Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.     

Novel lipophilic amidate oxorhenium and oxotechnetium complexes as potential brain agents: synthesis, characterization and biological evaluation

Novel oxorhenium and oxotechnetium complexes based on the tetradentate 1-(2-hydroxybenzamido)-2-(pyridinecarboxamido)benzene, H3L, ligand have been synthesized and characterized herein. Thus, by reacting equimolar quantities of the triply deprotonated ligand L3- with the suitable MO3+ precursor, the following neutral MOL complexes could be easily produced following similar synthetic routes: M = Re (1), M = 99gTc (2), and M = 99mTc (3). Complexes 1 and 2, prepared in macroscopic amounts, were chemically characterized and their structure determined by single-crystal X-ray analysis. They are isostructural metal chelates, adopting a distorted square pyramidal geometry around the metal. The N3O donor atom set of the tetradentate ligand defines the basal plane and the oxygen atom of the M = O core occupies the apex of the pyramid. Complex 3 forms quantitatively at tracer level by mixing the H3L ligand with Na99mTcO4 generator eluate in aqueous alkaline media and using tin chloride as reductant in the presence of citrate. Its structure was established by chromatographic comparison with prototypic complexes 1 and 2 using high-performance liquid chromatographic techniques. When challenged with excess glutathione in vitro, complex 3 is rapidly converted to hydrophilic unidentified metal species. Tissue distribution data after administration of complex 3 in vivo revealed a significant uptake and retention of this compound in brain tissue.     

The relationship between anatomy and photosynthetic performance of heterobaric leaves

Heterobaric leaves show heterogeneous pigmentation due to the occurrence of a network of transparent areas that are created from the bundle sheaths extensions (BSEs). Image analysis showed that the percentage of photosynthetically active leaf area (Ap) of the heterobaric leaves of 31 plant species was species dependent, ranging from 91% in Malva sylvestris to only 48% in Gynerium sp. Although a significant portion of the leaf surface does not correspond to photosynthetic tissue, the photosynthetic capacity of these leaves, expressed per unit of projected area (Pmax), was not considerably affected by the size of their transparent leaf area (At). This means that the photosynthetic capacity expressed per Ap (P*max) should increase with At. Moreover, the expression of P*max could be allowing the interpretation of the photosynthetic performance in relation to some critical anatomical traits. The P*max, irrespective of plant species, correlated with the specific leaf transparent volume (lambda(t)), as well as with the transparent leaf area complexity factor ((CF)A(t)), parameters indicating the volume per unit leaf area and length/density of the transparent tissues, respectively. Moreover, both parameters increased exponentially with leaf thickness, suggesting an essential functional role of BSEs mainly in thick leaves. The results of the present study suggest that although the Ap of an heterobaric leaf is reduced, the photosynthetic performance of each areole is increased, possibly due to the light transferring capacity of BSEs. This mechanism may allow a significant increase in leaf thickness and a consequent increase of the photosynthetic capacity per unit (projected) area, offering adaptive advantages in xerothermic environments.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Bacterial synergism or antagonism in a gel cassette system

The growth and the metabolic activity of Shewanella putrfaciens, Brochothrix thermosphacta, and Pseudomonas sp., when cultured individually or in all possible combinations in gel cassettes system supplemented with 0.1% glucose at 5 degrees C, were investigated. The overall outcome was that the coexistence of the above-mentioned microorganisms affected not only each growth rate but also their type of metabolic end products compared to the control cultures. These effects were varied and depended on the selection of the combination of the tested bacteria. For example, the growth of Pseudomonas sp. strains cocultured with either B. thermosphacta or S. putrefaciens strains resulted in different effects: a promoting one for the first and an inhibitory one for the second. Moreover, the production of formic acid and two unidentified organic acids (peaks a and b) was characteristic in all cases in which S. putrefaciens was cultured.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Mapping functional associations in the entire genome of Drosophila melanogaster using fusion analysis

We have previously shown that the detection of gene fusion events can contribute towards the elucidation of functional associations of proteins within entire genomes. Here we have analysed the entire genome of Drosophila melanogaster using fusion analysis and two additional constraints that improve the reliability of the predictions, viz. low sequence similarity and low degree of paralogy of the component proteins involved in a fusion event. Imposing these constraints, the total number of unique component pairs is reduced from 18 654 to a mere 220 cases, which are expected to represent some of the most reliably detected functionally associated proteins. Using additional information from sequence databases, we have been able to detect pairs of functionally associated proteins with important functions in cellular and developmental pathways, such as spermatogenesis and programmed cell death.