Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.     

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.     

Endogenous IL-18 in experimentally induced asthma affects cytokine serum levels but is irrelevant for clinical symptoms

T cells and T cell derived cytokines are involved in the complex pathogenesis of asthma. The role of the cytokine IL-18 however, is not clearly defined so far. On the one hand side IL-18 induces Th1-type cytokines and thereby might counter-regulate Th2-mediated allergic asthma. On the other hand IL-18 also bears pro-inflammatory effects possibly enhancing experimental asthma. In order to elucidate the role of IL-18 in allergic pulmonary inflammation typical symptoms were compared after induction of experimental asthma in IL-18^−/− and in wild type mice. Asthma was induced using ovalbumin (OVA) as allergen for sensitization and challenge. Sham sensitized and OVA challenged mice served as controls. Bronchoalveolar lavage-fluid cytology, leukocyte infiltration in lung tissues, serum levels of OVA-specific IgE and cytokines, and lung function were analyzed. Clear differences could be observed between control and asthmatic mice, both in wild type and IL-18^−/− animals. Surprisingly, no differences were found between asthmatic wild type and IL-18^−/− mice. Thus, in contrast to conflicting data in the literature IL-18 did not suppress or enhance the pulmonary allergic immune response in a murine experimental model of asthma.     

Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10-Instituted Intrinsic Immunity

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi''s Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.     

Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Autoantibodies are believed to be maintained by either the continuous generation of short-lived plasma cells in secondary lymphoid tissues or by long-lived plasma cells localized in bone marrow and spleen. Here, we show in a mouse model for the autoimmune blistering skin disease epidermolysis bullosa acquisita (EBA) that chronic autoantibody production can also be maintained in inflamed lymph nodes, by plasma cells exhibiting intermediate lifetimes. After EBA induction by immunization with a mCOL7c-GST-fusion protein, antigen-specific plasma cells and CD4 T cells were analyzed. Plasma cells were maintained for months in stable numbers in the draining lymph nodes, but not in spleen and bone marrow. In contrast, localization of mCOL7c-GST -specific CD4 T cells was not restricted to lymph nodes, indicating that availability of T cell help does not limit plasma cell localization to this site. BrdU-incorporation studies indicated that pathogenic mCOL7c- and non-pathogenic GST-specific plasma cells resemble intermediates between short-and long-lived plasma cells with half-lives of about 7 weeks. Immunization with mCOL7c-GST also yielded considerable numbers of plasma cells neither specific for mCOL7c- nor GST. These bystander-activated plasma cells exhibited much shorter half-lives and higher population turnover, suggesting that plasma cell lifetimes were only partly determined by the lymph node environment but also by the mode of activation. These results indicate that inflamed lymph nodes can harbor pathogenic plasma cells exhibiting distinct properties and hence may resemble a so far neglected site for chronic autoantibody production.     

Molecular dynamics simulations indicate a possible role of parallel beta-helices in seeded aggregation of poly-Gln

The molecular structures of amyloid fibers characterizing neurodegenerative diseases such as Huntington's or transmissible spongiform encephalopathies are unknown. Recently, x-ray diffraction patterns of poly-Gln fibers and electron microscopy images of two-dimensional crystals formed from building blocks of prion rods have suggested that the corresponding amyloid fibers are generated by the aggregation of parallel beta-helices. To explore this intriguing concept, we study the stability of small beta-helices in aqueous solution by molecular dynamics simulations. In particular, for the Huntington aggregation nucleus, which is thought to be formed of poly-Gln polymers, we show that three-coiled beta-helices are unstable at the suggested circular geometries and stable at a triangular shape with 18 residues per coil. Moreover, we demonstrate that individually unstable two-coiled triangular poly-Gln beta-helices become stabilized upon dimerization, suggesting that seeded aggregation of Huntington amyloids requires dimers of at least 36 Gln repeats (or monomers of approximately 54 Gln) for the formation of sufficiently stable aggregation nuclei. An analysis of our results and of sequences occurring in native beta-helices leads us to the proposal of a revised model for the PrP(Sc) aggregation nucleus.     

Improved enantioselective analysis of polyunsaturated hydroxy fatty acids in psoriatic skin scales using high-performance liquid chromatography

Enantioselective analysis is used as a valuable tool for determining the biological origin of chiral derivatives of arachidonic, 11,14-eicosadienoic and linoleic acid in psoriatic skin scales and for clarifying their role in pathogenesis. This paper reports on a simple and rapid enantioselective determination (without any derivatization) of the fatty acid derivatives 13(R,S)-hydroxyoctadecadienoic acid [13(R,S)-HODE], 9(R,S)-hydroxyoctadecadienoic acid [9(R,S)-HODE] and 12(R,S)-hydroxyeicosatetraenoic acid [12(R,S)-HETE], using high-performance liquid chromatography (HPLC) with Chiralpak AD as the chiral selector and electrospray ionisation mass spectrometry (ESI-MS). The enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE in psoriatic skin scales of untreated patients (untreated during the last 4 weeks before sampling) was evaluated in comparison to psoriatic skin scales of patients underlying systemic treatment. The enantiomeric distribution of 12-HETE and 9-HODE showed no remarkable differences, whilst samples of patients under systemic treatment exhibited a lower predominance of 13(S)-HODE than samples of untreated patients. Furthermore, the effect of UVB phototherapy on the enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE was studied and a semiquantitation of these compounds in psoriatic skin scales performed. The detected amounts of 9-HODE in samples of untreated patients were remarkably lower than those in samples of patients underlying systemic treatment. In the case of UVB phototherapy, no influence on the enantiomeric distribution could be observed.     

Migration of bone marrow-derived cells and improved perfusion after treatment with erythropoietin in a murine model of myocardial infarction

Erythropoietin (EPO) was shown to have protective effects after myocardial infarction (MI) by neovascularization and antiapoptotic mechanisms. Beside direct receptor-dependent mechanisms, mobilization and homing of bone marrow-derived cells (BMCs) may play a pivotal role in this regard. In this study, we intended to track different subpopulations of BMCs and to assess serially myocardial perfusion changes in EPO-treated mice after MI. To allow tracking of BMCs, we used a chimeric mouse model. Therefore, mice (C57BL/6J) were sublethally irradiated, and bone marrow (BM) from green fluorescent protein transgenic mice was transplanted. Ten weeks later coronary artery ligation was performed to induce MI. EPO was injected for 3 days with a total dose of 5000 IU/kg. Subpopulations (CD31, c-kit, CXCR-4 and Sca-1) of EGFP(+) cells were studied in peripheral blood, bone marrow and hearts by flow cytometry. Myocardial perfusion was serially investigated in vivo by pinhole single-photon emission computed tomography (SPECT) at days 6 and 30 after MI. EPO-treated animals revealed an enhanced mobilization of BMCs into peripheral blood. The numbers of these cells in BM remained unchanged. Homing of all BMCs subpopulations to the ischaemic myocardium was significantly increased in EPO-treated mice. Among the investigated subpopulations, EPO predominantly affected migration of CXCR-4(+) (4.3-fold increase). Repetitively SPECT analyses revealed a reduction of perfusion defects after EPO treatment over time. Our study shows that EPO treatment after MI enhances the migration capacity of BMCs into ischaemic tissue, which may attribute to an improved perfusion and reduced size of infarction, respectively.     

The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.