ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC. 4.1.1.49) is an enzyme for gluconeogenic pathway from lipid to sugar in C3 oil-seed plants ...
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Biochemical Society Transactions (200 I ) Volume 30, Part I
70 Phosphoenolpyruvate carboxykinase gene (cspck) from cucumber (Cucumis sativus L.) D.-J. Kim School of Science Education, College of Education, Chungbuk National University, 48 Gaeshin-Dong, Heungduk-Gu, Cheong Ju, 361-763, Chungbuk, Korea
72 The barrier properties of in vitro generated normal human urothelium
W.R.Cross, LEardley, H.J.Leese, J.Southgate Department of Biology, University of York, Heslington, Yotk, YO10 5 YW, Department of Urology, St. James’s University Hospital, Leeds, LJK
ATP-dependent phosphoenolpyruvate carboxykinase (PCK; E C 4.1.1.49) is an enzyme for gluconeogenic pathway from lipid to sugar in C 3 oil-seed plants during early germination. PCK is synthesised and accumulated rapidly in cotyledons during the few days immediately after seed imbibition, together with isocitrate lyase (ICL) and malate synthase (MS). Furthermore, PCK also acts during senescence in senescing cotyledons and leaves for recycling of nutrients. Full-length pck c D N A sequence had been reported first from cucumber earlier in 1994 by Kim and Smith. Since, this cspck gene is the first isolation from C 3 plants although the genes were isolated from C 4 grass by Finnegan et al., in 1999. Full genomic sequence of pck has been determined from cucumber. The sequences enable us to identify putative control regions at 5’ end of the gene, introns, and possible polyadenylation sites at the 3’ end. The cucumber genome contains a single cspck gene according to libraries screening and genomic southern blot analysis. Structure of cucumber cspck gene with putative cis-acting elements was identified and compared with ms and icl which play major roles lipid mobilisation in oil-seed plants during germination and senescence.
The human bladder and associated urinary tract is lined by urothelium, a transitional epithelium that acts as a permeability barrier. We are developing a human in vitro urothelial tissue model that mimics the in vivo system, in order to study the pathophysiology and pharmacology of urothelium. In this study we have assessed the barrier properties of in vitro propagated human urothelium. Normal human urothelial ( N H U ) cell cultures were established from surgical samples and propagated in serum-free and supplemented media. At confluency, urothelial barrier function was assessed by measuring the permeability to FITC-Dextran. Transurothelial ion transport was delineated by measuring transepithelial potential difference and short circuit current, and using specific ion channel inhibitors. NHU cells propagated in a serum-free medium formed monolayers, exhibited a low transepithelial electrical resistance and a high FITCDextran permeability. The addition of serum to the culture system resulted in urothelial stratification, intercellular tight junction formation, a high transepithelial electrical resistance, a low FITCDextran permeability and the expression of amiloride sensitive sodium channels. In conclusion, we have developed a human in vitro urothelial tissue model that expresses many of the morphological and functional properties of the in vivo system.
71 Barrels & corks in a computer - modelling bacterial iron
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transport systems M,Baade n, M. S. P. Sansom Laboratory of Molecular Biophysics, Oxford University, South Parks Road, Oxford OX1 3QU, U K The crystal structures of the outer membrane proteins FhuA and FepA from E. coli have recently been determined (Ferguson et al., Locher et al. and Buchanan et al.). These iron transporters pose an intriguing challenge to our current understanding of the Fe transport mechanism as the beta barrel is occluded by a globular Nterminal domain, the ‘cork’. Whether this part of the protein ‘unplugs’ during transport or undergoes a massive conformational change is unknown. Molecular dynamics simulations are a useful tool to probe the conformational dynamics of such systems, but they require a complete all-atom model as starting structure. The work presented shows how such a model can be devised using a number of computational techniques to model the protein and its environment accurately enough to reproduce the essential features of the system. First simulation results will also be presented. This work is supported by an E C Marie Curie Fellowship. References S. K. Buchanan et al.; 1999. Nature Struct. Biol. 6:56-63. A. D. Ferguson et al.; 1998. Science 282:2215-2220. K. P. Locher et al.; 1998. Cell 95:771-778.
0 2002 Biochemical Society
Caenorhabditis elegans as a model organism in the study of nucleoside transporters Appleford, M. Griffiths, E.G. Chomey, S.Y.M. Yao, D. MacGregor, R.E. Isaac, LA. Hope, D. Coates, C.E. Cass, J.D. Young, S.A. Baldwin University of Leeds,Leeds LS2 9J7; U.K. and University of Alberta, Edmonton, T6G 2H7, Canada
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The model organism, C. elegans, possesses six genes encoding putative equilibrative nucleoside transporters (ENTs). As the first step towards understanding the rationale underlying this diversity of transporters, we have expressed one of these, ZK809.4, in Xenopus oocytes; it exhibits a broad substrate specificity for purine and pyrimidine nucleosides and also transports antiviral nucleoside analogues such as 3’-azido-3’-deoxythymidine(AZT). cDNAs for two other E N T genes, CeENT2 and CeENT3, have also been expressed in Xenopus oocytes. We have demonstrated that these are able to transport nucleosides, with adenosine being a good substrate for CeENT2 and adenosine and uridine being substrates for CeENT3. In an attempt to elucidate the biological roles of these and other NTs, we have used G F P reporter constructs to investigate the temporal-spatial expression patterns of these genes. Additional clues to the biological roles of NTs have been obtained by doublestranded R N A interference (dsRNAi). RNAi using dsRNA corresponding to C e E N T l failed to yield a discernible phenotype. Similarly, injection of animals with dsRNA corresponding to CeENT2 yielded n o obvious phenotype. However, the progeny of worms co-injected with a mixture of C e E N T l and CeENT2 RNAs exhibited an everted vulva1 phenotype, suggesting that C e E N T l and CeENT2 exhibit a redundant function and have important roles during development.
