Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.     

Extremitätentransplantationen an Anuren

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Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels

The biochemistry and leaf anatomy of plants using C₄ photosynthesis promote the concentration of atmospheric CO₂ in leaf tissue that leads to improvements in growth and yield of C₄ plants over C₃ species in hot, dry, high light, and/or saline environments. C₄ plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C₄ photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C₃ photosynthesis. In addition to C₃ and C₄ species, some plant lineages contain closely related C₃–C₄ intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C₃ plants and species using C₄ photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C₄ photosynthesis. It is now clear that great variation exists in C₄ leaf anatomy, and diverse molecular mechanisms underlie C₄ biochemistry and physiology. However, all these different paths have led to the same destination—the expression of a C₄ CO₂ concentrating mechanism. Further identification of C₄ leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.     

Extremitätentransplantationen an Anuren

Ohne Zusammenfassung     

Extremitätentransplantationen an Anuren

Ohne Zusammenfassung     

IDENTIFICATION OF THE CLASS PRYMNESIOPHYCEAE AND THE GENUS PHAEOCYSTIS WITH RIBOSOMAL RNA–TARGETED NUCLEIC ACID PROBES DETECTED BY FLOW CYTOMETRY1

Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hybridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.