The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.     

Efficient Transformation Procedure of a Newly Isolated <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TN58 Strain Producing Antibacterial Activities

A new aerobic Gram-positive bacterium designated TN58 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. The nucleotide sequence of the 16S rRNA gene (1516 bp) of the TN58 strain showed high similarity (96–98%) to the Streptomyces 16S rRNA genes, especially with that of Streptomyces lavendulae which produces the anti-tumor compound mitomycin C, and the cyclic peptide antibiotic, complestatin. Cultural characteristic studies, alignment data of the 16S rRNA gene, and analysis of the nucleotide sequence of a 2.2 kb genomic DNA fragment from TN58 strongly suggested that this strain could be an actinomycete and most probably belongs to the genus Streptomyces. Study of the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of potassium. In analytical conditions, the application to supernatant culture of the TN58 strain of various extraction and purification steps led to the isolation of two pure active molecules having a retention time of 38.6 and 50.2 min, respectively. TN58 strain was untransformable with the Streptomyces cloning vector pIJ702 via classical polyethylene glycol (PEG) protoplast transformation and previously described Streptomyces electroporation procedures. Transformation was rendered possible by the electroporation technique only after utilization of a preculture medium without sucrose and a regeneration plate containing a low sucrose concentration.     

Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis,Molecular and Docking Studies

We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB₁ and AmyB₂ reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)₈ barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.     

A new Lactobacillus plantarum strain, TN8, from the gastro intestinal tract of poultry induces high cytokine production

This study aimed to determine the probiotic potential of 100 strains of Lactic Acid Bacteria (LAB) isolated from different intestinal segments of indigenous poultry in Tunisia. The strains were submitted to a battery of standard tests and criteria commonly used for determining their probiotic properties and attributes. The findings revealed that 19 of the isolates exhibited antimicrobial activity against 4 pathogenic bacteria, and that 4 (TN1, TN8, TN7, and TN13) showed good resistance to pH 3 and 5% bovine bile. Three isolates, namely TN1, TN8, and TN13, showed sensitivity to several antibiotics and were, therefore, selected for further enzymatic activity assays. Two isolates, namely TN1 and TN8, showed high efficacy of adhesion to chicken enterocytes. The cytokines released after stimulation by the two isolates showed high anti-inflammatory profiles, with an increased rate of Interleukin-10 (IL-10) production for the TN8 strain. Showing the highest performance, TN8 was submitted to 16S rRNA gene sequencing, which revealed that the strain was of the species Lactobacillus plantarum. Overall, the findings indicate that the Lactobacilli from poultry intestine has a number of promising properties that make it candidate for application as a probiotic additive in poultry industry.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.     

Sex‐specific genetic differences in endurance swimming of Trinidadian guppies

Swim performance is considered a main fitness‐determining trait in many aquatic organisms. Swimming is generally the only way most aquatic prey can escape predation, and swimming capacity is directly linked to food capture, habitat shifts, and reproduction. Therefore, evolutionary studies of swim performance are important to understand adaptation to aquatic environments. Most studies, however, concentrate on the importance of burst‐swim responses to predators, and little is known about its effect on endurance. Even fewer studies associate differences in organism swim capabilities to key gender‐specific responses. In this experiment, we assess the gender‐specific genetic basis of swimming endurance among four different populations of Trinidadian guppies adapted to different predation regimes. Our results show that second‐generation common‐garden females adapted to a low‐predation environment show longer swim endurance than fish adapted to a high‐predation environment. We also find an expected effect of lowered swimming endurance during pregnancy, but interestingly, it did not matter whether the females were in advanced stages of pregnancy, which severely changes body morphology, versus mid‐pregnancy. Males did not show the same trends across populations, and overall had lower swim endurances than female fish combined even when accounting for size differences. Populations recently transplanted from high‐ to low‐predation environments showed similar endurance to natural low‐predation environments in one population but not the other. This study highlights the importance of endurance in the adaptation of aquatic organisms to different predation regimes.     

ADPglucose pyrophosphorylase's N-terminus: structural role in allosteric regulation

We studied the functional role of the Escherichia coli ADPglucose pyrophosphorylase's N-terminus in allosteric regulation, and the particular effects caused by its length. Small truncated mutants were designed, and those lacking up to 15-residues were active and highly purified for further kinetic analyses. Ndelta3 and Ndelta7 did not change the kinetic parameters with respect to the wild-type. Ndelta11 and Ndelta15 enzymes were insensitive to allosteric regulation and highly active in the absence of the activator. Co-expression of two polypeptides corresponding to the N- and C-termini generated an enzyme with activation properties lower than those of the wild-type [C.M. Bejar, M.A. Ballicora, D.F. Gómez Casati, A.A. Iglesias, J. Preiss, The ADPglucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains, FEBS Lett. 573 (2004) 99-104]. Here, we characterized a Ndelta15 co-expression mutant, in which the allosteric regulation was restored to wild-type levels. Unusual allosteric effects caused by either an N-terminal truncation or co-expression of individual domains may respond to structural changes favoring an up-regulated or a down-regulated conformation rather than specific activator or inhibitor sites' disruption.     

Biochemical characterization,cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain

New β-1,4-d-xylan xylanohydrolase (XAn11) belonging to the xylanase 11 family was purified to homogeneity from a newly soil-isolated Aspergillus niger US368 strain. The pure xylanase is a glycosylated monomer having a molecular mass of about 26 kDa. The N-terminal sequence of the purified enzyme was determined and compared to some Aspergillus xylanases N-terminal ones. The gene encoding the XAn11 was cloned and sequenced.The maximal xylanase activity was obtained at pH 5.0 and 55 °C. The XAn11 was found to be stable in a wide range of pH (3–9) and in presence of some detergents and organic solvents. A specific activity of about 805.6 U/mg or 334 U/mg was measured using birchwood xylan or oatspelt xylan as substrate, respectively. A structural explanation of the difference between experimental and theoretical molecular mass as well as the stability of the enzyme against acidic pH was proposed by molecular modeling.     

Production, purification and characterization of two α-amylase isoforms from a newly isolated Aspergillus Oryzae strain S2

A new fungal strain that was isolated from old sweet soy sauce was identified, based on subsequent microscopic studies and analyses of rRNA18S gene sequence, intergenic region rRNA 18S-23S, and aflatoxins production tests, as an Aspergillus oryzae strain. The latter was noted to produce two extracellular α-amylases, namely AmyA and AmyB. The monitoring of alpha-amylase production in the presence and absence of various protease inhibitors indicated that AmyB could be formed from the proteolysis of AmyA. The enzymes were purified to homogeneity through fractional acetone precipitation, size exclusion, and anion exchange chromatography. The molecular masses estimated for AmyA and AmyB by SDS-PAGE were 50 and 42 kDa, respectively. The NH₂-terminal of the purified proteins showed the same amino acid sequences. Further biochemical characterization assays revealed that both enzymes attained maximal activity at pH 5.6 and 50 °C. They were activated and stabilized by Ca²⁺ and were noted to produce maltose and maltotriose as major starch hydrolysis end products. Overall, the findings of the present study indicate that both AmyA and AmyB exhibit a number of promising properties that make them potential strong candidates for application as additives in the bread making industry.