Using of electrochemical methods for studying of metallothionein content in the human blood serum of a patient poisoned by lead and treated by platinum

Metallothioneins belong to the group of intracellular, high molecular and cysteine-rich proteins whose content increase with increasing concentration of a heavy metal. Here we applied the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothionein in human blood serum of patient poisoned by lead and/or treated by platinum. The increased metallothionein concentrations in both cases were observed.     

5-benzylidenerhodanine and 5-benzylidene-2-4-thiazolidinedione based antibacterials

Herein we outline the antibacterial activity of amino acid containing thiazolidinediones and rhodanines against Gram-positive bacteria Staphylococcus aureus ATCC 31890, Staphylococcus epidermidis and Bacillus subtilis ATCC 6633. The rhodanine derivatives were generally more active than the analogous thiazolidinediones. Compounds of series 5 showed some selectivity for Bacillus subtilis ATCC 6633, the extent of which is enhanced by the inclusion of a non-polar amino acid at the 5-position of the core thiazolidinediones and rhodanines scaffolds. SAR data of series 8 demonstrated improved activity against the clinically more significant Staphylococci with selectivity over Bacillus subtilis ATCC 6633 induced by introduction of a bulky aryl substituent at the 5-position of the core scaffolds.     

Functional analysis of the single Est1/Ebs1 homologue in Kluyveromyces lactis reveals roles in both telomere maintenance and rapamycin resistance

Est1 and Ebs1 in Saccharomyces cerevisiae are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast Kluyveromyces lactis (named KlEst1). We show that similar to other Est1s, KlEst1 is required for normal telomere maintenance in vivo and full telomerase primer extension activity in vitro. KlEst1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of KlEst1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between KlEst1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to S. cerevisiae Ebs1 (ScEbs1) (but not ScEst1), KlEst1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of KlEst1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.     

Complexes of Streptavidin-Fused Antigens with Biotinylated Antibodies Targeting Receptors on Dendritic Cell Surface: A Novel Tool for Induction of Specific T-Cell Immune Responses

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.     

Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia

Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Bcc (Burkholderia cepacia complex) is the major cause of morbidity and mortality among CF (cystic fibrosis) patients. PA-IIL (lecB gene), a soluble lectin from Ps. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologues have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centres, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA (B. cenocepacia lectin A), was obtained. The presence of native BclA in B. cenocepacia extracts was checked using a proteomic approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with methyl alpha-D-mannoside demonstrates a dissociation constant (K(d)) of 2.75 x 10(-6) M. The X-ray crystal structure of the complex with methyl alpha-D-mannoside was determined at 1.7 A (1 A=0.1 nm) resolution. The lectin forms homodimers with one binding site per monomer, acting co-operatively with the second dimer site. Each monomer contains two Ca2+ ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site and oligomerization mode indicate that the proteins should have different roles in the bacteria.