Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Glycosaminoglycans (GAGs) play an important role in many biological processes in the extracellular matrix. In a theoretical approach, structures of monosaccharide building blocks of natural GAGs and their sulfated derivatives were optimized by a B3LYP6311ppdd//B3LYP/ 6-31+G(d) method. The dependence of the observed conformational properties on the applied methodology is described. NMR chemical shifts and proton-proton spin-spin coupling constants were calculated using the GIAO approach and analyzed in terms of the method's accuracy and sensitivity towards the influence of sulfation, O1-methylation, conformations of sugar ring, and ω dihedral angle. The net sulfation of the monosaccharides was found to be correlated with the 1H chemical shifts in the methyl group of the N-acetylated saccharides both theoretically and experimentally. The ω dihedral angle conformation populations of free monosaccharides and monosaccharide blocks within polymeric GAG molecules were calculated by a molecular dynamics approach using the GLYCAM06 force field and compared with the available NMR and quantum mechanical data. Qualitative trends for the impact of sulfation and ring conformation on the chemical shifts and proton-proton spin-spin coupling constants were obtained and discussed in terms of the potential and limitations of the computational methodology used to be complementary to NMR experiments and to assist in experimental data assignment.

To investigate and assess bone regeneration in sheep in combination with new implant materials classical histological staining methods as well as immunohistochemistry may provide additional information to standard radiographs or computer tomography. Available published data of bone defect regenerations in sheep often present none or sparely labeled histological images. Repeatedly, the exact location of the sample remains unclear, detail enlargements are missing and the labeling of different tissues or cells is absent. The aim of this article is to present an overview of sample preparation, staining methods and their benefits as well as a detailed histological description of bone regeneration in the sheep tibia. General histological staining methods like hematoxylin and eosin, Masson-Goldner trichrome, Movat’s pentachrome and alcian blue were used to define new bone formation within a sheep tibia critical size defect containing a polycaprolactone-co-lactide (PCL) scaffold implanted for 3 months (n = 4). Special attention was drawn to describe the bone healing patterns down to cell level. Additionally one histological quantification method and immunohistochemical staining methods are described.

Objectives. Polyamide-12 (PA) is a flexible material suited for denture bases and clasping. This study investigated its potential aging effects with a focus on surface roughness, color stability, and elasticity. Methods. PA specimens (Valplast) of 40 × 10 × 2mm and equally measuring PMMA specimens (Palapress) as control were fabricated. Color changes after storage in air, water, coffee, and red wine (n = 10) were measured using the CIE L*a*b color specification. Elasticity after thermocycling (1000, 3000, and 7000 cycles, n = 15) was measured by three-point bending testing. Mean surface roughness (Ra) was determined after storage in the liquids mentioned above and thermocycling (n = 10). Results. Tukey’s HSD test (P < 0.05) revealed statistically significant color changes of PA in red wine (ΔE = 4.27 after 12 days, EΔE = 6.90 after 12 days) and coffee (ΔE = 3.93 after 36 days) but no color changes in PMMA. Elastic modulus of PA was 845MPa and not affected by thermocycling (Tukey’s HSD test, P > 0.81). Dry specimens showed significantly decreased elasticity (P < 0.001). Mean surface roughness (PA 0.20 μm, PMMA 0.28 μm) did not change significantly after thermocycling or storage (Mann-Whitney U-test, 0.16 < P < 0.65). Significance. PA exhibited a higher susceptibility to discoloration than PMMA. Neither surface roughness nor elasticity of PA was altered by artificial aging.

Oleanolic and ursolic acid (OA and UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from S. officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator (PGR) combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the Respiration Activity MOnitoring System® (RAMOS®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The μmax values for these suspension cultures ranged from 0.20 to 0.37°d-1, their OA and UA contents were greater than 1.3 and 1.2 mg g-1, respectively, and they afforded maximum volumetric yields of 21.0 mg l-1 for OA and 32.8 mg l-1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.

This data article contains data related to the research article entitled, "in vitro characterization of bone marrow stromal cells from osteoarthritic donors" [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.