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Additional Residues (additional + residue)
Selected AbstractsHuman prion strain selection in transgenic mice,ANNALS OF NEUROLOGY, Issue 2 2010Kurt Giles DPhil Objective: Transgenic (Tg) mice expressing chimeras of mouse and human prion proteins (PrPs) have shorter incubation periods for Creutzfeldt-Jakob disease (CJD) prions than mice expressing full-length human PrP. Increasing the sequence similarity of the chimeric PrP to mouse PrP, by reverting human residues to mouse, resulted in a Tg line, denoted Tg22372, which was susceptible to sporadic (s) CJD prions in ,110 days. Methods: Mice expressing chimeric mouse/human PrP transgenes were produced. The mice were inoculated intracerebrally with extracts prepared from the brains of patients who died of CJD. Onset of neurological dysfunction marked the end of the incubation time. After sacrifice of the Tg mice, their brains were analyzed for PrPSc and neuropathological changes. Results: Reversion of 1 additional residue (M111V) resulted in a new Tg line, termed Tg1014, susceptible to sCJD prions in ,75 days. Tg1014 mice also have shorter incubation periods for variant (v) CJD prions, providing a more tractable model for studying this prion strain. Transmission of vCJD prions to Tg1014 mice resulted in 2 different strains, determined by neuropathology and biochemical analysis, which correlated with the length of the incubation time. One strain had the biochemical, neuropathological, and transmission characteristics, including longer incubation times, of the inoculated vCJD strain; the second strain produced a phenotype resembling that of sCJD prions including relatively shorter incubation periods. Mice with intermediate incubation periods for vCJD prions had a mixture of the 2 strains. Both strains were serially transmitted in Tg1014 mice, which led to further reduction in incubation periods. Conversion of vCJD-like to sCJD-like strains was favored in Tg1014 mice more than in the Tg22372 line. The single amino acid difference therefore appears to offer selective pressure for propagation of the sCJD-like strain. Interpretation: These 2 Tg mouse lines provide relatively rapid models to study human prion diseases as well as the evolution of human prion strains. ANN NEUROL 2010;68:151,161 [source] Molecular physiology of SLC4 anion exchangersEXPERIMENTAL PHYSIOLOGY, Issue 1 2006Seth L. Alper Plasmalemmal Cl,,HCO3, exchangers regulate intracellular pH and [Cl,] and cell volume. In polarized epithelial cells, they contribute also to transepithelial secretion and reabsorption of acid,base equivalents and of Cl,. Members of both the SLC4 and SLC26 mammalian gene families encode Na+ -independent Cl,,HCO3, exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl,,anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl, conductance, but definitive evidence for mediation of Cl, conductance is lacking. However, a single missense mutation allows AE1 to mediate both electrogenic SO42,,Cl, exchange or electroneutral, H+ -independent SO42,,SO42, exchange. In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues reported to bind carbonic anhydrase II are dispensable for Cl,,Cl, exchange, but required for Cl,,HCO3, exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H+, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain. Individual missense mutations within this and adjacent regions identify additional residues which acid-shift pHo sensitivity. These regions together are modelled to form contiguous surface patches on the AE2 cytoplasmic domain. In contrast, the N-terminal variant AE2c polypeptide exhibits an alkaline-shifted pHo sensitivity, as do certain transmembrane domain His mutants. AE2-mediated anion exchange is also stimulated by ammonium and by hypertonicity by a mechanism sensitive to inhibition by chelation of intracellular Ca2+ and by calmidazolium. This growing body of structure,function data, together with increased structural information, will advance mechanistic understanding of SLC4 anion exchangers. [source] Identification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of AlphastatinJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2007C. A. STATON Summary., Background :,We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo. Objectives:, The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity. Methods:, The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas. Results:, Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg,1 (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity. Conclusions:, Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin. [source] Structure of Arabidopsis chloroplastic monothiol glutaredoxin AtGRXcpACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Lenong Li Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are conserved across species. Arabidopsis thaliana monothiol glutaredoxin AtGRXcp is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been determined at 2.4,Å resolution. AtGRXcp has a glutaredoxin/thioredoxin-like fold with distinct structural features that differ from those of dithiol Grxs. The structure reveals that the putative active-site motif CGFS is well defined and is located on the molecular surface and that a long groove extends to both sides of the catalytic Cys97. Structural comparison and molecular modeling suggest that glutathione can bind in this groove and form extensive interactions with conserved charged residues including Lys89, Arg126 and Asp152. Further comparative studies reveal that a unique loop with five additional residues adjacent to the active-site motif may be a key structural feature of monothiol Grxs and may influence their function. This study provides the first structural information on plant CGFS-type monothiol Grxs, allowing a better understanding of the redox-regulation mechanism mediated by these plant Grxs. [source] | ||||||||||