HLA-DQA2 and HLA-DQB2 genes are specifically expressed in human Langerhans cells and encode a new HLA class II molecule

The precise role of human epidermal Langerhans cells (LCs) in immune response is highly controversial. While studying the gene expression profile of these cells, we were intrigued to identify the HLA-DQB2 gene as potentially expressed in LCs. Despite a strong evolutionary conservation of their sequences, the concomitant expression of the poorly polymorphic HLA-DQA2/HLA-DQB2 genes, paralogous to the HLA-DQA1/HLA-DQB1 genes, has never been detected in any cell type. We confirmed by RT-PCR that the HLA-DQA2 and -DQB2 genes are both expressed in LCs, but not in monocyte-derived dendritic cells, or in blood CD1c(+) or plasmacytoid dendritic cells. The presence of the HLA-DQβ2 chain in LCs could be demonstrated by Western blotting, whereas immunofluorescence revealed its localization in early endosomes. As in the case of other HLA class II molecules, the HLA-DQα2 and -DQβ2 chains formed heterodimers that had to associate with the invariant chain to reach endosomal compartments. HLA-DQα2/β2 heterodimers were expressed at the cell surface, where they could mediate staphylococcal superantigen stimulation of T cells. Interestingly, HLA-DQα2 and HLA-DQβ1 chains formed mixed heterodimers which efficiently left the endoplasmic reticulum. These observations strongly suggest that the poorly polymorphic HLA-DQA2 and -DQB2 genes should be considered to be of immunological importance. The HLA-DQα2/β2 molecules could influence the complexity of the repertoire of Ags presented by LCs.     

Identification of phenylalkane derivatives when Mycobacterium neoaurum and Rhodococcus erythropolis were cultured in the presence of various phenylalkanes

Phenylalkanes are ubiquitously found in nature as pollutants originating from oil, gas oil and petrol. Rising commercial demand for mineral oil fractions has led to the increased prevalence of environmental contamination, whereby these particular hydrocarbons are encountered by bacteria which have subsequently developed sophisticated metabolic routes for purposes of degradation. Herein a detailed analysis of these metabolic pathways in the degradation of phenylalkanes by Mycobacterium neoaurum and Rhodococcus erythropolis highlighted preponderance for the formation of certain metabolites of which 17 were identified and whereby striking differences were noticed depending specifically upon the length of the substrate’s alkyl chain. Although the degradation of even-numbered phenylalkane substrates was assumed to result in the generation of phenylacetic acid formed due to substrate terminal oxidation and subsequent β-oxidation, cultures of M. neoaurum and R. erythropolis were determined in an extracellular accumulation of odd-numbered acidic metabolites, suggesting a simultaneous presence of sub-terminal degradation mechanisms. However, results obtained from biotransformation assays containing even-chained phenylalkanoic acid intermediates as substrates revealed exclusive β-oxidative mechanisms and no generation of odd-numbered degradation products. R. erythropolis in contrast to M. neoaurum also proved viable for hydroxylation of the aromatic ring of metabolites. Interestingly, the generation of phenylacetic acid and subsequently 2-hydroxyphenyl acetic acid was monitored and entailed the presence of the lactone intermediate 2-coumaranone. These results enhance our understanding of the degradation of phenylalkanes and illustrate the potential application of such species in the bioremediation of these common environmental pollutants and in the strains’ diverse abilities to transform mineral oil compounds to new valuable products.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

Induction of reactive oxygen species and cell survival in the presence of advanced glycation end products and similar structures

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains and the terminal amino group are supposed to be involved in the pathogenesis of several diseases and therefore the effects of AGEs on cells are the objective of numerous investigations. The effects of AGEs on cells are commonly assumed to be transduced via the receptor for AGEs (RAGE) but there are also other receptors known to interact with AGEs and they are likely to be involved in signal transduction. The primary cellular effect of AGEs on cultured cells was found to be the formation of reactive oxygen species (ROS). For the present study one murine and three human cell lines were used. The effects of a set of different highly modified AGEs and AGE-like compounds derived from the incubation of different modifiers with BSA were tested for their effects on these cells. Almost all AGEs tested induced the production of reactive oxygen species (ROS) in the different cell lines although the intensity of the detected signals varied considerably between the cell lines and are strongly dependent on the AGE used for cell activation. The most highly modified BSA-species were shown to inhibit cell growth in all cell lines, whereas a moderately modified glucose derived BSA-AGE and BSA-GA(red) did not show any inhibitory effect on cell growth even when a high ROS formation was detected.     

Insights into succession processes using temporally repeated habitat models: results from a long‐term study in a post‐mining landscape

Questions: The early phases of primary succession are governed by chance events and dispersal‐related processes in an environment that is largely free of competition. Thus, the predictability of vegetation patterns using environmental site factors can be expected to be low and spatial autocorrelation to be high. We asked whether the match between vegetation and environment becomes better in the course of succession, and whether vegetation types shift their realized niche with time. Location: Lignite mining region in Central Germany, the post‐mining landscape “Goitzsche” (Saxony‐Anhalt). Methods: Vegetation types were mapped in a 10‐m grid (total area 4.8 ha), starting in 1995, at 3‐year intervals until 2007. We used a temporal comparison of habitat models. We applied: GLS regression to partition the variation in coverage of vegetation types into environmental (soil pH) and spatial components; logistic regression to model the presence/absence of vegetation types along a soil acidity gradient; and autologistic regression allowing for soil acidity and neighbourhood effects. Results: For most vegetation types, the proportion of variation explained by space was high but declined during succession. The outcome of autologistic models suggests that soil acidity often plays a minor role compared to neighbourhood effects in the earlier phase of succession than 12 years later. For four vegetation types, the pH range in which the type was expected to be dominant clearly became smaller with time. These trends support the view that the role of processes related to chance and dispersal decrease with time, while those related to environmental filtering mediated by biotic interactions increase. Conclusions: We conclude that temporal comparisons of spatially explicit habitat models provide insights into changing biotic community processes and their effects on habitat specificity of species or their communities. Thus, this approach may be particularly important for analysis of ecological systems that are not in equilibrium with their environmental drivers.     

Purification and multimer formation of allurin, a sperm chemoattractant from Xenopus laevis egg jelly

Allurin, a sperm chemoattractant isolated from Xenopus laevis egg jelly, can be purified in one step from an extract of diffusible jelly proteins ("egg water") using a FPLC or HPLC anion exchange column and a multi-step NaCl gradient. Allurin homomultimers were detected by Western blotting with antibodies prepared against the purified protein or peptides within the protein. Allurin multimers were stable and resisted dissociation by SDS and beta-mercaptoethanol. Alkylation of allurin provided evidence for two free sulfhydryl groups but did not eliminate multimer formation, suggesting that intermolecular disulfide bond formation is not required for allurin aggregation. Concentration of egg water was accompanied by a reduction of chemoattractant activity that could not be fully accounted for by homomultimer formation. Rather, the presence of a multiphasic dose-activity curve upon partial purification and formation of hetero-allurin complexes during concentration suggested that egg water may contain allurin-binding proteins that reduce multimer formation and activity.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.     

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.     

Occurrence and activity of human intestinal bacteria involved in the conversion of dietary lignans

The human intestinal microbiota is necessary for the production of enterolignans from the dietary lignan secoisolariciresinol diglucoside (SDG). However, little is known about the bacteria that contribute to SDG conversion. Therefore, we aimed at describing the occurrence and activity of SDG metabolising bacteria. The data showed differences in conversion efficiency between SDG deglycosylating species, but SDG was completely deglycosylated within 20 h by five of six strains. The strain Clostridium sp. SDG-Mt85-3Db showed the highest initial rate of SDG deglycosylation. Furthermore, we found that Bacteroides distasonis and B. fragilis made up 0.5% and 3.3% of total faecal bacteria, respectively. However, Clostridium sp. SDG-Mt85-3Db was detected within the dominant microbiota of only two out of 20 faecal samples. Bacteria involved in the demethylation step of SDG conversion also demethylated a variety of compounds other than SDG. In particular, Peptostreptococcus productus demethylated the lignans pinoresinol, lariciresinol and matairesinol. Finally, Eggerthella lenta catalysed the reduction of pinoresinol and lariciresinol to secoisolariciresinol.     

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.