The primary cilium orchestrates chondrocyte mechanotransduction

Purpose: to understand the role of the chondrocyte primary cilium in mechanotransduction. The primary cilium is a singular organelle protruding outward from the mature centriole into the extracellular space, decorated with many receptors and integrins. The tubulin scaffold, ciliary membrane and proteome are constructed and maintained by dynein motors and intraflagellar transport cargos. In many cell types such as kidney epithelia, vascular endothelium and osteocytes, primary cilia are involved in mechanotransduction, often through the activities of polycystin 1 and 2 located on the cilium. Physiologically cartilage experiences mechanical signals, altering cell behaviour, that include compression and fluid flow. It is thought aberrant mechanotransduction may be one factor in the development of tissue pathology. Here we test the hypothesis that the cilium is essential for chondrocyte mechanotransduction and the up-regulation of proteoglycan synthesis through an established purinergic pathway involving the release of ATP and subsequent activation of Ca2+ signalling.Methods: to test this we used a hypomorphic mutation of Tg737, which encodes for IFT88, and abolishes genesis and growth of the cilium and which has been shown in vivo to result in murine matrix patterning defects. A well-established 3D agarose culture system was implemented to allow compressive loading of murine WT and Tg737 chondrocytes in culture followed by the quantification of ATP release with a luciferase assay, calcium transients by means of Fluo-4 imaging, and matrix production by qPCR and biochemical assay. Additionally expression of purinergic receptors (P2R) and polycystins (PC) 1 and 2 were assessed by western blot and immunocytochemistry.Results: compression of WT chondrocytes increased calcium transients (p<0.05) and matrix production at gene and protein levels (p<0.05) however these mechanosensitive responses were not present in Tg737 chondrocytes. Mechanosensitive ATP release (p<0.01) was maintained between WT and Tg737 cells implying that the cilium is required for ATP reception or transduction. Indeed, exogenous addition of ATP up-regulated Ca2+ transients in WT (p<0.001) but did not in Tg737 cells, although there were no differences in P2R expression. In Tg737 cells PC-1 expression was altered such that the full size protein product was absent.Conclusions: we conclude that the primary cilium is essential for chondrocyte mechanotransduction through the regulation of purinergic Ca2+ signalling. We speculate that this may be attributed to a role for the cilia protein polycystin-1. This demonstrates the central role for the chondrocyte primary cilium in cartilage physiology in the context of the chondrocyte response to mechanical stimuli

Shell environment for developing connectionist decision support systems

Expert Systems114225-234EXSY

Inheritance of tolerance to leaf iron deficiency chlorosis in tomato

By using two tomato genotypes, "227/1" (Fe chlorosis susceptible) and "Roza" (Fe chlorosis tolerant), and their reciprocal F-1, F-2 and BC1 generations, the inheritance of tolerance to leaf Fe deficiency chlorosis of Roza was studied. Plants were grown in a nutrient solution and subjected to 2.0 x 10(-6) M Fe EDDHA and 10 mM NaHCO3 to induce Fe deficiency stress by stabilization of pH to 7.8-8.2. A rating scale of 1-3 for chlorophyll was used and both monogenic and polygenic inheritance hypotheses were tested. Better responses to Fe deficiency, as measured by SPAD meter values, were obtained from the cross "Roza x 227/1" than from the reciprocal cross. Data from F-2 and BC1 suggest Fe chlorosis tolerance of Roza is to be controlled by polygenic loci with a relatively high additive effect