Separation of metalloprotein complexes in serum by size-exclusion chromatography: Optimisation of the separation parameters retention behaviour and recovery employing radiotracers

A suitable procedure was developed for speciation analysis of metalloprotein complexes in serum using directly coupled size-exclusion chromatography and an element-specific detector. Two column matrices used for size-exclusion chromatography (TSK G 3000 SW and Asahipak GS 520) were investigated with respect to the recovery and retention behaviour for metalloprotein complexes. Optimisation of the separation parameters (buffer type, concentration, pH) was achieved by means of metalloprotein complexes marked with radiotracers. For speciation of serum the matrix in the Asahipak GS column is more efficient. Given optimal eluent characteristics (100 mM Tris, pH 7.4) the recovery of the elements investigated (sodium, calcium, iron and zinc) was 100%. Further, the retention behaviour (retention time, ratios of the peak areas) remained unchanged for several successive separations.     

Effect of some plant growth regulator treatments on apple fruit ripening

The activity of IAA oxidase (IAAox), peroxidases (POD), and polyphenoloxidases (PPO), as affected by different pre-harvest growth regulator treatments (ABA, AVG, NAA, PDJ), was determined in on-tree ripening apples (cv. Golden Delicious) before and during the ethylene climacteric. The production of ethylene was inhibited by AVG and delayed by NAA, whereas ABA and PDJ treatments caused, in the on-tree remaining fruits, a marked fruit drop and a decrease or a slight increase in ethylene levels respectively. While all treatments reduced POD activity, jasmonate increased IAAox and PPO activity. The inhibitory effect of NAA on all enzyme activity seems related to interference with C₂H₂ action or to a reduced sensitivity of the fruit abscission zone tissues to the hormone. The observed high fruit drop induced by ABA treatment made it impossible to detect differences in enzyme activity. AVG-treated fruits showed no substantial effects on IAAox and PPO activity in comparison to the control, a finding that seems to be related to a delay in all senescence processes caused by the very low level of the inhibited ethylene production. In control fruits IAAox activity increased during the initial ripening stages and decreased thereafter, POD activity increased throughout ripening and PPO showed little variation.     

Photosynthetic electrogenic events in native membranes of Chloroflexus aurantiacus. Flash-induced charge displacements in the acceptor quinone complex of the photosynthetic reaction center

Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. Tertiary structure modeling suggests that the redox-sensitivity of the chloroplast malate dehydrogenase (EC 1.1.1.82) is due to disulfide crosslinking of the carbon substrate and nucleotide-binding domains. Consistent with this suggestion, introduction of Cys residues in opposition to one another on the two domains of the Escherichia coli enzyme results in redox-sensitivity [Muslin EH et al. (1995) Biophys J 68: 2218-2223]. We have now substituted Cys residues into the bacterial malate dehydrogenase (EC 1.1.1.37) in positions that correspond more exactly to those postulated to be responsible for the redox-sensitivity of the chloroplast enzyme. The introduction of one pair of Cys residues renders the enzyme redox-sensitive, but the introduction of the alternate pair does not. Energy minimization calculations suggest that the difference in redox-sensitivity is consistent with differences in the energy required for formation of the disulfide bond.     

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three‐ to tenfold improvements over wild‐type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single‐chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16‐fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180‐fold improvement over previous reports in the secretion of full‐length, functional, glycosylated human IgG₁. The production of full‐length IgG₁ at milligram per liter titers in a simple, laboratory‐scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. Biotechnol. Bioeng. 2009;103: 1192–1201. © 2009 Wiley Periodicals, Inc.     

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.     

Hydrodynamics of vertical subsurface flow constructed wetlands: Tracer tests with rhodamine WT and numerical modelling

Typical unsteady unsaturated conditions can profoundly affect the hydrodynamics of vertical subsurface flow constructed wetlands. In this study we analyzed the hydrodynamics of a 33 m² vertical flow pilot plant, treating municipal secondary effluents. Three different saturation conditions were analyzed under several constant flux regimes: complete saturation, partial saturation with the free water table 20 cm over the bottom of the bed, and complete drainage. Tracer tests were performed in steady state conditions by dosing rhodamine WT as square input signals. Breakthrough curves were analyzed by means of both a classical residence time distribution analysis and an originally developed numerical plug-flow model with longitudinal dispersion adapted to the unsaturated conditions. We found that the degree of global mixing in the vertical flow constructed wetland increased as the water content increased; this effect was controlled by the hydraulic residence time of the system. Conversely, the degree of local mixing was inversely affected by water content; the dispersivity was 4.5, 10, and 14 cm for fully saturated, partially saturated and draining conditions, respectively. We explain the dependency of dispersivity on water content in physical terms; however, further studies are needed to mathematically include this relationship in numerical models that describe the behaviour of vertical flow constructed wetlands.     

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 °C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N -acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided.     

Differential intracellular distributions of inositol trisphosphate and ryanodine receptors within and among hematopoietic cells.

To better understand the mechanism(s) of leukocyte Ca(2+) signaling, we have studied the intracellular locations of two Ca(2+)-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca(2+) signaling.     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.