Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration.     

Cytochemical localization of alkaline phosphatase and ouabain-sensitive K+-dependent p-nitrophenylphosphatase activities in brain capillaries of the newt

The ultrastructural localization of alkaline phosphatase and K+-NPPase was investigated in brain capillaries of newt by a cytochemical study using whole brain perfusion. The alkaline phosphatase activity was present in both luminal and antiluminal membranes of the endothelial cells. By contrast, the K+-NPPase was located only in antiluminal membranes of the brain capillaries. This distinct enzymatic distribution suggested that the luminal and antiluminal membranes are functionally different. The role of alkaline phosphatase and K+-NPPase in the blood brain barrier is discussed.     

A recombination map of the human X-chromosome

Summary A family with 11 normal boys has been typed with DNA probes spanning the whole of the X-chromosome to observe directly the recombination events in 11 meioses from one female. This has (a) identified apparent recombination hot-spots on the X-chromosome; (b) shown the positions and numbers of cross-overs that have occurred in the p and q arms; (c) not shown any cross-overs in the centromeric region and (d) enabled the calculation of the genetic length of the X-chromosome.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Vacuolation induced by VacA toxin of Helicobacter pylori requires the intracellular accumulation of membrane permeant bases, Cl(-) and water.

The protein vacuolating toxin A (VacA) of Helicobacter pylori converts late endosomes into large vacuoles in the presence of permeant bases. Here it is shown that this phenomenon corresponds to an accumulation of permeant bases and Cl(-) in HeLa cells and requires the presence of extracellular Cl(-). The net influx of Cl(-) is due to electroneutral, Na(+), K(+), 2Cl(-) cotransporter-mediated transport. Cell vacuolation leads to cell volume increase, consistent with water flux into the cell, while hyper-osmotic media decreased vacuole formation. These data represent the first evidence that VacA-treated cells undergo an osmotic unbalance, reinforcing the hypothesis that the VacA chloride channel is responsible for cell vacuolation.     

Purification and characterization of glutathione transferase of human thyroid

An anionic (pI 4.6) isoenzyme of glutathione transferase was purified to homogeneity from human thyroid by affinity chromatography followed by isoelectric focusing. The content of enzyme was calculated to constitute about 0.2% of soluble proteins. The enzyme is formed by two identical subunits of 23,000 daltons approximately. The thyroid transferase did not catalyze the reduction of peroxides. Physical, catalytic and immunological analyses demonstrated extensive similarities between the thyroid transferase and the transferase from placenta, erythrocytes and breast. On the other hand, the thyroid transferase appears catalytically different from transferase 7-7, even if both cross-react with the antibodies raised against human placenta transferase.     

Influence of charged groups on the conformational stability of succinoglycan in dilute aqueous solution

Viscosity, optical activity, and differential scanning calorimetry data clearly point out that partial and/or total removal of charged substituent groups, i.e. succinate and pyruvyl residues, from succinoglycan lead to water soluble derivatives exhibiting a higher stability order → disorder conformational changes with respect to the native polysaccharide. The new succinoglycan derivatives also exhibit very little, if any, hysteresis upon ‘renaturation’ (cooling) as opposed to the case of the parent polymer. The absence of ionized groups is thus beneficial, thermodynamically and kinetically, to the attainment in dilute aqueous solution of an ordered conformation by the uncharged succinoglycan backbone, as allowed by the regular enchainment of its constituent sugar residues.