An exponential model of isometric muscular fatigue as a function of age and muscle groups.

Despite the recommendations that an important design criterion is not to exceed 15% of an operator's maximum voluntary contraction (MVC) with any muscle that must be used for a long period of time, many tasks involve the exertion of much higher forces coupled with long-term contractions. Many studies have investigated the force-time relationship of isometric muscle contractions to determine the endurance time of a given relative force. To date, however, direct studies of muscle performance throughout fatiguing tasks have not been conducted to the same degree. This research was concerned with studying the effects of different muscle groups (biceps vs quadriceps) of subjects with different age groups (20-29 vs 50-59 years of age) on long-term muscular isometric contractions at different levels of %MVC (20%, 40%, 60%, 80%, and 100% MVC), and modelling the functional data to describe the time course of strength decrement. The data revealed that the time course of strength decrement was best modelled by the function: [formula: see text] An experiment, using 20 subjects with each subject performing 10 conditions (two muscle groups x five levels of %MVC), showed that this function accounted for over 95% of the variance of strength decrement. All parameter estimates were statistically significant.     

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.     

Acquisition of linguistic and cognitive skills by children with cleft palate

This study compared the early cognitive and linguistic development of young children with cleft palate (N = 28) to that of noncleft children (N = 29). Measures included the Mental scale of the Bayley Scales of Infant Development, the Minnesota Child Development Inventory, Mean Length of Utterance, and words acquired by 24 months. Children with cleft palate, although well within the normal range, performed significantly below the children in the control group on the Mental Scale of the Bayley Scales of Infant Development, some subscales of the Minnesota Child Development Inventory, and words acquired by 24 months. Differences observed in the cognitive development of children with and without cleft palate were verbal as opposed to nonverbal (i.e., linguistic in nature) and were related to hearing status at 12 months and velopharyngeal adequacy.     

A minisatellite sequence within the propeptide region of the vacuolar carboxypeptidase Y gene of Schizosaccharomyces pombe

We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast Schizosaccharomyces pombe. The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene. The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of S. pombe (S. pombe var. pombe and S. pombe var. malidevorans). The minisatellite sequence, however, appeared to be species specific and was absent in other members of the Schizosaccharomyces genus. This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.     

Lactols in hydrolysates of DNA treated with alpha-acetoxy-N-nitrosopyrrolidine or crotonaldehyde

alpha-Acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) is a stable precursor to alpha-hydroxyNPYR, the initial product of metabolism and proposed proximate carcinogen of N-nitrosopyrrolidine (NPYR). Crotonaldehyde (2-butenal) is a metabolite of NPYR and also a mutagen and carcinogen. Both alpha-acetoxyNPYR and crotonaldehyde form DNA adducts, but these reactions have not been completely characterized. In previous studies, we detected substantial amounts of unidentified radioactivity in hydrolysates of DNA that had been treated with radiolabeled alpha-acetoxyNPYR. In this study, we have characterized these products as 2-hydroxytetrahydrofuran, the cyclic form of 4-hydroxybutanal, and paraldol, the dimer of 3-hydroxybutanal. These products were identified by comparison to standards and by conversion to 2,4-dinitrophenylhydrazones. 2-Hydroxytetrahydrofuran is the major product in neutral thermal hydrolysates of alpha-acetoxyNPYR-treated DNA and is derived predominantly from N2-(tetrahydrofuran-2-yl)deoxyguanosine 8. Paraldol is present to a lesser extent than 2-hydroxytetrahydrofuran in these reactions and is formed from paraldol-releasing adducts, which in turn are produced in the reaction of crotonaldehyde, a solvolysis product of alpha-acetoxyNPYR, with DNA. Other products in hydrolysates of alpha-acetoxyNPYR-treated DNA are N7-substituted guanines 5 and 6, cyclic N7-C8 guanines 4, 11, and 12, and 1, N2-propanodeoxyguanosines 9 and 10. Paraldol is a major product in hydrolysates of crotonaldehyde-treated DNA, being present in amounts 100 times greater than those of previously identified adducts 9 and 10. The results of this study provide a more complete picture of the reactions of alpha-acetoxyNPYR with DNA and yield some new insights about possible endogenous DNA adducts formed from crotonaldehyde.     

Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).