Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.     

Adapt or die—Response of large herbivores to environmental changes in Europe during the Holocene

Climate warming and human landscape transformation during the Holocene resulted in environmental changes for wild animals. The last remnants of the European Pleistocene megafauna that survived into the Holocene were particularly vulnerable to changes in habitat. To track the response of habitat use and foraging of large herbivores to natural and anthropogenic changes in environmental conditions during the Holocene, we investigated carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope composition in bone collagen of moose (Alces alces), European bison (Bison bonasus) and aurochs (Bos primigenius) in Central and Eastern Europe. We found strong variations in isotope compositions in the studied species throughout the Holocene and diverse responses to changing environmental conditions. All three species showed significant changes in their δ¹³C values reflecting a shift of foraging habitats from more open in the Early and pre‐Neolithic Holocene to more forest during the Neolithic and Late Holocene. This shift was strongest in European bison, suggesting higher plasticity, more limited in moose, and the least in aurochs. Significant increases of δ¹⁵N values in European bison and moose are evidence of a diet change towards more grazing, but may also reflect increased nitrogen in soils following deglaciation and global temperature increases. Among the factors explaining the observed isotope variations were time (age of samples), longitude and elevation in European bison, and time, longitude and forest cover in aurochs. None of the analysed factors explained isotope variations in moose. Our results demonstrate the strong influence of natural (forest expansion) and anthropogenic (deforestation and human pressure) changes on the foraging ecology of large herbivores, with forests playing a major role as a refugial habitat since the Neolithic, particularly for European bison and aurochs. We propose that high flexibility in foraging strategy was the key for survival of large herbivores in the changing environmental conditions of the Holocene.     

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.     

Expression of bacterial biphenyl-chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.     

A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1(lacZ/lacZ) die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1(lacZ) knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1(lacZ) offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1(lacZ/+)-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.