Analysis of the paired TCR α- and β-chains of single human T cells

Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV) and multiple sclerosis (MS). In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+) T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+) T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.     

Abundance,population structure and vertical distribution of dominant calanoid copepods on the eastern Weddell Sea shelf during a spring phytoplankton bloom

The main emphasis of this study was to analyse the short-term development of abundance, population structure and vertical distribution of the dominant calanoid copepods during a phytoplankton bloom in the coastal area of the eastern Weddell Sea in December 2003. Microcalanus pygmaeus was by far the most abundant calanoid species. Metridia gerlachei, Ctenocalanus citer, Calanoides acutus, Calanus propinquus and the ice-associated Stephos longipes were also present in considerable proportions. The observed changes in the population characteristics and parameters of these species are described in detail and discussed in the context of the spring phytoplankton bloom. A conspicuous event occurring during the final stage of the study was the development of a strong storm. While the results suggest that this storm did not have any considerable influence on the populations of all other investigated copepod species, it very likely caused pronounced changes in the S. longipes population present in the water column. Before the storm, S. longipes was found primarily in the upper 100 m of the water column, and its population was dominated by adults (mean proportion = 41%) and the copepodite stage I (mean proportion = 30%). After the storm, the abundance increased considerably, and the copepodite stage I contributed by far the largest proportion (53%) of the total population indicating that the early copepodite stages probably had been released from the sea ice into the under ice water layer due to ice break-up and ice melt processes caused by the storm.     

Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.     

Contact-free cold atmospheric plasma treatment of Deinococcus radiodurans

In this study we investigated the sensitivity of Deinococcus radiodurans to contact-free cold atmospheric plasma treatment as part of a project to establish new efficient procedures for disinfection of inanimate surfaces. The Gram-positive D.?radiodurans is one of the most resistant microorganisms worldwide. Stationary phases of D.?radiodurans were exposed to cold atmospheric plasma for different time intervals or to ultraviolet?C (UVC) radiation at dose rates of 0.001-0.0656?J?cm(-2), respectively. A methicillin-resistant Staphylococcus aureus strain (MRSA) served as control for Gram-positive bacteria. The surface microdischarge plasma technology was used for generation of cold atmospheric plasma. A plasma discharge was ignited using ambient air. Surprisingly, D.?radiodurans was sensitive to the cold atmospheric plasma treatment in the same range as the MRSA strain. Survival of both bacteria decreased with increasing plasma exposure times up to 6 log(10) cycles (>99.999?%) within 20?s of plasma treatment. In contrast, UVC radiation of both bacteria demonstrated that D.?radiodurans was more resistant to UVC treatment than MRSA. Cold atmospheric plasma seems to be a promising tool for industrial and clinical purposes where time-saving is a critical point to achieve efficient disinfection of inanimate surfaces and where protection from corrosive materials is needed.     

Ribosome binding of a single copy of the SecY complex: implications for protein translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.     

The effects of sulphide on growth and behaviour of the thiotrophic Zoothamnium niveum symbiosis

Zoothamnium niveum (Ciliophora, Oligohymenophora) is a giant, colonial marine ciliate from sulphide-rich, shallow-water habitats, obligatorily associated with the ectosymbiotic, chemoautotrophic, sulphide-oxidizing bacterium 'Candidatus Thiobios zoothamnicoli'. The aims of this study were to characterize the natural habitat and investigate growth, reproduction, survival and maintenance of the symbiosis from Corsica, France (Mediterranean Sea) using a flow-through respirometer providing stable chemical conditions. We were able to successfully cultivate the Z. niveum symbiosis during its entire lifespan and document reproduction, whereby the optimum conditions were found to range from 3 to 33 micromol l(-1) sigmaH2S in normoxic seawater. Starting with an inoculum of 13 specimens, we found up to 173 new specimens that were asexually produced after only 11 days. Observed mean lifespan of the Z. niveum colonies was approximately 11 days and mean colony size reached 51 branches, from which rapid host division rates of up to every 4.1 hours were calculated. Comparing the ectosymbiotic population from Z. niveum colonies collected from their natural habitat with those cultivated under optimal conditions, we found significant differences in the bacterial morphology and the frequency of dividing cells on distinct host parts, which is most likely caused by behaviour of the host ciliate. Applying different sulphide concentrations we revealed that the symbiosis was not able to survive without sulphide and was harmed by high sulphide conditions. To our knowledge, this study reports the first successful cultivation of a thiotrophic ectosymbiosis.     

Molecular mechanism of action of monocyclam versus bicyclam non-peptide antagonists in the CXCR4 chemokine receptor

AMD3465 is a novel, nonpeptide CXCR4 antagonist and a potent inhibitor of HIV cell entry in that one of the four-nitrogen cyclam rings of the symmetrical, prototype bicyclam antagonist AMD3100 has been replaced by a two-nitrogen N-pyridinylmethylene moiety. This substitution induced an 8-fold higher affinity as determined against (125)I-12G5 monoclonal CXCR4 antibody binding, and a 22-fold higher potency in inhibition of CXCL12-induced signaling through phosphatidylinositol accumulation. Mutational mapping of AMD3465 and a series of analogs of this in a library of 23 mutants covering the main ligand binding pocket of the CXCR4 receptor demonstrated that the single cyclam ring of AMD3465 binds in the pocket around AspIV:20 (Asp(171)), in analogy with AMD3100, whereas the N-pyridinylmethylene moiety mimics the other cyclam ring through interactions with the two acidic anchor-point residues in transmembrane (TM)-VI (AspVI:23/Asp(262)) and TM-VII (GluVII:06/Glu(288)). Importantly, AMD3465 has picked up novel interaction sites, for example, His(281) located at the interface of extracellular loop 3 and TM-VII and HisIII:05 (His(113)) in the middle of the binding pocket. It is concluded that the simple N-pyridinylmethylene moiety of AMD3465 substitutes for one of the complex cyclam moieties of AMD3100 through an improved and in fact expanded interaction pattern mainly with residues located in the extracellular segments of TM-VI and -VII of the CXCR4 receptor. It is suggested that the remaining cyclam ring of AMD3465, which ensures the efficacious blocking of the receptor, in a similar manner can be replaced by chemical moieties allowing for, for example, oral bioavailability.     

Mutation in the AP4M1 Gene Provides a Model for Neuroaxonal Injury in Cerebral Palsy

Cerebral palsy due to perinatal injury to cerebral white matter is usually not caused by genetic mutations, but by ischemia and/or inflammation. Here, we describe an autosomal-recessive type of tetraplegic cerebral palsy with mental retardation, reduction of cerebral white matter, and atrophy of the cerebellum in an inbred sibship. The phenotype was recorded and evolution followed for over 20 years. Brain lesions were studied by diffusion tensor MR tractography.Homozygosity mapping with SNPs was performed for identification of the chromosomal locus for the disease. In the 14 Mb candidate region on chromosome 7q22, RNA expression profiling was used for selecting among the 203 genes in the area. In postmortem brain tissue available from one patient, histology and immunohistochemistry were performed. Disease course and imaging were mostly reminiscent of hypoxic-ischemic tetraplegic cerebral palsy, with neuroaxonal degeneration and white matter loss. In all five patients, a donor splice site pathogenic mutation in intron 14 of the AP4M1 gene (c.1137+1G→T), was identified. AP4M1, encoding for the μ subunit of the adaptor protein complex-4, is involved in intracellular trafficking of glutamate receptors. Aberrant GluRδ2 glutamate receptor localization and dendritic spine morphology were observed in the postmortem brain specimen. This disease entity, which we refer to as congenital spastic tetraplegia (CST), is therefore a genetic model for congenital cerebral palsy with evidence for neuroaxonal damage and glutamate receptor abnormality, mimicking perinatally acquired hypoxic-ischemic white matter injury.