The MntC crystal structure suggests that import of Mn2+ in cyanobacteria is redox controlled.

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 A resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended alpha-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.

Electrophysiological modulation of cardiomyocytic tissue by transfected fibroblasts expressing potassium channels: a novel strategy to manipulate excitability.

BACKGROUND: Traditional pharmacological therapies aiming to modify the abnormal electrophysiological substrate underlying cardiac arrhythmias may be limited by their relatively low efficacy, global cardiac activity, and significant proarrhythmic effects. We suggest a new approach, in which transfected cellular grafts expressing various ionic channels may be used to manipulate the local electrophysiological properties of cardiac tissue. To examine the feasibility of this concept, we tested the hypothesis that transfected fibroblasts expressing the voltage-sensitive potassium channel Kv1.3 can modify the electrophysiological properties of cardiomyocytic cultures. METHODS AND RESULTS: A high-resolution multielectrode mapping technique was used to assess the electrophysiological and structural properties of primary cultures of neonatal rat ventricular myocytes. The transfected fibroblasts, added to the cardiomyocytic cultures, caused a significant effect on the conduction properties of the hybrid cultures. These changes were manifested by significant reduction in extracellular signal amplitude and by the appearance of multiple local conduction blocks. The location of all conduction blocks correlated with the spatial distribution of the transfected fibroblasts assessed by vital staining. All electrophysiological changes were reversed after the application of Charybdotoxin, a specific Kv1.3 blocker. In contrast, conduction remained uniform in the control hybrid cultures when nontransfected fibroblasts were used. CONCLUSIONS: Transfected fibroblasts are able to electrically couple with cardiac myocytes, causing a significant local and reversible modification of the tissue's electrophysiological properties. More broadly, this study suggests that transfected cellular grafts expressing various ionic channels may be used to modify cardiac excitability, providing a possible future novel cell therapy strategy.

Getting out of a wheelchair: an uncommon insertion mutation in exon 19 of EGFR responsive to erlotinib.

BACKGROUND: Most patients with non-small cell lung cancer (NSCLC) present with advanced disease and have poor long-term prognosis. Advanced NSCLC that contains characteristic mutations in epidermal growth factor receptor (EGFR) are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs). EGFR exon 19 insertions mutations are rare, and response to TKIs is still unclear. CASE DESCRIPTION: A young Arab patient was diagnosed with metastatic disease of NSCLC harboring an exon 19 insertion of 18 nucleotides. The patient showed a very impressive clinical and radiological response within few weeks treatment with TKI agent. DISCUSSION AND EVALUATION: To our best knowledge, This case is the first case in Arab woman and one of few cases described in the literature with this rare mutation responding to TKIs. CONCLUSIONS: Treatment with TKIs should be the standard choice in patients with metastatic disease NSCLC.

The dimeric structure of the Cpn60.2 chaperonin of Mycobacterium tuberculosis at 2.8 Å reveals possible modes of function.

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF.

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF +/- internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 microg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF-IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF-IRES (-) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.