[Data quality of unsupervised endothelial cell counting vs. reading centre analysis in multicentric clinical trials].

Purpose: The aim of this study was to assess data quality from unsupervised endothelial cell counting in the multicentric setting.

Patients And Methods: We performed an endothelial cell counting trial with two fictitious trial sites. The trial protocol simply demanded for marking 30 cells for analysis.

The use of medications with known or potential anticholinergic activity in patients with dementia receiving cholinesterase inhibitors.

Objectives: To assess the prevalence of prescribed medications with anticholinergic activity given concurrently with acetylcholinesterase-inhibitor therapy in long-term care residents with dementia and to recommend dose adjustment or discontinuation of these medications with anticholinergic activity.

Design: Prospective case series.

Setting: Long-term care facilities in Indiana.

Identification of the sites of interaction between c-Raf-1 and Ras-GTP.

Specific sites of protein-protein interaction were identified in the 51-149 region of c-Raf-1 using contact epitope scanning and site-directed mutagenesis. Nineteen overlapping peptides based upon the primary sequence of the Ras binding domain of c-Raf-1 were tested for the ability to competitively inhibit complex formation between Ras-GTP and the c-Raf-1 N-terminus. A peptide containing c-Raf-1 residues 91-105 as well as five overlapping peptides covering a region extending from residues 118 to 143 interfered with Ras association, defining these sites as potential contact surfaces with Ras.

Structural analysis of the Ras GTPase activating protein catalytic domain by semirandom mutagenesis: implications for a mechanism of interaction with Ras-GTP.

The bovine complementary DNA encoding the catalytic domain of Ras GTPase activating protein was mutagenized semirandomly using a variation of the polymerase chain reaction. Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase activating function. All of the inactivating single mutations affected the structure of the catalytic fragment as assessed by large decreases in soluble protein when expressed in Escherichia coli.

Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity.

Substrate characterization of the Saccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase.

The in vitro substrate preferences of recombinant S. cerevisiae protein farnesyltransferase and type-I protein geranylgeranyl-transferase were determined. Proteins ending in 16 different CaaX sequences (C = cysteine, a = aliphatic amino acid, X = variable amino acids) were used to determine the protein substrate preferences of these S.

Characterization of Ras effector mutant interactions with the NF1-GAP related domain.

The GTPase activating proteins Ras GAP and the neurofibromatosis-type 1 (NF1) gene product have been implicated as both potential mediators and regulators of the mitogenic effects of the ras proteins. In this study, the interactions of selected Ras effector mutants with the NF1-GAP related domain (NF1-GRD) were investigated. The NF1-GRD was unable to stimulate the GTPase of Ras[Asn33], Ras[Ser35] or Ras[Asn38], all transformation defective mutants.