ABSTRACT
In this work, we describe a microfluidic three-dimensional (3D) chondrocyte culture mimicking in vivo articular chondrocyte morphology, cell distribution, metabolism, and gene expression. This has been accomplished by establishing a physiologic nutrient diffusion gradient across the simulated matrix, while geometric design constraints of the microchambers drive native-like cellular behavior. Primary equine chondrocytes remained viable for the extended culture time of 3 weeks and maintained the low metabolic activity and high Sox9, aggrecan, and Col2 expression typical of articular chondrocytes. Our microfluidic 3D chondrocyte microtissues were further exposed to inflammatory cytokines to establish an animal-free, in vitro osteoarthritis model. Results of our study indicate that our microtissue model emulates the basic characteristics of native cartilage and responds to biochemical injury, thus providing a new foundation for exploration of osteoarthritis pathophysiology in both human and veterinary patients.
ABSTRACT
The graded expression of transcription factor interferon regulatory factor 4 (IRF4) regulates B cell development and is critical for plasma cell differentiation. However, the mechanisms, by which IRF4 elicits its crucial tasks, are largely unknown. To characterize the molecular targets of IRF4 in B cells, we established an IRF4-deficient DT40 B cell line. We found that in the absence of IRF4, the expression of several molecules involved in BCR signalling was altered. For example, the expression of B cell adaptor for PI3K (BCAP) was upregulated, whereas the SHIP (SH2-containing Inositol 5?-Phosphatase) expression was downregulated. These molecular unbalances were accompanied by increased BCR-induced calcium signalling, attenuated B cell linker protein (BLNK) and ERK activity and enhanced activity of PI3K/protein kinase B (Akt) pathway. Further, the IRF4-deficient cells showed dramatically diminished cytoskeletal responses to anti-IgM cross-linking. Our results show that IRF4 has an important role in the regulation of BCR signalling and help to shed light on the molecular mechanisms of B cell development and germinal centre response.
Subject(s)
Avian Proteins/metabolism , B-Lymphocytes/physiology , Interferon Regulatory Factors/metabolism , Receptors, Antigen, B-Cell/metabolism , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Avian Proteins/genetics , Calcium Signaling/genetics , Cell Line , Chickens , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/genetics , Gene Knockout Techniques , Interferon Regulatory Factors/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/genetics , Protein-Tyrosine Kinases/metabolism , Syk KinaseABSTRACT
The culture medium of Streptomyces xanthochromogenes JH903 was found to show selective activity against DNA-repair-deficient Escherichia coli CM871 strain. In this report we describe a simple method to locate and isolate DNA binding compounds from the fermentation broth. The method is based on the retention of DNA-reacting compounds in cellulose complexed with DNA, and purification of these compounds with thin-layer chromatography. Screening of microbial metabolites from chloroform extracts of fermentation media resulted in detection of five genotoxic fractions.
