Effect of ADP-ribosylation and phosphorylation on the interaction of elongation factor 2 with guanylic nucleotides.

Samples of unmodified EF-2, EF-2 ADP-ribosylated with diphtheria toxin and NAD, and/or phosphorylated using ATP and the Ca(2+)-calmodulin dependent kinase III partially purified, were irradiated at 254 nm with 32P-labeled GDP or GTP, and analyzed by one- and two-dimensional gel electrophoresis. By this method we showed that unmodified EF-2 formed a stable complex with GDP but not with GTP, whereas phosphorylated EF-2 and ADP-ribosylated + phosphorylated EF-2 formed stable complexes even in the absence of irradiation, with GTP but not GDP. ADP-ribosylated EF-2 did not form stable complexes with either GDP or GTP. Prior ADP-ribosylation of EF-2 increased its ability to the phosphorylated. These results show that the structures of the two domains containing diphtamide 715 and the phosphorylatable threonines (between Ala 51 and Arg 60) are interdependent; modifications of these residues induce different conformational changes of EF-2 which alter the interactions of the factor with guanylic nucleotides as well with ribosomes.     

Synthesis,Antimicrobial Activity and Molecular Docking Study of Novel α‐(Diphenylphosphoryl)‐ and α‐(Diphenylphosphorothioyl)cycloalkanone Oximes

A series of novel α‐(diphenylphosphoryl)‐ and α‐(diphenylphosphorothioyl)cycloalkanone oximes have been synthesized in search for novel bioactive molecules. Their structures were characterized by various spectroscopic methods including IR, NMR (¹H, ³¹P, ¹³C), mass spectrometry and single crystal X‐ray diffraction. The newly synthesized phosphorus‐containing oximes were screened for their in vitro antimicrobial activity against Gram‐positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram‐negative bacteria (Escherichia coli and Salmonella typhimurium) and fungal strains (Candida albicans and Candida glabrata). The biological assays showed that all the studied compounds exhibited high antibacterial and antifungal activities at only 0.1–2.1 μg/mL. In silico molecular docking studies in FabH enzyme active site were performed in order to predict the possible interaction modes and binding energies of the drug candidates at the molecular level.     

The role of myoglobin in retarding oxygen depletion in anoxic heart

The present study explores the role of myoglobin (Mb) in retarding the development of anoxia in the perfused working rat heart. We examine this phenomenon by analyzing the behavior and the kinetics of Mb oxygenation and cytochrome aa3 (cytaa3) redoxation. Absorbance changes, measured at wavelength pairs specific to Mb and cytaa3, show parallelism between the Mb oxygenation status and the redox states of cytaa3. Induction of anoxia leads to early and accelerated Mb deoxygenation whereas cytaa3 reduction marks a slight delay and its rate is twice slower than that of Mb. Then, when Mb is desatured above 50%, the cytaa3 reduction becomes accelerated. With the reoxygenated perfusion following the anoxia, the rate of Mb reoxygenation is twice faster than that of the cytaa3 reoxidation. When the oxygen-binding function of Mb, in situ in the heart, is abolished by treatment with sodium nitrite (NaNO2), the redox kinetics of cytaa3 show significant perturbations. Induction of anoxia leads to a precocious and accelerated reduction of cytaa3, compared to the same anoxic heart before the treatment. At reoxygenation, the reoxidation rate of cytaa3 decreases significantly, compared to that before the treatment. Similarly, in the nitrite treated heart, the phosphocreatine (PCr) level decreases to 60% of the control, whereas the inorganic phosphate (Pi) level increases to 300%. ATP concentration, however, remains constant. We conclude from these results that Mb may support mitochondrial respiration at the critical levels of the myocardial O2 supply.     

Decolorization of an industrial effluent by free and immobilized cells of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> AAP56<Emphasis Type="Italic">.</Emphasis> Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD₅ of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h⁻¹. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.     

β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K _m and V _max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.     

Efficient synthesis of gluco-oligosaccharides and alkyl-glucosides by transglycosylation activity of β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

An extracellular β-glucosidase (β-glu x) from Sclerotinia sclerotiorum was used as catalyst for the synthesis of gluco-oligosaccharides (GOSs) and alkyl-glucosides. The purified β-glu x was not regiospecific for β(1→4) linkages in either hydrolysis or transglycosylation catalysed-reactions. It efficiently synthesized GOSs from cellobiose, gentiobiose and methyl β-d-glucoside by transglycosylation. At optimal conditions, 119 mg/ml of GOSs (∼ ∼33%) were formed over 9 h from cellobiose as substrate. Alkyl-glucosides were also efficiently synthesized by transglycosylation of cellobiose in presence of different alcohols in biphasic media. However, their concentrations decreased as the size of the alcohol chain increased.     

Cloning and molecular characterization of a new fungal xylanase gene from Sclerotinia sclerotiorum S2

Sclerotinia sclerotiorum fungus has three endoxylanases induced by wheat bran. In the first part, a partial xylanase sequence gene (90 bp) was isolated by PCR corresponding to catalytic domains (β 5 and β 6 strands of this protein). The high homology of this sequence with xylanase of Botryotinia fuckeliana has permitted in the second part to amplify the XYN1 gene. Sequence analysis of DNA and cDNA revealed an ORF of 746 bp interrupted by a 65 bp intron, thus encoding a predicted protein of 226 amino acids. The mature enzyme (20.06 kDa), is coded by 188 amino acid (pI 9.26). XYN1 belongs to G/11 glycosyl hydrolases family with a conserved catalytic domain containing E(86) and E(178) residues. Bioinformatics analysis revealed that there was no Asn-X-Ser/Thr motif required for N-linked glycosylation in the deduced sequence however, five O-glycosylation sites could intervene in the different folding of xylanses isoforms and in their secretary pathway.     

Concepts on charge transfer through naturally vibrating DNA molecule

Delocalization of charges thorough DNA occurs due to the natural and continuous movements of molecule which stimulates the charge transfer through the molecule. A model is presented showing that the mechanism of electrical conduction occurs mainly by thermally-activated drift motion of holes under control of the localized carriers; where electrons are localized in the conduction band. These localized (stationary-trapped) electrons control the movements of the positive charges and do not play an effective role in the electrical conduction itself. It is found that the localized charge-carriers in the bands have characteristic relaxation times at 5×10(^-2)s, 1.94×10(^-4)s, 5×10(^-7)s, and 2×10(^-11)s respectively which are corresponding to four intrinsic thermal activation energies 0.56eV, 0.33eV, 0.24eV, and 0.05eV respectively. The ac-conductivity of some published data are well fitted with the presented model and the total charge density in DNA molecule is calculated to be n=1.88×10(^19)cm(^-3) at 300K which is corresponding to a linear electron density n=8.66×10(^3)cm(^-1) at 300K. The model shed light on the role of transfer and/or localization of charges through DNA which has multiple applications in medical, nano-technical, bio-sensing and different domains. So, repair DNA by adjusting the charge transport through the molecule is future challenges to new medical applications.     

Enzymatic synthesis of fructooligosaccharides from date by-products using an immobilized crude enzyme preparation of <Emphasis Type="Italic">β</Emphasis>-D-fructofuranosidase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis> NBRC 4033

Aqueous extracts from date by-products of the sucrose-rich variety “Deglet Nour” were used as a starting substrate to achieve the enzymatic synthesis of fructooligosaccharides (FOS) commonly used as prebiotics. A crude β-fructofuranosidase (Ffase) preparation from Aspergillus awamori NBRC4033 was immobilized on chitosan by covalent binding through glutaraldehyde linkages (Yi = 88%, Ya = 54%), and used for this purpose. The effect of water-extraction volume on the FOS synthesis by transfructosylation was studied. It was found that 150 mL/100 g of date by-products gave the best FOS concentration and productivity (123 g/L and 18.5 g/h/100 g respectively), related to an optimal sucrose conversion of 53.26%. The main FOS product was purified via a biogel-P2 gel filtration column. Its structure was determined as 1-kestose: α-Dglucopyranosyl-( 1→2)-β-D-fructofuranosyl-(2→1)-β-Dfructofuranoside by combination of ¹H, ¹³C and 2D-NMR techniques. Our results provide new insights into the enzymatic synthesis of FOS from an alternative source of sucrose, namely date by-products.     

Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds

Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 μM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.