Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking

The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking.     

Identification of HLA-A*0201-presented T cell epitopes derived from the oncofetal antigen-immature laminin receptor protein in patients with hematological malignancies

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies.     

Fine mapping of collagen-induced arthritis quantitative trait loci in an advanced intercross line

The generation of advanced intercross lines (AIL) is a powerful approach for high-resolution fine mapping of quantitative trait loci (QTLs), because they accumulate much more recombination events compared with conventional F2 intercross and N2 backcross. However, the application of this approach is severely hampered by the requirements of excessive resources to maintain such crosses, i.e., in terms of animal care, space, and time. Therefore, in this study, we produced an AIL to fine map collagen-induced arthritis (CIA) QTLs using comparatively limited resources. We used only 308 (DBA/1 x FVB/N)F11/12 AIL mice to refine QTLs controlling the severity and onset of arthritis as well as the Ab response and T cell subset in CIA, namely Cia2, Cia27, and Trmq3. These QTLs were originally identified in (DBA/1 x FVB/N)F2 progeny. The confidence intervals of the three QTLs were refined from 40, 43, and 48 Mb to 12, 4.1, and 12 Mb, respectively. The data were complemented by the use of another QTL fine-mapping approach, haplotype analysis, to further refine Cia2 into a 2-Mb genomic region. To aid in the search for candidate genes for the QTLs, genome-wide expression profiling was performed to identify strain-specific differentially expressed genes within the confidence intervals. Of the 1396 strain-specific differentially expressed genes, 3, 3, and 12 genes were within the support intervals of the Cia2, Cia27, and Trmq3, respectively. In addition, this study revealed that Cia27 and Trmq3 controlling anti-CII IgG2a Ab and CD4:CD8 T cell ratio, respectively, also regulated CIA clinical phenotypes.     

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.     

Influence of a salinity gradient on the vessel characters of the mangrove species Rhizophora mucronata

BACKGROUND AND AIMS: Although mangroves have been extensively studied, little is known about their ecological wood anatomy. This investigation examined the potential use of vessel density as a proxy for soil water salinity in the mangrove species Rhizophora mucronata (Rhizophoraceae) from Kenya. METHODS: In a time-standardized approach, 50 wood discs from trees growing in six salinity categories were investigated. Vessel densities, and tangential and radial diameters of rainy and dry season wood of one distinct year, at three positions on the stem discs, were measured. A repeated-measures ANOVA with the prevailing salinity was performed. KEY RESULTS: Vessel density showed a significant increase with salinity, supporting its use as a prospective measure of salinity. Interestingly, the negative salinity response of the radial diameter of vessels was less striking, and tangential diameter was constant under the varying environmental conditions. An effect of age or growth rate or the presence of vessel dimorphism could be excluded as the cause of the absence of any ecological trend. CONCLUSIONS: The clear trend in vessel density with salinity, together with the absence of a growth rate and age effect, validates the potential of vessel density as an environmental proxy. However, it can only be used as a relative measure of salinity given that other environmental variables such as inundation frequency have an additional influence on vessel density. With view to a reliable, absolute proxy, future research should focus on finding wood anatomical features correlated exclusively with soil water salinity or inundation frequency. The plasticity in vessel density with differing salinity suggests a role in the establishment of a safe water transport system. To confirm this hypothesis, the role of inter-vessel pits, their relationship to the rather constant vessel diameter and the underlying physiology and cell biology needs to be examined.     

Assessing the impact of errors in sorting and identifying macroinvertebrate samples

This study assesses the impact of errors in sorting and identifying macroinvertebrate samples collected and analysed using different protocols (e.g. STAR-AQEM, RIVPACS). The study is based on the auditing scheme implemented in the EU-funded project STAR and presents the first attempt at analysing the audit data. Data from 10 participating countries are analysed with regard to the impact of sorting and identification errors. These differences are measured in the form of gains and losses at each level of audit for 120 samples. Based on gains and losses to the primary results, qualitative binary taxa lists were deducted for each level of audit for a subset of 72 data sets. Between these taxa lists the taxonomic similarity and the impact of differences on selected metrics common to stream assessment were analysed. The results of our study indicate that in all methods used, a considerable amount of sorting and identification error could be detected. This total impact is reflected in most functional metrics. In some metrics indicative of taxonomic richness, the total impact of differences is not directly reflected in differences in metric scores. The results stress the importance of implementing quality control mechanisms in macroinvertebrate assessment schemes. Peter Haase, Andrea Sundermann: These authors contributed equally to this work.     

High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.     

Two-component systems of Corynebacterium glutamicum: deletion analysis and involvement of the PhoS-PhoR system in the phosphate starvation response

Corynebacterium glutamicum contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (Pi) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under Pi limitation, and therefore the genes lacking in this strain were named phoS (encoding the sensor kinase) and phoR (encoding the response regulator). DNA microarray analyses with the C. glutamicum wild type and the DeltaphoRS mutant supported a role for the PhoRS system in the adaptation to Pi starvation. In contrast to the wild type, the DeltaphoRS mutant did not induce the known Pi starvation-inducible (psi) genes within 1 hour after a shift from Pi excess to Pi limitation, except for the pstSCAB operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the pst operon. Primer extension analysis of selected psi genes (pstS, ugpA, phoR, ushA, and nucH) confirmed the microarray data and provided evidence for positive autoregulation of the phoRS genes.     

Hybridisation between South polar skua (Catharacta maccormicki) and Brown skua (C. antarctica lonnbergi) in the Antarctic Peninsula region

Hybridisation between South polar skua (C. maccormicki) and Brown skua (C. antarctica lonnbergi) in the area of the Antarctic Peninsula is known at least since the beginning of the last century but no survey has been done so far. This paper reviews information on the species composition of skua colonies of more than 10 pairs in the Antarctic Peninsula region, and the incidence of mixed pairs. Morphometrics, population size and breeding success were examined in detail at King George Island. The northward distribution of South polar skuas extended to King George Island (62°11′ S 59°00′ W), with a small outlying population on Signy Island (60°45′ S 45°36′ W), whereas Brown skuas did not breed further south than Anvers Island archipelago (64°46′ S 64°03′ W). The proportion of mixed pairs was highest at the northern end of the ∼500-km-wide hybrid zone. Body size distribution of sympatric skuas from King George Island is clearly bimodal but overlaps considerably and hybrids cannot be identified. Skua population sizes at Potter Peninsula/King George Island remained stable between 1994 and 2004. Numbers of mixed breeding pairs fluctuated more strongly than those of pure species pairs. Breeding success of Brown skuas varied the least.