Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase

The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144. Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis. The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation. Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity. The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.      

The pore-lining region of shaker voltage-gated potassium channels: comparison of beta-barrel and alpha-helix bundle models

Although there is a large body of site-directed mutagenesis data that identify the pore-lining sequence of the voltage-gated potassium channel, the structure of this region remains unknown. We have interpreted the available biochemical data as a set of topological and orientational restraints and employed these restraints to produce molecular models of the potassium channel pore region, H5. The H5 sequence has been modeled either as a tetramer of membrane-spanning beta-hairpins, thus producing an eight-stranded beta-barrel, or as a tetramer of incompletely membrane-spanning alpha-helical hairpins, thus producing an eight-staved alpha-helix bundle. In total, restraints-directed modeling has produced 40 different configurations of the beta-barrel model, each configuration comprising an ensemble of 20 structures, and 24 different configurations of the alpha-helix bundle model, each comprising an ensemble of 24 structures. Thus, over 1300 model structures for H5 have been generated. Configurations have been ranked on the basis of their predicted pore properties and on the extent of their agreement with the biochemical data. This ranking is employed to identify particular configurations of H5 that may be explored further as models of the pore-lining region of the voltage-gated potassium channel pore.     

Occupational exposures and amyotrophic lateral sclerosis. A population-based case-control study

This population-based case-control study was conducted in three countries in western Washington State to evaluate associations between workplace exposures and the risk of amyotrophic lateral sclerosis (ALS). Cases (n = 174) were all newly diagnosed with ALS by neurologists during 1990-1994, and controls (n = 348), who were matched according to age (+/-5 years) and sex, were identified via random-digit dialing or Medicare enrollment files. Four industrial hygienists blindly assessed detailed lifetime job histories for exposures to metals, solvents, and agricultural chemicals. Case-control comparisons were made for jobs held between 15 years of age and 10 years prior to the cases' dates of diagnosis. After adjustment for age and education, ever exposure to agricultural chemicals was associated with ALS (odds ratio (OR) = 2.0, 95% confidence interval (CI) 1.1-3.5); this association was observed separately in men (OR = 2.4, 95% CI 1.2-4.8) but not in women (OR = 0.9, 95% CI 0.2-3.8). Among men, the odds ratio for low exposure to agricultural chemicals (below the median level for exposed controls) relative to no exposure was 1.5 (95% CI 0.4-5.3), and for high exposure, it was 2.8 (95% CI 1.3-6.1) (p for trend = 0.03). Similar analyses based on the panel's assessment of exposures to metals and solvents showed no associations. These findings suggest an association between ALS and agricultural chemicals in men.     

Variables that initiate and maintain an early-onset trajectory for juvenile offending

A trajectory defined by three time-ordered events was offered as a useful adjunct to building a development theory about antisocial behaviors. A sequence was defined with significant linkages between antisocial childhood behavior and early arrest and between early arrest and chronic offending. The majority of chronic offenders traveled through all three events in the sequence. Each event in the sequence shared a common process of disrupted family process plus frequent family transitions and marked social disadvantage. The findings support the hypothesis that the process that leads to antisocial behaviors at grade four may also maintain the entire sequence. The level of disrupted process at initiation and a time-based measure of involvement with deviant peers predicted which individuals moved forward in the sequence and which did not. The findings are consistent with the idea that the majority of chronic offending juveniles follow a trajectory that can be explained by a single theory.