Effect of iron restriction on outer membrane protein composition of Pseudomonas strains studied by conventional and microchip electrophoresis.

In this study the outer membrane protein (OMP) composition of six Pseudomonas aeruginosa strains had been analyzed by conventional CE and microchip electrophoresis. Bacterial OMPs are important virulence factors and play a significant role in the pathogenesis of infectious diseases. Changes in their composition might refer to the altered pathogenic properties and antibiotic sensitivity of a certain strain. Pathogenic bacteria invading the human host have to multiplicate under iron-restricted conditions that induce changes in the OMP composition. High-molecular-weight OMPs have to be expressed, which serve as receptors for the iron-siderophore complexes. OMP patterns of bacteria obtained by the two different methods in this study were similar, all major proteins could be detected by both techniques, and the molecular weights showed good correlations, although direct comparison of the peak areas is not straightforward due to the different detection methods (UV and LIF). Changes in OMP composition under iron restriction could be detected, and appearance of a 92 kDa protein in all six P. aeruginosa strains and a 94 kDa protein in the KT 2 strain could be demonstrated. Besides that up- and downregulation of certain proteins could be also detected. The increased separation speed, picoliters of sample consumption, baseline separation achieved more frequently by this method--especially in the high-molecular-weight region--showed the advantages of microchip electrophoresis in the analysis of clinical samples.

The beta subunit of the type I Fcepsilon receptor is a target for peptides inhibiting IgE-mediated secretory response of mast cells.

Peptides originally derived from complement component C3a were earlier shown to inhibit the type I FcepsilonR (FcepsilonRI)-mediated degranulation of mucosal type mast cells. In the present study, we show that C3a7, a peptide with a natural sequence, and its modified derivative, C3a9, are powerful inhibitors of the above response of both serosal and mucosal type mastocytes. We demonstrate that these peptides inhibit FcepsilonRI-induced membrane proximal events, suppress phosphorylation of the FcepsilonRI beta subunit, the protein tyrosine kinase Lyn, as well as the transient rise in free cytosolic Ca2+ level. The late phase of cellular response was also inhibited, as demonstrated by the reduced TNF-alpha secretion. Experiments using two independent methods provided evidence that the interaction site of complement-derived peptides is the FcepsilonRI beta-chain. This was further supported by fluorescence confocal microscopic colocalization and resonance energy transfer measurements. Taken together, these results suggest the presence of distinct "activating" and "inhibitory" motifs in the C3a sequence. Response to both is in balance under physiologic conditions. Furthermore, present data predict that such inhibitory peptides may serve as potent agents for future therapeutic intervention.

Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins.

BACKGROUND: Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. METHODS: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. RESULTS: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach. CONCLUSIONS: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.

Regulation of mast cell activation by complement-derived peptides.

It is known for more than 25 years that the complement-derived anaphylatoxic peptides, C3a, C4a and C5a are potent activators of basophils and certain types of mast cells. Although tissue distribution of receptors for C3a and C5a well exceeds myeloid cells, apparently they are not expressed on mucosal type mast cells, consequently these cells are not activated by C3a and C5a. Our results do however demonstrate that C3a and peptides related to this complement activation product are able to inhibit FcRI-clustering induced activation of mucosal type mast cells-such as RBL-2H3 cells and bone-marrow derived mast cells. Based on the current results we propose the presence of separate "activator" and "inhibitor" sequence motifs in C3a which are in balance under physiologic conditions.

Mucosal type mast cells express complement receptor type 2 (CD21).

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

Bone marrow-derived mast cell differentiation is strongly reduced in histidine decarboxylase knockout, histamine-free mice.

Mast cells are differentiated in vitro from bone marrow precursors. In this study the development of bone marrow-derived mast cells was examined from histidine decarboxylase deficient (HDC-/-) and wild-type mice in the presence of IL-3. The number of non-adherent, tryptase- and c-kit-positive mast cells in bone marrow-derived cultures of HDC(-/-) mice was decreased compared to that of wild-type (HDC+/+) animals, but within the tryptase- and c-kit-positive cells there was no difference in the expression intensity of both markers between the two groups. Furthermore, less serine proteases mMCP5, mMCP6 and FcepsilonRIalpha mRNA were detected in bone marrow-derived cell cultures originating from HDC-/- mice. Antigen-provoked degranulation through high-affinity FcepsilonI receptor was also lower in HDC-/- mice. The colony assays in semisolid medium yielded a significantly lower ratio of mixed colonies and higher proportion of macrophage colonies from HDC-/- mice-derived bone marrow compared to the wild-type. In the course of the differentiation of HDC-/- --derived mast cells exogenously added histamine is unable to substitute the endogenously missing histamine. Concordantly, alpha-fluoromethyl-histamine, the specific inhibitor of HDC, revealed only a marginal inhibition on the differentiation of tryptase-positive mast cells from wild-type mice. These findings suggest that the effect of histamine on the IL-3-dependent development of bone marrow-derived mast cell differentiation during the early period is crucial and irreplaceable.