Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

The alternative oxidase pathway is involved in optimizing photosynthesis in Medicago truncatula infected by Fusarium oxysporum and Rhizoctonia solani

Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (v_alt) plays a role under fungal infection. When infected with R. solani, the increase of v_alt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced v_alt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.     

Secreted recombinant P53 protein from Pichia pastoris is a useful antigen for detection of serum p53: autoantibody in patients with advanced colorectal adenocarcinoma

The detection of P53 alteration by serological method is easier to perform, does not require tumor tissues and is of interest for patients monitoring. In this study, we described the development of a home made ELISA test based on recombinant human P53 protein produced in Pichia pastoris and used as antigen for the detection of serum p53-Abs in colorectal carcinoma patients. The human P53 was secreted as a His-tagged protein by recombinant KM71 strain (Kα21) via the peptide signal α of the Saccharomyces cerevisiae mating type gene. The recombinant P53-His was able to detect p53-Abs in 23.4 % of patients. Serum p53-Abs correlated significantly with surgical treatment (P = 0.007), relapse during follow-up (P = 0.036), depth of invasion (P = 0.036) and the level of CA19-9 (P = 0.034). Survival analysis showed that patients negative for serum p53-Abs exhibited a prolonged disease free survival period (P log rank = 0.012). In conclusion, the secreted recombinant human P53-His produced in P. pastoris seems to be a useful antigen for detection of serum p53 Abs in patients with colorectal carcinoma.     

Reproductive biology of the annular seabream,Diplodus annularis (Linnaeus, 1758), in the Gulf of Gabes (Central Mediterranean)

Annular seabream, Diplodus annularis (Linnaeus, 1758), were caught off the coast of the Gulf of Gabes (Southern Tunisia, Central Mediterranean) between April 2008 and March 2010 by commercial catches. With a total 2066 specimens the fish ranged in size from 7.7 cm to 18.5 cm total length and from 7.5 g to 123.3 g in weight. Changes in biological parameters (weight, length, gonadosomatic index, hepatosomatic index and condition factor) were examined in order to provide information on the spawning period and reproductive cycle in the gulf. Overall sex ratio was 1 : 1.52 in favour of females. The reproductive season extends from March to June, with a spawning activity peak in May. Total length at sexual maturity was 10.5 ± 0.22 cm (males) and 10.6 ± 0.3 cm (females). The results of this study could help to establish a recommended minimum capture size.     

Performances de la scintigraphie parathyroïdienne au 99mTc-Sestamibi dans l’hyperparathyroïdie secondaire (à propos de 20 cas)

Aim of the studyTo evaluate the performance of the ^99mTc-Sestamibi parathyroid scintigraphy and to compare it with the performance of cervical ultrasonography in patients with secondary hyperparathyroidism who are candidates for parathyroidectomy.Patients and methodsWe performed a retrospective study including 20 patients with severe secondary hyperparathyroidism who underwent parathyroid scintigraphy in the nuclear medicine department of Sfax, during the period between January 2009 and June 2012. Our two days protocol included dual-phase, MIBI/Tc subtraction and single photon emission photons (SPECT) techniques. We analyzed the results obtained from each technique alone, then from combinations thereof. For all patients, we have collected the surgical and histopathological data as well cervical ultrasound if available.ResultsThe subtraction technique was the best performing with a sensitivity of 47% and an accuracy of 55%. The combination of subtraction scintigraphy and SPECT has improved the sensitivity to 53%and accuracy to 57%. The combined lecture of ultrasound and scintigraphy has given the best performance with a sensitivity of 58%, a specificity of 83% and an accuracy of 66%.ConclusionParathyroid scintigraphy combining subtraction and SPECT showed better reliability. The coupling with ultrasound is essential to improve results. The poor performance of scintigraphy in secondary hyperparathyroidism implies that it should be required only to search for ectopic or supernumerary glands.     

Surface Nanostructure Control with Poly (ethylene glycol)(PEG) Spacer by Templateless Electropolymerization

Controlling the shape of surface nanostructures is fundamental for various potential applications for cxamples,in water harvesting systems,liquid transportation or oil/water separation membranes.In this paper,the creation of porous surface structures is made by a process called templateless electropolymerization,in which water(H₂0)is oxidizedreduced to form gas(O₂/H₂)bubbles onto the surfaces and acting as soft template for the polymer growth.Keeping the monomer(thieno[3,4-b]lhiophene)and the substituent(pyrene)constant,we demonstrate how a flexible PEG spacer can affect the structure shape.When the PEG spacer increases,the structures change frorm nanotubes(1D growth)to nanoribbons(2D)and after to hollow nanospheres(3D),which also affeets the wettig propertics.     

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated.In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.     

Reversibility of the effects of cadmium on the growth and nitrogen metabolism in the tomato(Lycopersicon esculentum)

In order to better understand the effects of heavy metals on the growth of plants, we decided to perform recovering experiments by following both chemical and physiological parameters in cadmium pre-stressed tomato seedlings after cadmium had been removed from the nutrient solution. The work shows that cadmium suppression results in resumption of growth activity. The biomass of leaves and stems rose steadily. The increase in root biomass exceeded those of leaves and stems. At the same time, nitrate content was increased to reach the level obtained with unstressed controls. In all the organs studied, the activities of the enzymes involved in the anabolic nitrogen primary assimilation pathways (nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) soared after that cadmium had been removed. While NAD(+)-dependent glutamate dehydrogenase (GDH-NAD+) activity also rose progressively during the recovering time, the cognate NADH-dependent glutamate dehydrogenase (GDH-NADH) activity decreased. This result allows us to propose that the ammonia produced by the stress-induced protein catabolism is detoxified and re-assimilated by the GDH-NADH isoenzyme. On the basis of these results, we will discuss the ability of the plant to dilute the effects of pollutants during the recovering period. An important outcome of this work is that a transient contamination of the culture medium by pollutants is not necessarily followed by a significant depreciation in product yield or quality.     

Mutation Spectrum of RB1 Gene in Unilateral Retinoblastoma Cases from Tunisia and Correlations with Clinical Features

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.