ABSTRACT
The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrP(C) (cellular prion protein), to a protease-resistant isoform, PrP(Sc) (prion protein scrapie isoform). The importance of the highly flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP(37-53), showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen-modifying enzyme prolyl 4-hydroxylase. Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrP(Sc) from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.
Subject(s)
Peptides/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Prions/chemistry , Prions/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , CHO Cells , Circular Dichroism , Cricetinae , Guanidine/pharmacology , Hydroxylation , Mice , Molecular Sequence Data , Osmolar Concentration , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptides/chemistry , PrPSc Proteins/genetics , Prions/genetics , Proline/metabolism , Protein Denaturation/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Temperature , TransfectionABSTRACT
The probability model of antimicrobial action is based on the definition of bactericidal activity as the probability, q, that any cell in the population will be killed during a division interval. Bacteriostatic activity is defined as a change in the division intervals (generation times) of the cells. A simplified, homogeneous model is used which assumes that, at a constant concentration of the drug, all cells have the same kill probability and the same generation times. Birth-death analysis techniques require that the combined bacteriostatic and bactericidal effects of the drug (they are not mutually exclusive) are accounted for. Moreover, to suitably reflect the combined effect, the rate of change of the viable population, i.e. the slope of a kill curve (activity), needs to be expressed not in terms of exposure time, but in units of drug-free generations (DFGs), obtained by dividing exposure time by a measured DFG time interval (growth rate). A Discrete MIC (DMIC) is defined as the zero slope kill curve, coinciding with the horizontal axis and dividing population change into a restrained (subinhibitory) growth region, below the DMIC, and population reduction above it. At the DMIC, the probability of a cell being killed is 0.5, resulting in no change from the initial inoculum concentration, since half the cells are killed but the remaining cells double. The DMIC is found to be a measure of bactericidal activity only, even though bacteriostatic activity may also be present. An antibiotic-organism activity profile includes measurement of the DMIC, rate of change of activity at the DMIC and normalized activity at a number of clinically relevant drug concentrations. An overall, quantitative efficacy value over a dosing interval can be obtained from the activity profile and expressed as the number of DFGs which are needed to achieve a 99.9% reduction of the viable population at the site of infection. These reference efficacy values can be used to derive interpretive standards (break-points) based upon a quantitative relationship between laboratory measurements and population reduction at the site of infection. Model-derived measures of efficacy also provide a basis for assessing drug combination activity, including quantitative criteria of synergy and antagonism.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/growth & development , Culture Media , Diffusion , Drug Interactions , Drug Therapy, Combination/pharmacology , Microbial Sensitivity Tests , Models, Statistical , ProbabilityABSTRACT
Inositol monophosphatase can be modified at two sites by pyrene maleimide. These sites have been identified as Cys141 and Cys218. Stoichiometric addition of pyrene maleimide allows the sole modification of Cys218. The fluorescence of the pyrene moiety on the modified protein can be excited directly or by resonance energy transfer. The fluorescence properties of the pyrene group on Cys218 allows the interaction of ligands with the enzyme to be monitored. This feature has allowed dissociation constants for various metal ions to be determined and allowed the formation of various enzyme/ligand complexes to be observed. These studies have demonstrated that Mg2+ is required to support Pi binding and that Li+ interacts with a post-catalytic complex which is only formed in the forward reaction.
