Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-kappaB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.     

Constituents from the stems of Actinodaphne lancifolia

Two C(16)-lactonic compounds, actinolides A-B (1-2), were isolated from the stems of Actinodaphne lancifolia, together with five known lactones (3-7) and three known lignans (8-10). Their structures were determined spectroscopically, which included 2D NMR spectroscopic analysis.     

Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression

In Saccharomyces cerevisiae, OLE1 encodes a Δ9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.     

A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.