Differences in hamstring muscle stretching of elite field hockey players and normal subjects.

OBJECTIVES: To investigate differences in stretching behaviour of hamstring muscles in elite field hockey players and normal subjects. METHODS: 16 normal healthy subjects (group A) and 16 elite field hockey players (group B). Stretching of the hamstrings was performed with a knee extension device. Two successive single stretches (1 and 2) until the maximum stretch tolerance was achieved were applied to each subject twice. Simultaneously range of movement (ROM), passive resistance to stretch (PRS) at 36 degrees ROM, and electromyographic (EMG) activity of the biceps femoris and semitendinosus muscles were recorded. RESULTS: ROM (p=0.02 for stretch 1; p=0.04 for stretch 2) and PRS (p=0.04 and p=0.03) were significantly higher in group B, as compared to group A. The EMG activity of the biceps femoris (p=0.02 for stretch 1; p=0.3 for stretch 2) and semitendinosus (p=0.03 and p=0.11) were significantly higher in group B only for stretch 1. From stretch 1 to 2, a significant increase in ROM was observed in both group A (p=0.002) and group B (p=0.0003), as well as a significant decrease of PRS (p=0.0002; p=0.002). EMG activity only showed a significant decrease in group B (biceps p=0.03;semitendinosus p=0.01), but not in group A (p=0.6; p=0.06). The EMG activity sets in when the subjects begin to perceptive pain in the stretched muscles. CONCLUSIONS: Significant differences exist. in the stretching behaviour of elite field hockey players and non-athletes. ROM, PRS and EMG activity are higher in athletes. Implications for treatment should be considered, but recommendations cannot be made on the basis of this study yet.

RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.

Rapamycin is a potent immunosuppressant that blocks the G1/S transition in antigen-activated T cells and in yeast. The similar effects of rapamycin in animal cells and yeast suggest that the biochemical steps affected by rapamycin are conserved. Using a two-hybrid system we isolated mammalian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin. Specific interactors, designated RAPT1, encode overlapping sequences homologous to yeast Tor, a putative novel phosphatidylinositol 3-kinase. A region of 133 amino acids of RAPT1 is sufficient for binding to the FKBP12/rapamycin complex. The corresponding region in yeast Tor contains the serine residue that when mutated to arginine confers resistance to rapamycin. Introduction of this mutation into RAPT1 abolishes its interaction with the FKBP12/rapamycin complex.

The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.

The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion. The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2. UPR elements present in other ER chaperone genes, such as yeast KAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2. As with KAR2, FKB2 mRNA levels are also elevated by heat shock. The similarities in the regulation of FKB2 and other ER chaperone genes suggest that FKBP-13 may play a role in protein trafficking in the ER.

Saccharomyces cerevisiae contains a homolog of human FKBP-13, a membrane-associated FK506/rapamycin binding protein.

FKB2 encodes a homolog of human FKBP-13, a membrane-associated binding protein for the immunosuppressants FK506 and rapamycin. FKB2 is located on the right arm of chromosome IV and contains an open reading frame of 135 amino acids, of which the first 17 residues comprise a putative hydrophobic leader peptide. Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity) and to FKBP-12, the major cytosolic receptor for FK506. In the alignment of FKBP-13 and FKBP-12 sequences, there are 28 invariant residues. Among these conserved residues are those that comprise the drug binding and peptidyl-prolyl cis-trans isomerase active site of FKBP-12. The phylogenetic conservation of the FKBP family suggest that the proteins are involved in a basic cellular function.

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.

Molecular analysis of a Neurospora crassa gene expressed during conidiation.

The asexual developmental pathway in the life cycle of the filamentous fungus Neurospora crassa culminates in the formation of spores called conidia. Several clones of genomic Neurospora DNA have been isolated that correspond to mRNA species expressed during conidiation and not during mycelial growth (V. Berlin and C. Yanofsky, Mol. Cell. Biol. 5:849-855, 1985). In this paper we describe the characterization of one of these clones, named pCon-10a. This clone contains two genes, con-10 and con-13, which are induced coordinately during the later stages of conidiation. The two genes are separated by 1.4 kilobases of DNA; they are located on linkage group IV and are transcribed from the same strand of DNA. The molecular organization and sequence of one of these genes, con-10, and its flanking regions are presented. Full-length cDNA clones for con-10 also were isolated and sequenced, and transcription-initiation and polyadenylation sites were defined. The con-10 gene contains an open reading frame interrupted by two small introns and encodes an 86-amino-acid residue polypeptide that is both hydrophilic and weakly acidic. Expression of the con-10 gene in various mutants defective at different stages of conidiation indicates that it plays a role after aerial hyphal development. Possible functions, organization, and regulation of conidiation-specific genes are discussed.

Protein changes during the asexual cycle of Neurospora crassa.

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.

Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa.

A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.

Release of transcript and template during transcription termination at the trp operon attenuator.

We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli. Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator. We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin. These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J. P., and Conaway, R. (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C. (1980) Nucleic Acids Res. 8, 3355-3369). We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template. In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription. It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template. Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.

Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen.

Adriamycin is reduced to a semiquinone-free radical by microsomal and nuclear enzymes. To investigate the effect of these radicals on DNA, an end-labeled DNA fragment of defined sequence was included in a reaction that contained NADPH, NADPH cytochrome P-450 reductase (EC 1.6.2.4), and adriamycin. Extensive DNA strand scission was observed. The breaks occurred at all nucleotides with equal probability regardless of nucleotide sequence. Single-stranded DNA was as good a substrate as double-stranded DNA for the strand scission reaction. DNA damage occurred during the early phase of the reaction. No strand breaks were observed upon addition of DNA to the reaction 60 min after enzyme activation despite the high concentration of stable free radicals present at this time. The extent of strand scission was greatly reduced when the reaction was carried out in a nitrogen atmosphere. We conclude that DNA breakage created by enzymatically derived adriamycin-free radicals is mediated by molecular oxygen, most probably by hydroxyl-free radicals and hydroxyl ions. We also conclude that the semiquinone-free radicals of adriamycin and radicals of the reduced forms of adriamycin need not intercalate into DNA to create strand breaks.