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101.
Bogani D Warr N Elms P Davies J Tymowska-Lalanne Z Goldsworthy M Cox RD Keays DA Flint J Wilson V Nolan P Arkell R 《Genesis (New York, N.Y. : 2000)》2004,40(2):109-117
Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype. 相似文献
102.
Mason AB Halbrooks PJ Larouche JR Briggs SK Moffett ML Ramsey JE Connolly SA Smith VC MacGillivray RT 《Protein expression and purification》2004,36(2):318-326
Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells. 相似文献
103.
Boraston AB Notenboom V Warren RA Kilburn DG Rose DR Davies G 《Journal of molecular biology》2003,327(3):659-669
Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs. 相似文献
104.
T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies 下载免费PDF全文
Lewicki H Tishon A Homann D Mazarguil H Laval F Asensio VC Campbell IL DeArmond S Coon B Teng C Gairin JE Oldstone MB 《Journal of virology》2003,77(6):3799-3808
CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP(-/-)) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1beta, IFN-gamma-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2(b) or H-2(d) molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K(b) tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) cytokines and were unable to lyse PrP(-/-) embryo fibroblasts or macrophages coated with (51)Cr-labeled mPrP peptide. These results suggest that the expression of PrP(sc) in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-gamma and TNF-alpha expression or release or lytic activity. 相似文献
105.
Partial molecular characterization of two simian immunodeficiency viruses (SIV) from African colobids: SIVwrc from Western red colobus (Piliocolobus badius) and SIVolc from olive colobus (Procolobus verus) 下载免费PDF全文
Courgnaud V Formenty P Akoua-Koffi C Noe R Boesch C Delaporte E Peeters M 《Journal of virology》2003,77(1):744-748
In order to study primate lentivirus evolution in the Colobinae subfamily, in which only one simian immunodeficiency virus (SIV) has been described to date, we screened additional species from the three different genera of African colobus monkeys for SIV infection. Blood was obtained from 13 West African colobids, and HIV cross-reactive antibodies were observed in 5 of 10 Piliocolobus badius, 1 of 2 Procolobus verus, and 0 of 1 Colobus polykomos specimens. Phylogenetic analyses of partial pol sequences revealed that the new SIVs were more closely related to each other than to the other SIVs and especially did not cluster with the previously described SIVcol from Colobus guereza. This study presents evidence that the three genera of African colobus monkeys are naturally infected with an SIV and indicates also that there was no coevolution between virus and hosts at the level of the Colobinae subfamily. 相似文献
106.
Salt IP Morrow VA Brandie FM Connell JM Petrie JR 《The Journal of biological chemistry》2003,278(21):18791-18797
Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose. 相似文献
107.
Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells 总被引:11,自引:0,他引:11
Morrow VA Foufelle F Connell JM Petrie JR Gould GW Salt IP 《The Journal of biological chemistry》2003,278(34):31629-31639
Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled. 相似文献
108.
109.
Cesura AM Pinard E Schubenel R Goetschy V Friedlein A Langen H Polcic P Forte MA Bernardi P Kemp JA 《The Journal of biological chemistry》2003,278(50):49812-49818
The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore. 相似文献
110.
Johansson K Bourhis JM Campanacci V Cambillau C Canard B Longhi S 《The Journal of biological chemistry》2003,278(45):44567-44573
Measles virus is a negative-sense, single-stranded RNA virus belonging to the Mononegavirales order which comprises several human pathogens such as Ebola, Nipah, and Hendra viruses. The phosphoprotein of measles virus is a modular protein consisting of an intrinsically disordered N-terminal domain (Karlin, D., Longhi, S., Receveur, V., and Canard, B. (2002) Virology 296, 251-262) and of a C-terminal moiety (PCT) composed of alternating disordered and globular regions. We report the crystal structure of the extreme C-terminal domain (XD) of measles virus phosphoprotein (aa 459-507) at 1.8 A resolution. We have previously reported that the C-terminal domain of measles virus nucleoprotein, NTAIL, is intrinsically unstructured and undergoes induced folding in the presence of PCT (Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S., and Canard, B. (2003) J. Biol. Chem. 278, 18638-18648). Using far-UV circular dichroism, we show that within PCT, XD is the region responsible for the induced folding of NTAIL. The crystal structure of XD consists of three helices, arranged in an anti-parallel triple-helix bundle. The surface of XD formed between helices alpha2 and alpha3 displays a long hydrophobic cleft that might provide a complementary hydrophobic surface to embed and promote folding of the predicted alpha-helix of NTAIL. We present a tentative model of the interaction between XD and NTAIL. These results, beyond presenting the first measles virus protein structure, shed light both on the function of the phosphoprotein at the molecular level and on the process of induced folding. 相似文献