Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Genistein inhibits calcium release by platelet-derived growth factor but not bradykinin or cadmium in human fibroblasts

Cd²⁺ provokes inositol trisphosphateproduction and releases stored Ca²⁺, apparently by binding to a zinc site in the external domain of an orphan receptor. One pM Cd²⁺ evokes an immediate spike in cytosolic free Ca²⁺, which is similar to that evoked by bradykinin. Platelet-derived growth factor (PDGF) also increases free Ca²⁺ in human dermalfibroblasts, but there is a distinct lag before free Ca²⁺ rises in response to PDGF. Genistein, which selectively inhibits tyrosine kinases, markedly inhibited Ca²⁺ mobilization evoked by PDGF. Calcium mobilization triggered by cadmium or bradykinin was relatively insensitive to genistein. The PDGF receptor is known to be a tyrosine kinase, whichphosphorylates and thereby activatesphospholipase C, whereas a G protein couples the bradykinin receptor to anotherphospholipase C isoform. These findings support the hypothesis that the orphan receptor triggered by cadmium is coupled to phospholipase C via a G protein.Abbreviations BSA bovine serum albumin - BK bradykinin - [Ca²⁺]_i cytosolic free calcium - DME Dulbecco's modified Eagle's medium - FBS fetal bovine serum - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - IC₅₀ concentration that produces 50% inhibition - PDGF platelet-derived growth factor - PSS physiological salts solution - SE standard error of the mean     

Approaches to breeding for salinity tolerance - a case study on Porteresia coarctata

Cereals are the world's major source of food for human nutrition. Among these, rice (Oryza sativa) is the most prominent and represents the staple diet for more than two-fifths (2.4 billion) of the world's population, making it the most important food crop of the developing world (Anon., 2000a). Rice production in vast stretches of coastal areas is hampered due to high soil salinity. This is because rice is a glycophyte and it does not grow well under saline conditions. In order to increase rice production in these areas there is a need to develop rice varieties suited to saline environments. Research has shown that Porteresia coarctata, a highly salt tolerant wild relative of rice growing in estuarine soils, is an important material for transferring salt tolerant characteristics to rice. It is quite possible that Porteresia may be used as a parent for evolving better and truly salt resistant varieties. The inadequate results and the difficulties associated with conventional breeding techniques necessitate the use of the tools of crop biotechnology in unravelling some of the characteristics of Porteresia that have been highlighted in this report. In view of the limited resources available for increasing salinity tolerance to the breeders to wild rice germplasm, Porteresia is undoubtedly one of the key source species for elevating salinity tolerance in cultivated rice.