évacués par drainage. Ceci nous amène à nous interroger sur les performances techniques et économiques d'un tel système de drainage souterrain et sur sa pérennité dans ce milieu vu sa sensibilité au colmatage, constatée sur le terrain, par ensablement et pénétration des racines

Unpublished Mediterranean records of marine alien and cryptogenic species

Good datasets of geo-referenced records of alien species are a prerequisite for assessing the spatio-temporal dynamics of biological invasions, their invasive potential, and the magnitude of their impacts. However, with the exception of first records on a country level or wider regions, observations of species presence tend to remain unpublished, buried in scattered repositories or in the personal databases of experts. Through an initiative to collect, harmonize and make such unpublished data for marine alien and cryptogenic species in the Mediterranean Sea available, a large dataset comprising 5376 records was created. It includes records of 239 alien or cryptogenic taxa (192 Animalia, 24 Plantae, 23 Chromista) from 19 countries surrounding the Mediterranean Sea. In terms of records, the most reported Phyla in descending order were Chordata, Mollusca, Chlorophyta, Arthropoda, and Rhodophyta. The most recorded species was Caulerpa cylindracea, followed by Siganus luridus, Magallana sp. (cf. gigas or angulata) and Pterois miles. The dataset includes records from 1972 to 2020, with the highest number of records observed in 2018. Among the records of the dataset, Dictyota acutiloba is a first record for the Mediterranean Sea. Nine first country records are also included: the alga Caulerpa taxifolia var. distichophylla, the cube boxfish Ostracion cubicus, and the cleaner shrimp Urocaridella pulchella from Israel; the sponge Paraleucilla magna from Libya and Slovenia; the lumpfish Cyclopterus lumpus from Cyprus; the bryozoan Celleporaria vermiformis and the polychaetes Prionospio depauperata and Notomastus aberans from Malta