The first sex-specific molecular marker discovered in the moss Pseudocalliergon trifarium

Most dioecious plants do not exhibit discernible sexual dimorphism before sexual maturity. Therefore, it is impossible to address any sex-related questions during the prereproductive phase unless a genetic sex marker is available for gender determination. The aim of the present study was to develop a genetic sex marker for the moss Pseudocalliergon trifarium to allow gender and sex ratio determination at any stage in the life cycle. A high proportion of P. trifarium populations do not express sex. The screening of genomic DNA with inter simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. A presumably female-specific band was found, excised from the gel, cloned, and sequenced. A sequence-walking method was used to characterize the same region in males. A primer pair was designed to allow the amplification of a 159-bp portion of the female-specific DNA region. All tested material, up to 16-year-old herbarium specimens, provided unambiguous amplification products. This study successfully provides, for the first time in a moss, a sex-specific DNA marker. It allows reliable determination of gender and sex ratios. The short length of the amplification product is an advantage as satisfactory PCR products are more likely when the targeted sequence is short. The amount of variation in the DNA region shared by both sexes was relatively high. If the male sequence can be better characterized, the sex-specific regions could possibly be used to evaluate sex-specific phylogeographic patterns.     

Murine mCLCA6 is an integral apical membrane protein of non-goblet cell enterocytes and co-localizes with the cystic fibrosis transmembrane conductance regulator.

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine.     

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.     

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.     

Acetal linked oligoethylenimines for use as pH-sensitive gene carriers

Two types of acid-degradable nonviral gene carriers, OEI-MK and OEI-BAA, were synthesized by polymerizing oligoethylenimine of 800 Da (OEI800) with the pH-sensitive acetone ketal cross-linker 2,2-bis(N-maleimidoethyloxy) propane (MK) or the 4-methoxybenzaldehyde bisacrylate acetal cross-linker 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). Corresponding acid-insensitive counterparts (OEI-BM and LT-OEI-HD) were synthesized as well, representing control polymers. Kinetics of hydrolysis were measured and confirmed the pH-dependent degradation profile of the acetal functions, with short half-lives of 3 min at pH 5.0, and 5 h (OEI-MK) or 3.5 h (OEI-BAA) at physiological pH 7.4 and 37 degrees C. DNA polyplexes of a luciferase expression plasmid were tested for gene transfer efficiency and biocompatibility in two cell lines (B16F10 and Neuro2A). Polyplexes with acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low cation/plasmid (c/p) w/w ratios the transfection efficiency of pH-sensitive polymers was slightly reduced, but it became similar or superior to the efficiency of acid-stable polymers at higher c/p ratios. An improved in vivo biocompatibility of the acid-degradable polymers over the stable control polymers was confirmed by liver histology after systemic administration of polymers in Balb/c mice.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.     

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepa-tocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.