The iQ200 Microscopic Analyzer is valuable tool for evaluation of urinary sediment at transplanted patients

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Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase from Rhodospirillum rubrum

Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG

Prion and doppel proteins bind to granule cells of the cerebellum

We reported that expression of the cellular prion protein (PrP(C)) rescues doppel (Dpl)-induced cerebellar degeneration in mice. To search for protein(s) that mediate this process, we fused the C-termini of mouse (Mo) PrP and Dpl to the Fc portion of an IgG. Although both MoPrP-Fc and MoDpl-Fc bound to many regions of the brain, we observed restricted binding to granule cells in the cerebellum, suggesting a scenario in which granule cells express a protein that mediates Dpl-induced neurodegeneration. Because granule cells do not express PrP(C), it seems unlikely that MoPrP-Fc binding reflects a ligand that is involved in the conversion of PrP(C) into PrP(Sc), the disease-causing isoform. In contrast, the dominant-negative MoPrP(Q218K)-Fc not only binds to granule cells but also binds to neurons of the molecular layer where PrP(C) is expressed. These findings raise the possibility that the cells of the molecular layer express an auxiliary protein, provisionally designated protein X, which is involved in prion formation and is likely to be distinct from the protein that mediates Dpl-induced degeneration. Although the binding of the dominant-negative MoPrP(Q218K)-Fc to cells in the molecular layer expressing PrP(C) is consistent with a scenario for the binding of MoPrP(Q218K)-Fc to protein X, the absence of PrP(Sc) deposition in the molecular layer requires that PrP(Sc), once formed there, be readily transported to the cerebellar white matter where PrP(Sc) is found. Identifying the ligands to which PrP-Fc, Dpl-Fc, and dominant-negative PrP bind may provide new insights into the functions of PrP(C) and Dpl as well as the mechanism of PrP(Sc) formation

Structural basis of catalysis by monometalated methionine aminopeptidase

Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts