The composition of the intracellular domains of the B cell antigen receptor complex studied from the cytoplasmic side of the membrane.

Immunoglobulins of the classes M and D function as antigen receptors on B lymphocytes. They are linked to other proteins to form B cell antigen receptor (BCR) complexes which transduce the signal triggered by the binding of antigen. In order to study the components that interact with BCR complexes in the cell it is essential that they are accessible to biochemical studies. Therefore, we have developed a simple and rapid method that allows the purification and labelling of B lymphocyte plasma membranes. For this, B cells are attached to polyacrylamide beads. Upon disruption of the cells, bead-bound membranes are obtained which expose the cytoplasmic side into the medium. The membrane proteins can then be radioiodinated and eluted with detergents. The combination of the improved methods for the preparation of bead-attached membrane patches and radiolabelling of the proteins has allowed for the first time an investigation into the cytoplasmic side of the BCR complex. All the subunits that had been previously described could be detected in 2D autoradiographs. Furthermore, it could be shown that the protein Ig-beta, which is part of an Ig-associated heterodimer, is predominantly labelled at the extracellular domain. The second component, Ig-alpha, is labelled to a higher degree at its intracellular domain. In addition, further proteins could be detected exclusively at the cytoplasmic side of the membrane. Results from 2D autoradiographs show that they may form heterodimers. These proteins are candidates for the interaction of BCR complexes with further members of the signalling cascade, such as protein tyrosine kinases and/or G proteins.

Initial in vitro toxicity testing of functional foods rich in catechins and anthocyanins in human cells.

Functional foods need to be assessed for beneficial effects to support claims, but also for toxic effects. This report describes two examples of how complex food samples are initially characterized in human cells in vitro. Water extracts of green tea (GT) and black carrots (BC) were analyzed for key ingredients (catechins and anthocyanidins, respectively). Extracts, reconstituted mixtures of the major ingredients or individual compounds [(-)-epigallocatechin gallate or cyanidin, respectively] were evaluated in parallel using human colon cells (HT29 clone 19A). End points of cytotoxicity included determination of membrane integrity, proliferation inhibition, and genetic damage. Cells were pretreated with plant compounds at sub-toxic concentrations, and their resistance to toxicity of H2O2 was evaluated as a parameter of protection. The extracts reduced cell viability (BC) and cell growth (BC, GT) and caused DNA damage (BC, GT). They were more toxic than their key ingredients. Neither GT-samples nor BC protected against H2O2-induced DNA damage, whereas cyanidin did. In vitro analysis of extracts from functional foods firstly aims at defining the sub-toxic concentrations at which protective activities are then further characterized. It also allows comparing responses of complex samples and individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components.

Investigation of the distribution of deoxynivalenol and ochratoxin A contamination within a 26 t truckload of wheat kernels.

A study was conducted to determine the distribution of deoxynivalenol (DON) and ochratoxin A (OTA) in a lot of 261 of wheat kernels. Within this study, two different sampling and sample preparation strategies were carried out. On the one hand, following the official commission regulation 401/2006/EC, an aggregate sample out of 100 incremental samples was build, homogenized and prepared for laboratory analysis. On the other hand each individual subsample was investigated for its deoxynivalenol and ochratoxin A content. The determined concentration of DON in the individual samples was in a range from 830 up to 2655 (µg/kg, for OTA results ranged from < 0.2 up to 8.6 µg/kg. Thus, a coefficient of variance of 25% for DON and 200% for OTA was achieved. From this, a spot formation for OTA was observed and the average value of the 100 incremental samples did not correspond to the achieved value for the aggregate sample. While the DON contamination at this concentration range seems to be more even, consequently the result of the aggregate sample was in accordance with the average value.In addition a sample communition study was performed to answer the question whether the time consuming process of grinding of the whole aggregate sample is necessary or not. The results of this study show that contamination of whole wheat kernels with DON is at the same level within a 1 kg sample (CV 16%), while OTA contamination shows high variability (CV 94%). At least for OTA this study indicated that an extensive and complete sample communition of the high volume aggregate sample is necessary.

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

Changes in the expression of Ig-associated proteins on B lymphocytes activated by anti-IgM antibodies.

Binding of antigen to receptor complexes on B cells elicits a cascade of intracellular signalling events leading to proliferation and, together with T-cell help, Ig secretion. Components of the antigen receptor (AgR) complex have been demonstrated to be either covalently bound or associated with surface Ig (sIg) molecules. The function of these proteins is still unknown. In order to address this question, we have stimulated B cells with anti-mu antibodies and have studied possible changes in the expression of AgR complexes. After anti-mu stimulation, the IgM molecules disappeared rapidly from the cell surface together with the covalently bound proteins. The IgM molecules were internalized and probably degraded. The IgM-associated heterodimer Ig-alpha/Ig-beta was also removed from the cells, leaving the IgD-associated heterodimer unaffected. Two proteins showed an enhanced association with sIg after 15 min and then were gradually removed from the cell surface. Two other proteins became increasingly attached to sIg. This association remained stable for the rest of the culture period (up to 4 h). Further studies are underway to characterize these proteins more closely and to examine possible interactions with downstream members of the signalling cascade.

Molecular targets of CD45 in B cell antigen receptor signal transduction.

Expression of the phosphotyrosine phosphatase CD45 is essential for B cell Ag receptor (BCR)-mediated p21ras activation and calcium mobilization. To examine the molecular basis of this requirement, we analyzed signaling events following BCR ligation in CD45-deficient (CD45-) and CD45-reconstituted (CD45+) variants of J558Lmicrom3 cells. Ag stimulation resulted in tyrosine phosphorylation of cellular proteins in both cells. However, the spectrum of proteins phosphorylated in the CD45+ cells was qualitatively and/or quantitatively distinct from that in the CD45- cells. Among the protein tyrosine kinases examined, the Src family kinases Fyn and Blk were inducibly tyrosine phosphorylated and activated by receptor ligation only in CD45+ cells. While Ag-induced Btk tyrosine phosphorylation occurred in both cells, its activation was greatly diminished in the CD45- cells. Analysis of specific effector molecules revealed that tyrosine phosphorylation of Shc, but not rasGAP or Vav, correlated with the unique ability of BCR ligation to trigger p21ras activation in CD45+ cells. BCR-mediated Shc phosphorylation and recruitment of Grb2 depended on CD45 expression. Thus, Shc tyrosine phosphorylation may be the primary CD45-dependent mechanism by which Ag receptors are coupled to the p21ras pathway in J558Lmicrom3. In addition, phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 were tyrosine phosphorylated upon Ag stimulation in CD45- cells, despite much reduced inositol trisphosphate production and lack of calcium mobilization. These findings suggest that CD45 may modulate events other than PLCgamma phosphorylation, which regulate phosphoinositide hydrolysis and the calcium mobilization response following BCR ligation.

The immunosuppressive peptide of HIV-1 inhibits T and B lymphocyte stimulation.

The transmembrane glycoproteins of all retroviruses contain a conserved region composed of a leucine zipper, an immunosuppressive domain, and an immunodominant Cys-Cys loop. The amino acid sequence of the immunosuppressive domain of gp41 of human immunodeficiency virus type 1 (HIV-1; amino acids 583-599) is closely related, but not identical, to the immunosuppressive domains of type C and D retroviruses. A synthetic peptide corresponding to the immunosuppressive domain of HIV-1 (immunosuppressive peptide, ISU-peptide) inhibits mitogen and lymphokine stimulation of T lymphocytes. It is interspecies reactive and inhibits both human and mouse lymphocytes. The inhibitory effect is not based on direct cytotoxicity and the peptide is immunosuppressive only when conjugated to a carrier protein. The ISU-peptide of HIV-1 also inhibits B lymphocyte stimulation by the B cell mitogen lipopolysaccharide and by specific antibodies against delta and mu chains of cell surface immunoglobulins. These data suggest that the immunosuppressive domain of gp41 may play an important role in the immunopathogenesis of AIDS.

Fc gammaRIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation.

The B cell receptor for immunoglobulin G, Fc gammaRIIB1, is a potent transducer of signals that block antigen-induced B cell activation. Coligation of Fc gammaRIIB1 with B lymphocyte antigen receptors (BCR) causes premature termination of phosphoinositide hydrolysis and Ca2+ mobilization and inhibits proliferation. This inhibitory signal is mediated in part by phosphorylation of Fc gammaRIIB1 and recruitment of phosphatases; however, the molecular target(s) of effectors is unknown. Here we report that Fc gammaRIIB1 inhibition of BCR signaling is mediated in part by selective dephosphorylation of CD19, a BCR accessory molecule and coreceptor. CD19 dephosphorylation leads to failed CD19 association with phosphatidylinositol 3-kinase, and this in turn leads to termination of inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and Ca2+ influx. The results define a molecular circuit by which Fc gammaRIIB signals block phosphoinositide hydrolysis.