Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD – nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N₂ fixation using the δ¹⁵N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N₂-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.     

Agricultural wastes as substrates for β-glucosidase production by Talaromyces thermophilus: Role of these enzymes in enhancing waste paper saccharification

In the present study, we investigated a potent extracellular β-glucosidases secreted by the thermophilic fungal strain AX4 of Talaromyces thermophilus, isolated from Tunisian soil samples. This strain was selected referring to the highest thermostability of its β-glucosidases compared to the other fungal isolates. The β-glucosidase production was investigated by submerged fermentation. The optimal temperature and initial pH for maximum β-glucosidase production were 50°C and 7.0, respectively. Several carbon sources were assayed for their effects on β-glucosidase production, significant yields were obtained in media containing lactose 1% (3.0?±?0.36?U/ml) and wheat bran 2% (4.0?±?0.4?U/ml). The combination of wheat bran at 2% and lactose at 0.8% as carbon source enhanced β-glucosidase production, which reached 8.5?±?0.28?U/ml. Furthermore, the β-glucosidase-rich enzymatic juice of T. thermophilus exhibited significant synergism with Trichoderma reesei (Rut C30) cellulases for pretreated waste paper (PWP) hydrolysis. Interestingly, the use of this optimal enzymatic cocktail increased 4.23 fold the glucose yield after saccharification of waste paper. A maximum sugar yield (94%) was reached when using low substrate (2%) and enzyme loading (EC1).     

Reuse of Textile Wastewater after Treatment with Isolated Bacteria from Oued Hamdoun River

Industrialization is a boon for developing countries such as Tunisia. However, textile effluents that are being discharged are environmental pollutants, extremely toxic to plants and other living organisms, including humans. The current study was conducted to determine the phytotoxic effect of textile effluents, collected near an industrial zone in the center of Tunisia (Ksar Helal), on the germination and various growth indices of durum wheat (Triticumaestivum L.). Results showed that textile effluent treatments reduced significantly the percentage of seed germination and slowed its kinetic as compared with control. Roots and leaves were also significantly reduced. The phytotoxicity was highly reduced from textile effluents after aerobic biotreatment with bacteria. It can be concluded that the biological treatment process of textile wastewater might serve as a fertilizer production that is able to improve the growth of plants. These results are encouraging in the context of developing a low-budget technology for the effective management of these effluents.     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Partial purification of a Bacillus licheniformis levansucrase producing levan with antitumor activity

The extracellular fructosyltransferase (FTase) of a novel strain of Bacillus licheniformis capable of producing fructooligosaccharides (FOS) and a polysaccharide type levan was obtained and partially purified. The purification was achieved by ammonium sulfate precipitation, DEAE cellulose and gel filtration chromatographies. The enzyme was partially purified as determined by SDS-PAGE, and the specific activity reached was 67.5, representing a purification factor of 177 and yield of 40%. Levan was isolated from the cultures of B. licheniformis. The levan was composed mainly of fructose residues when analyzed by TLC after acid hydrolysis and NMR analysis. In a previous study, the levan produced exhibited a hypoglycemiant activity. The present paper deals with the study of the antitumor and anti-cytotoxic effect of levan produced by B. licheniformis strain. In the in vitro antitumor activity test of levan against some tumor cell lines, relatively the significantly high activity was observed against the HepG(2).     

Physiological and ultrastructural responses of sour orange (Citrus aurantium L.) clones to water stress

Water stress is a major abiotic constraint leading to serious crop losses. Recently, in the Mediterranean region, water stress has become markedly sensed, especially in Citrus orchards. This study investigated the physiological responses of local sour orange (Citrus aurantium L.) clones to severe water stress. Water stress was applied by withholding irrigation during weeks, followed by a rewatering phase during three months. Under water stress, sour orange clones decreased their stomatal conductance, net photosynthetic rate, and transpiration rate. On the contrary, biomass was stable, especially in the Kliaa clone. In addition, reduced leaf water potentials (-3 MPa) and water contents were measured in most of the clones, except Kliaa which kept the highest water potential (-2.5 MPa). After rewatering, all clones recovered except of the Ghars Mrad (GM) clone. Ultrastructural observations of leaf sections by transmission electron microscopy did not reveal marked alterations in the mesophyll cells and chloroplast structure of Kliaa in comparison to the sensitive clone GM, in which palisade parenchyma cells and chloroplasts were disorganized. This contrasting behavior was mainly attributed to genetic differences as attested by molecular analysis. This study highlighted GM as the drought-sensitive clone and Kliaa as the tolerant clone able to develop an avoidance strategy based on an efficient stomatal regulation. Although a high percentage of polyembryony characterizes C. aurantium and justifies its multiplication by seeds, heterogeneous water-stress responses could be observed within sour orange plants in young orchards.     

Traumatismes fermés des bourses: stratégie de prise en charge

Introduction

Blunt scrotal trauma is increasingly frequent, but surgical exploration of these cases of trauma remains controversial. The objectives of this study were to assess the diagnostic value of clinical examination and ultrasound in testicular trauma and to analyse the complications of the various treatments proposed (surgical and medical treatments), in order to more clearly define the place of medical or surgical treatment in this form of trauma in young adults.

Patients and Methods

130 cases of blunt scrotal trauma were managed between 1993 and 2004. In the absence of clinical and ultrasound criteria of severity (haematocele, very large intratesticular haematoma, rupture of the tunica albuginea), medical treatment consisting of rest, anti-inflammatory drugs, and testicular support was instituted. Surgical exploration was performed when serious lesions of the testis were suspected. Scrotal ultrasound was performed in 68 patients and 46 of them underwent scrotal exploration. The sensitivity and specificity of scrotal ultrasound were determined by comparing radiological findings with definitive intraoperative findings. The immediate morbidity and long-term sequelae were analysed.

Results

The mean age of the patients was 24 years (range: 4 to 73 years). The clinical features were dominated by pain (73.8%) and scrotal swelling (89.2%). The sensitivity of testicular ultrasound was 34.7% for testicular rupture, 100% for testicular contusions, 33.3% for intratesticular hematoma and 76.9% for haematocele. Medical and surgical treatments were instituted in 29.2% and 70.7% of cases, respectively. With a mean follow-up of 8 months, chronic pain and testicular atrophy were observed in 18% and 5.5% of cases, respectively.

Conclusion

In the absence of signs of severity, medical treatment with regular surveillance remains justified. However, in the presence of doubtful clinical or ultrasound findings, surgical exploration must be performed as soon as possible     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.     

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities

A marine snail digestive phospholipase A₂ (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A₂, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 °C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6 mM CaCl₂. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases.Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA₂. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A₂ from venoms and another class of non toxic pancreatic phospholipase A₂ from mammals.     

An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.