An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation.

To dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility.

The molecular location of Ehrlich chromogen and pyridinoline cross-links in bovine perimysial collagen.

Collagenous peptides containing the Ehrlich chromogen (EC), a trifunctional cross-link of proposed pyrrolic structure, were selectively isolated from a tryptic digest of bovine perimysial collagen by coupling to a diazotised support. Peptides containing pyridinoline (Pyr), another trifunctional cross-link but based on a 3-hydroxypyridinium ring, were isolated from the uncoupled material. The isolated cross-linked peptides were purified by chromatographic procedures and subsequently characterised by amino acid and sequence analyses. EC occurred in stoichiometric amounts in three-chained peptides derived from type I collagen cross-link regions. In contrast, Pyr was found in non-stoichiometric amounts in three-chained peptides where two of the chains were identified as the 76 amino-terminal residues of the α1 (III) collagen chain. The third chain in these Pyr cross-linked peptides was derived from the C-terminal helical cross-link region of either type III collagen or the corresponding region of type I collagen, with the former region predominating. These findings suggest that EC and Pyr cross-links of perimysial collagen are associated mainly with type I and type III collagen respectively.

Identification of the loci of the collagen-associated Ehrlich chromogen in type I collagen confirms its role as a trivalent cross-link.

Collagenous peptides containing the Ehrlich chromogen (EC) were selectively isolated from a tryptic digest of bovine tendon by coupling to a diazotized polyacrylamide support. The isolated p-phenol-azo-EC peptides were purified and characterized by amino acid and sequence analyses. EC occurred in stoichiometric amounts in trimeric cross-linked chains originating from the known cross-link regions of type-I collagen. The major locus of the EC was alpha 2(I)Hyl-933 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N but it was also shown to occur at the loci alpha 1(I)Hyl-87 x alpha 1(I)Lys(Hyl)-16C x alpha 1(I)Lys(Hyl)-16C and alpha 1(I)Hyl-930 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N. After sequence analyses of the C-terminal helical cross-link region alpha 2(I)928-963, corrections are presented for residues 927, 930, 932 and 933 of the bovine alpha 2(I) chain. The collagen-associated EC is postulated to be a trisubstituted pyrrole formed by the reaction of the aldehyde form of a telopeptidyl lysine residue with a bifunctional keto amino cross-link. It is also proposed that when the telopeptidyl lysine residue is hydroxylated the above reaction will result in pyridinoline formation.

Children and chess expertise: the role of calibration.

Three studies of calibration are reported. Calibration refers to the accuracy with which one can predict one's own performance. In the first study child chess players, non-chess playing parents, and statistics students were asked to predict chances of winning chess games against hypothetical opponents. These subjective probabilities were compared to the actual probabilities, based on the Elo rating system. Better players' predictions were better calibrated. Confidence and ratings are negatively correlated, indicating that with lower ratings, players re overconfident. Skilled child players' predictions were better calibrated than any of the adults'. In the second study subjects were asked to estimate chances of winning in conjunctive situations, e.g., winning all the rounds in a tournament. Again, better child players were more accurate in their predictions and more accurate than adults. In the third study, child players were asked to predict their chances of winning in a non-chess domain after hearing a hypothetical win/loss history. Higher-rated players' predictions were again better calibrated, even though the domain was outside their expertise. The motivational and cognitive implications of calibration are discussed.

The estimation of the age of cattle by the measurement of thermal stability of tendon collagen.

Two measures of the thermal stability of tendon collagen (thermal transition temperature and isometric tension) have been studied as a function of age of cattle. The aim was to assess their potential for estimating the age of an animal from which a sample of meat originated. The variation in thermal transition temperature between animals was too large for reliable estimation of animal age by this method. The maximum isometric force generated during heating of strips of tendon from the longissimus dorsi muscle was found to increase linearly with age. Measurements of isometric tension at pH 6·0 had the least variation between animals from 2 to 16 years of age, but were not suitable for younger animals. At pH 7·5, the entire age range could be measured, and although the variation between animals was increased, the accuracy of the age determination still compared favourably with that of dentition.

The relationship between animal age and the thermal stability and cross-link content of collagen from five goat muscles.

The thermal stability of intramuscular collagen, as determined using differential scanning calorimetry, was measured in five muscles from 75 goats with known birth dates ranging in age from one day to 13 years. The collagen cross-link pyridinoline, and the collagen-associated, and putative cross-link, Ehrlich Chromogen were also measured. Five different muscles were examined and the effects of age compared to those found in the tendon of the longissimus dorsi muscle. The differences between intramuscular collagen and tendon collagen were found to be much greater than those between the intramuscular collagens of different muscles. Intramuscular collagen is more thermally stable than tendon collagen due to higher levels of heat-stable cross-links. However the increase in thermal stability of intramuscular collagen with age could not be explained simply in terms of the cross-links measured.

The use and abuse of minor tranquillisers.

The author briefly outlines the benefits of benzodiazepines and also notes their risks. The usefulness of tricyclic antidepressants as an alternative to long-term benzodiazepines is emphasised.

Collagen crosslinks and their relationship to the thermal properties of calf tendons.

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

Biochemical properties of purified transverse tubules isolated from skeletal muscle triads.

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [gamma-32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.

Effects of high pressure treatment of rabbit Longissimus dorsi muscles on the microsomal membranes.

High pressure treatment (150 MPa for 10 min at 35°C) of rabbit longissimus dorsi muscles reduced the yield of a heavy microsomal preparation (8000-28 000 g) by approximately 90%. This was shown to be due to the loss of the sarcoplasmic reticulum, the major component of microsomal preparations, from untreated muscles. While microsomal preparations from pressure-treated muscles were shown to have lost all of their (Ca(2+), Mg(2+))-ATPase activity they retained all their Mg(2+)-ATPase (basal) activity. Sucrose density gradient centrifugation of these preparations yielded two fractions. By reason of its low density, high cholesterol content, high Mg(2+)-ATPase activity, SDS-gel protein profile and solubility in lysolecithin, the lightest fraction appeared to be derived from the transvers tubules. The heavier fraction, present in greater quantity but with a lower Mg(2+)-ATPase activity than the light fraction, was probably derived from the sarcolemma.

A fluorometric assay for the potassium-dependent phosphatase activity of the (Na+ + K+)-adenosine triphosphatase.

A fluorometric assay for the K+-dependent phosphatase activity of the (Na+ + K+)-ATPase in both purified and membrane-bound forms is described. The assay utilizes 3-O-methylfluorescein phosphate as substrate and measures the fluorescence of the 3-O-methylfluorescein produced by hydrolysis of the substrate. The assay described is an order of magnitude more sensitive than the assay employing p-nitrophenylphosphate, the substrate most commonly used to measure this activity. The assay is also suitable for the specific measurement of (Na+ + K+)-ATPase activities in membranes which contain high levels of other ATPase activities.

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients.

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.

Effects of muscle pH on pressure-induced changes in the sarcoplasmic reticulum.

The effect of muscle pH on high pressure treatment of rabbit longissimus dorsi muscles has been studied by the use of pre-slaughter injections of epinephrine in order to vary the glycogen content and thus the extent of post-slaughter glycolysis in the muscles. Following pressure treatment the pH of the muscles varied between 5·9 and 7·1 while the control muscles varied between 6·6 and 7·1. The effects of the high pressure treatment on the ATPase activities, protein yield and protein composition of the sarcoplasmic reticulum (SR) were measured. The basal ATPase activity was not affected over this pH range while the extra ATPase activity, protein yield and protein composition of the SR were affected. The effects increased with decreasing pH.

Post-mortem glycolysis in rabbit Longissimus dorsi muscles following electrical stimulation.

The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, ATPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although only high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorylase a activity, apparently by increasing the activity of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yield of SR and increased its basal ATPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the ATPase activities, probably the myofibrillar ATPase.