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Biochemical Society Transactions (2000) Volume 28, part 3
69
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The concept of engineered extracellular matrices that can aid in the repair of damaged tissues is a common theme in the tissue engineering field. Central to this theme is the rationale that matrices, which mimic the composition and architecture of native tissues, can support and signal appropriate host cell repair responses. The design of such matrices draws upon concepts emerging from the cell biology, biomaterial and bioengineering fields. We have incorporated expertise from each of these areas into the development of matrices and matrix/growth factor combinations for the repair of bone and cartilage tissue using biomaterials such as type I collagen and hyaluronan. Type I collagen fibers coated with resorbable hydroxyapatite and crosslinked form an osteoconductive matrix that performs equivalent t o autogenous bone when combined with a bone marrow aspirate o r a recombinant human bone morphogenetic protein (GDF-5). The combination of recombinant human basic fibroblast g r o r t h factor (FGF-2) with a hyaluronan gel results in a percutaneously injectable formulation that enhances fracture repair. More recent matrix formulations utilize a modified hyaluronan t o coat and crosslink type I collagen fibers into a 3-D matrix that supports both osteogenesis and chondrogenesis in combination with growth factors such as GDF-5. The studies to be presented highlight the progress made in our understanding of cell, matrix, and growth factor interactions as they relate to skeletal tissue repair.
Phosphorous (P) is not only a structural component of essential biological molecules such as ATP, nucleic acids and phospholipids, but is also a regulator of key enzymatic reactions and metabolic pathways. Consequently this element is essential for growth and development and its concentration is carefully regulated at both the cellular and whole plant levels. Plants may acquire P in the form of phosphate (Pi) directly from the soil solution via their roots, but it can also be delivered to the cortex of the root via arbuscular mycorrhizal (AM) fungi with which they form stable symbioses. In a similar manner to other ions, phosphate movement in and out of the individual cells of a plant or AM fungus is mediated via transporters, or possibly channels in the plasma membrane of the cell. Phosphate uptake studies in yeast and Neurorpora crassa indicate the presence of two phosphate uptake systems with distinct kinetics; a high affinity, saturable, uptake system that is induced during growth in low phosphate conditions, and a low affinity system that appears to be constitutively expressed. A number of years ago, transport proteins responsible for the high affinity kinetic component were cloned from both yeast and N . crassa; however, the nature of the proteins that mediates low affinity kinetic component are still unknown. Over the past few years, sequence information from yeast and N . crassa has enabled the cloning of phosphate transporters from a range of plant species and also from AM fungi. Characterization of these proteins is providing insight into the mechanisms underlying phosphate acquisition by plant roots in the presence and absence of an arbuscular mycorrhizal symbiosis.
Extracellular Matrices and MatrixIGrowth Factor Combinations for Bone and Cartilage Repair LC.Spirp, L.S. Liu, M.A. Heidaran, A.Y. Thompson, C.K. Ng, J. Pohl, and J.W. Poser Orquest, Inc., 365 Ravendale Drive, Mountain View, California, 94043, Biopharm Gmbh, Czernyring 22, 0-69115, Heidelberg, Germany
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Calcium signalling and calcium channels in plants and yeast D.Sanders, L. Navazio, M. Bewell, A.Siddiqua, G. Dickinson, M. Fischer Biology Department, University of York, York YO10 SYW
Elevation of cytosolic free Ca plays a central role in transducing many metabolic and developmental stimuli in plants. Pathways for mobilisation of Ca from vacuoles include channels which are gated by inositol 1,4,5trisphosphate (IP3) and cyclic ADP-ribose (cADPR). By contrast much less is known about the properties of calcium-permable channels at the ER. We investigated the possibility that ligand-gated Ca release occurs in ERenriched vesicles derived from cauliflower inflorescences. cADPR-gated calcium release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cyt c reductase activity. Calcium release could also be evoked by the N A D P metabolite nicotinic acid adenine dinucleotide phosphate (NAADP). This pathway, not previously described in plants, was insensitive to inhibitors of IP3- and cADPR-elicited calcium release, and, unlike the IP3- and cADPR-gated pathways, was located exclusively at the ER. Production of NAADP from N A D P by homogenates of cauliflower was detected using a sea urchin microsome bioassay. The existence of multiple Ca mobilisation pathways might contribute to the generation of stimulus-specific calcium signals in plants. In yeast, the alpha-factor induced mating response requires external C a and C a uptake is mediated by interaction of two proteins. Cchl and Midl. The phenotype of deletion mutants suggest roles for these Ca channel forming subunits in a variety of additional physiological processes, including (1) cell viability at low Ca; (2) the adaptation of yeast growth to temperature; (3) the adaptation/resistance t o divalent ions.
0 2000 Biochemical Society
Phosphate transport in roots and arbuscular mycorrhizas M.J.Harrison The Samuel Roberts Noble Foundation, Plant Biology Division, 2510 Sam Noble Parkway, Ardmore, OK 73402, USA
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Cyclic nucleotide- and calcium/calmodulin-regulatedchannels in plants: targets for manipulating heavy metal tolerance, and possible physiological roles R. Sunkar', B. Kaplan', T.Arazi', D. Dolev' and H,'J 'The Weizmann Institute of Science, Rehovot, fsraeland LfBA Centre for Plant Sciences, University of Leeds, Leeds LS2 9J7;
[email protected]
Using radiolabelled recombinant calmodulin as a probe to screen cDNA expression libraries, we have discovered a tobacco protein (NtCBP4) that can modulate plant tolerance to heavy metals'. Structurally, NtCBP4 is similar t o mammalian cyclic nucleotide gated non-selective cation channels, and t o channel proteins recently isolated from various plant species. NtCBP4 contains six putative transmembrane domains, a predicted pore region, a conserved cyclic nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic nucleotide-binding domainz. Transgenic tobacco expressing NtCBP4, which was localised in the plasma membrane, exhibit improved tolerance to Ni2+ and hypersensitivity to Pb2+,associated with reduced Nif+ and enhanced Pb2+ accumulation, respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains were removed, have a different phenotype, which provides further evidence for the function of NtCBP4 in metal uptake across the plasma membrane. The various transgenic plants should prove useful for elucidating the physiological roles of NtCBP4 and related plant proteins. Physiological responses known to be mediated by calcium and cyclic nucleotides are being investigated. 'Arazi T, Sunkar R, Kaplan B & Fromm H (1999) Plant J. 20: 171-182. 'Arazi T, Kaplan B & Fromm H (2000) Plant Mol. Biol. (in press).
