Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin™ is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin™ could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin™ were generated. Immunoblot experiments showed that Solulin™ accumulated to maximum levels of 115 and 27 μg g⁻¹ plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin™ was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin™ from the BY2 culture supernatant. The sequence was identical to that of Solulin™ produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.     

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.     

Interpretability of bi-level variable selection methods

Variable selection is usually performed to increase interpretability, as sparser models are easier to understand than full models. However, a focus on sparsity is not always suitable, for example, when features are related due to contextual similarities or high correlations. Here, it may be more appropriate to identify groups and their predictive members, a task that can be accomplished with bi-level selection procedures. To investigate whether such techniques lead to increased interpretability, group exponential LASSO (GEL), sparse group LASSO (SGL), composite minimax concave penalty (cMCP), and least absolute shrinkage, and selection operator (LASSO) as reference methods were used to select predictors in time-to-event, regression, and classification tasks in bootstrap samples from a cohort of 1001 patients. Different groupings based on prior knowledge, correlation structure, and random assignment were compared in terms of selection relevance, group consistency, and collinearity tolerance. The results show that bi-level selection methods are superior to LASSO in all criteria. The cMCP demonstrated superiority in selection relevance, while SGL was convincing in group consistency. An all-round capacity was achieved by GEL: the approach jointly selected correlated and content-related predictors while maintaining high selection relevance. This method seems recommendable when variables are grouped, and interpretation is of primary interest.     

Hyperbaric oxygen induces apoptosis via a mitochondrial mechanism

During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization, caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen.     

Selection based on the folding properties of proteins with ribosome display

Ribosome display is a powerful tool for selecting and evolving protein functions through ligand-binding. Here, this in vitro system was used to perform selection based on the folding properties of proteins, independent of specific ligand-binding. The selection is based on two properties of misfolded proteins: (1) increased sensitivity to proteolysis and (2) greater exposure of hydrophobic area. By targeting these properties, we show that compactly folded and soluble proteins can be enriched over insoluble and random coil proteins. This approach may be especially useful for selection and evolution of folded proteins from random sequence libraries.     

A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in streptomyces coelicolor A3(2)

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient limitation is likely to be a major signal for the onset of their development, resulting in spore formation by specialized aerial hyphae. Streptomycetes grow on numerous carbon sources, which they utilize in a preferential manner. The main signaling pathway underlying this phenomenon is carbon catabolite repression, which in streptomycetes is totally dependent on the glycolytic enzyme glucose kinase (Glk). How Glk exerts this fascinating dual role (metabolic and regulatory) is still largely a mystery. We show here that while Glk is made constitutively throughout the growth of Streptomyces coelicolor A3(2), its catalytic activity is modulated in a carbon source-dependent manner: while cultures growing exponentially on glucose exhibit high Glk activity, mannitol- grown cultures show negligible activity. Glk activity was directly proportional to the amount of two Glk isoforms observed by Western blot analysis. The activity profile of GlcP, the major glucose permease, correlated very well with that of Glk. Our data are consistent with a direct interaction between Glk and GlcP, suggesting that a Glk-GlcP permease complex is required for efficient glucose transport by metabolic trapping. This is supported by the strongly reduced accumulation of glucose in glucose kinase mutants. A model to explain our data is presented.     

NUMB does not impair growth and differentiation status of experimental gliomas

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.     

Investigation of a Family with Autosomal Dominant Dilated Cardiomyopathy Defines a Novel Locus on Chromosome 2q14-q22

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and the most frequent indication for heart transplantation in young patients. Probably >25% of DCM cases are of familial etiology. We report here genetic localization in a three-generation German family with 12 affected individuals with autosomal dominant familial DCM characterized by ventricular dilatation, impaired systolic function, and conduction disease. After exclusion of known DCM loci, we performed a whole-genome screen and detected linkage of DCM to chromosome 2q14-q22. Investigation of only affected individuals defines a 24-cM interval between markers D2S2224 and D2S2324; when unaffected individuals are also included, the critical region decreases to 11 cM between markers D2S2224 and D2S112, with a peak LOD score of 3.73 at recombination fraction 0 at D2S2339. The identification of an additional locus for familial autosomal dominant DCM underlines the genetic heterogeneity and may assist in the elucidation of the causes of this disease.