Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and characterization of the proBA operon of Streptococcus bovis.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.     

An "artificial tongue" for calibrating solution flow characteristics of taste stimulus delivery systems

An "artificial tongue" is described which can be used to calibratethe stimulus delivery characteristics of flow systems for liquidtaste stimuli. The calibrator, which functions on the basisof conductance measurements, is used to ascertain the behaviourof certain test solutions in the flow system. The test solutionsare so prepared that they mimic the hydraulic properties ofthe stimulus solutions to be used. The special apparatus requiredis neither complex nor costly, and the procedures involved arestraightforward.     

Are drought-resistance promoting bacteria cross-compatible with different plant models?

The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.     

The intrinsic stability of the human prion β-sheet region investigated by molecular dynamics

Human prion diseases are neurodegenerative disorders associated to the misfolding of the prion protein (PrP). Common features of prion disorders are the fibrillar amyloid deposits and the formation of prefibrillar oligomeric species also suggested as the origin of cytotoxicity associated with diseases. Although the process of PrP misfolding has been extensively investigated, many crucial aspects of this process remain unclear. We have here carried out a molecular dynamics study to evaluate the intrinsic dynamics of PrP β-sheet, a region that is believed to play a crucial role in prion aggregation. Moreover, as this region mediates protein association in dimeric assemblies frequently observed in prion crystallographic investigations, we also analyzed the dynamics of these intermolecular interactions. The extensive sampling of replica exchange shows that the native antiparallel β-structure of the prion is endowed with a remarkable stability. Therefore, upon unfolding, the persistence of a structured β-region may seed molecular association and influence the subsequent phases of the aggregation process. The analysis of the four-stranded β-sheet detected in the dimeric assemblies of PrP shows a tendency of this region to form dynamical structured states. The impact on the β-sheet structure and dynamics of disease associated point mutations has also been evaluated.     

The behaviour of deoxyhexose trihaloacetimidates in selected glycosylations

Armed deoxyhexose glycosyl donors are very reactive and sometimes too uncontrollably activated in glycosylation reactions; yields can be thereby reduced, especially when unreactive glycosyl acceptors are involved. In this paper, the behaviour of a range of deoxyhexose trihaloacetimidate (trichloro- and N-phenyl trifluoro-) donors is compared in some selected glycosylations towards biologically relevant targets. The selected N-phenyl trifluoroacetimidates often afforded best results in terms of both donor synthesis and glycosylation yield.     

The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.