2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

Background  

Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.     

Concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 ± 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 ± 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 ± 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 ± 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations.     

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson's disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.     

Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects

Cell-penetrating peptides (CPPs) are carriers developed to improve mammalian cell uptake of important research tools such as antisense oligonucleotides and short interfering RNAs. However, the data on CPP uptake into non-mammalian cells are limited. We have studied the uptake and antimicrobial effects of the three representative peptides penetratin (derived from a non-mammalian protein), MAP (artificial peptide) and pVEC (derived from a mammalian protein) using fluorescence HPLC in four common model systems: insect cells (Sf9), gram-positive bacteria (Bacillus megaterium), gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). We demonstrate that non-mammalian cells internalize CPPs and a comparison of the uptake of the peptides show that the intracellular concentration and degradation of the peptides varies widely among organisms. In addition, these CPPs showed antimicrobial activity.     

Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.     

Preparative capillary electrophoresis based on adsorption of the solutes (proteins) onto a moving blotting membrane as they migrate out of the capillary.

A micropreparative capillary electrophoresis apparatus equipped with a new type of fraction collection device is described: solutes, such as proteins, are adsorbed onto a moving blotting membrane (for instance a polyvinylidene difluoride membrane) as they migrate electrophoretically out of the capillary. The adsorbed proteins are visualized by conventional protein staining methods or by fluorescent labeling. Specific identification of separated components by an immunological technique is demonstrated. The method also offers the potential to analyze proteins and peptides collected on the membrane by gas phase sequencing and mass spectrometry.     

Synthesis of oligodeoxyribonucleotides on silica gel support

A rapid solid phase method of oligonucleotide synthesis based on monomeric protected nucleosides has been developed.     

Compaction and particle segregation in myelin membrane arrays

Compacted membrane arrays are formed in the nerve myelin sheath by lowering the water activity (through evaporation or immersion in hypertonic solutions of nonelectrolytes or monovalent salts) or by binding specific cations (Ca(++), La(+++), and tetracaine at concentrations above 5-10 mM). X-ray diffraction observations on intact, hydrated nerves treated to induce compaction provide a control to assess the significance of structural changes seen by freeze-fracture electron microscopy. Compaction inevitably leads to lateral segregation of particles away from the closely packed membrane arrays into contiguous normal, or slightly expanded, period arrays. In the particle-enriched layers, the E fracture face is more particle-dense than the P face, whereas no particles are found on either face in the compacted layers. Morphologically, compaction induced by the all-or-nothing, relatively irreversible action of specific cations cannot be distinguished from compaction to the same extent induced by the graded, reversible effects of nonelectrolytes. Compaction by sodium chloride resembles that by specific- cation binding in that the repeat period is independent of reagent concentration; but, like dehydration by nonelectrolytes, the extent of compaction is reversibly related to reagent concentration. Sodium chloride-compacted myelin can be distinguished morphologically by a lack of the elongated border particles at the boundary between smooth and particle-enriched membrane observed for other compacting treatments. Fracture faces in compacted arrays are not always smooth, but the unusual appearances can be duplicated in purified myelin lipid multilayers subjected to similar treatments, which indicates that the particle-free membrane fracture faces are uninterrupted lipid hydrocarbon layers. Correlation of x-ray diffraction and electron microscopy observations provides a direct basis for identifying the intramembrane particles with transmembrane protein. The transmembrane protein appears to play a significant role in maintaining the normal membrane separation; swelling of the particle-enriched arrays in myelin compacted by tetracaine at low ionic strength provides information about the charge distribution on the transmembrane protein. Swelling of the compacted arrays following irreversible particle segregation shows that the interaction properties of the particle-free membranes are similar to those of pure lipid multilayers. Compaction and the consequent particle segregation in lyelin results from conditions stabilizing close apposition of the lipid bilayers. Particle segregation in areas of close contact between other cell membranes may also be driven by interbilayer attractive forces.     

Nutrient stocks in managed and natural humid tropical fallows

Managed fallows which recover nutrients more rapidly than natural secondary vegetation may improve the performance of shifting agriculture systems operating under inadequately long fallow cycles. Our objective was to construct nutrient balances for the soil, vegetation, and litter compartments of six planted leguminous fallows and natural secondary vegetation during 53 months. The fallows were planted on a previously cultivated Ultisol (Acrisol) in the Peruvian Amazon and included:Centrosema macrocarpum (Centrosema),Pueraria phaseoloides (Pueraria),Stylosanthes guianensis (Stylosanthes),Desmodium ovalifolium (Desmodium),Cajanus cajan (Cajanus), andInga edulis (Inga). In addition, in the natural fallow treatment secondary vegetation was allowed to establish and grow naturally. Quantities of extractable P, K, Ca, and Mg, total N, and organic C in soil to a 45 cm depth, and macrouttrients in aboveground biomass, roots, and litter were estimated at fallow planting, at 8, 17, and 29 months afterward, and at fallow clearing (53 months). Total N stocks increased by 10% in the Stylosanthes, Desmodium, Pueraria, and Inga treatments, but changed little in the Cajanus, Centrosema and natural fallows. This difference was largely due to greater net increases in both soil and vegetation compartments in the former group of treatments. In the Inga, Desmodium, and natural fallows, total stocks of P and K at 53 months were about 40% to 80% greater and 12% greater, respectively, than initial values, but Ca and Mg stocks were reduced by 25% to 40%. In the other treatments, there was generally little change in P stocks, but large (30% to 60%) reductions in K, Ca, and Mg during the course of the fallow. Although there were net decreases of stocks of P, K, Ca, and Mg in soil in all treatments during the fallow, storage of P and K in vegetation and litter in the Inga, Desmodium, and natural fallows offset losses of these nutrients from soil. These treatments also tended to accumulate more Ca and Mg in biomass and litter than the other treatments. These results suggest that leguminous fallow vegetation that accumulates large amounts of biomass may increase N, P, and K stocks, but that incomplete recuperation of Ca and Mg may limit the sustainability of short-rotation fallow-based systems on acidic, infertile soils. ei]Section editor: G R Stewart