Genetic diversity and population structure of Tunisian wild Kermes oak (Quercus coccifera L.): Assessment by ISSR molecular markers and implication for conservation

Molecular Biology Reports - In Tunisia, Kermes oak (Quercus coccifera L.) populations are severely destroyed due to deforestation. Nowadays, no preservation programs are attempted, yet, to conserve...     

Involvement of cysteine 306 and alanine 63 in the thermostability and oligomeric organization of glucose isomerase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. SK

The implication of the original alanine 63 (Ala63) and the unique cysteine 306 (Cys306) residues in the thermostability of the Streptomyces sp. SK glucose isomerase (SKGI) were investigated by site-directed mutagenesis and homology modelling. The Cys306 to Ala mutation within SKGI dramatically affected its thermal stability by decreasing the half-life from 80 to 15 min at 90°C while the Ala63 to Ser replacement shifted this half-life to 65 min. The electrophoretic analysis proves that the residue Cys306 participates in oligomerization of the SKGI. Its stabilizing role is materialized by hydrogen bonds established with arginines at positions 284 and 259, as deduced from the constructed three-dimensional model. We have also shown that the presence of an Ala63 instead of Ser63 seems to be more suitable for enzyme thermostability by maintaining hydrophobic pocket that contributes to the protection of the enzyme active site.     

Structuring detergents for extracting and stabilizing functional membrane proteins

Background

Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.

Methodology/Principal Findings

Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by ¹H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.

Conclusion/Significance

These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.     

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process.     

Involvement of alanine 103 residue in kinetic and physicochemical properties of glucose isomerases from Streptomyces species

The Ala103 to Gly mutation, introduced within the glucose isomerase from Streptomyces sp. SK (SKGI) decreased its catalytic efficiency (k(cat)/K(m)) toward D-glucose from 7.1 to 3 mM(-1) min(-1). The reverse counterpart replacement Gly103Ala introduced into the glucose isomerase of Streptomyces olivochromogenes (SOGI) considerably improved its catalytic efficiency to be 6.7 instead of 3.2 mM(-1) min(-1). This later mutation also increased the half-life time of the enzyme from 70 to 95 min at 80 degrees C and mainly modified its pH profile. These results provide evidence that the residue Ala103 plays an essential role in the kinetic and physicochemical properties of glucose isomerases from Streptomyces species.     

Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain

The araA gene encoding L-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 degrees C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 degrees C. At temperatures above 65 degrees C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The V(max) values were calculated to be 41.3 U/mg for L-arabinose and 8.9 U/mg for D-galactose. Their catalytic efficiencies (k(cat)/K(m)) for l-arabinose and D-galactose were, respectively, 71.4 and 8.46 mM(-1) min(-1). L-AI US100 converted the d-galactose into D-tagatose with a high conversion rate of 48% after 7 h at 70 degrees C.     

Molecular recognition by gold, silver and copper nanoparticles

The intrinsic physical properties of the noble metal nanoparticles,which are highly sensitive to the nature of their local molecular environment,make such systems ideal for the detection of molecular recognition events.The current review describes the state of the art concerning molecular recognition of Noble metal nanoparticles.In the first part the preparation of such nanoparticles is discussed along with methods of capping and stabilization.A brief discussion of the three common methods of functionalization:Electrostatic adsorption;Chemisorption;Affinity-based coordination is given.In the second section a discussion of the optical and electrical properties of nanoparticles is given to aid the reader in understanding the use of such properties in molecular recognition.In the main section the various types of capping agents for molecular recognition;nucleic acid coatings,protein coatings and molecules from the family of supramolecular chemistry are described along with their numerous applications.Emphasis for the nucleic acids is on complementary oligonucleotide and aptamer recognition.For the proteins the recognition properties of antibodies form the core of the section.With respect to the supramolecular systems the cyclodextrins,calix[n]arenes,dendrimers,crown ethers and the cucurbitales are treated in depth.Finally a short section deals with the possible toxicity of the nanoparticles,a concern in public health.     

Synergistic Effects of Efflux Pump Modulators on the Azole Antifungal Susceptibility of Microsporum canis

The microbiologic and clinical resistance of dermatophytes is seldom reported, and the mechanisms associated with resistance are not well known. This study investigated the effect of efflux pump modulators (EPMs) (i.e., haloperidol HAL and promethazine PTZ) and their inhibiting activity on the minimum inhibitory concentrations of itraconazole (ITZ) and fluconazole (FLZ) against selected M. canis strains. M. canis strains with low (≤?1 μg/ml itraconazole and?<?64 μg/ml fluconazole) and high (>?1 μg/ml itraconazole and?≥?64 μg/ml fluconazole) azole MIC values were tested using Checkerboard microdilution assay. The disk diffusion assay, the minimum fungicidal concentration and the time-kill assay were also performed in order to confirm the results of checkerboard microdilution assay. The MIC values of ITZ and FLZ of M. canis decreased in the presence of subinhibitory concentrations of HAL and PTZ, the latter being more effective with a greater increased susceptibility. Synergism was observed in all strains with high azole MICs (FICI?<?0.5) and no synergism in the strains with low azole MICs. A fungicidal activity was observed after 48 h of incubation when ITZ and FLZ were tested in combination with HAL or PTZ. These results suggest that the drug efflux pumps are involved in the defense mechanisms to azole drugs in M. canis strains. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Complementary studies on M. canis resistance are advocated in order to investigate the molecular mechanisms of this phenomenon.