Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

Anaerobic energy release in skeletal muscle during electrical stimulation in men

The quadriceps femoris muscles of seven men were electrically stimulated under extended anaerobic conditions to quantitate anaerobic energy release and the contribution of the glycolytic system to total ATP production. Muscles were intermittently stimulated 64 times at 20 Hz while leg blood flow was occluded. Each contraction lasted 1.6 s and was followed by 1.6 s of rest. The total contraction time was 102.4 s. Muscle biopsies were taken at rest and following 16, 32, 48, and 64 contractions. The ATP turnover rates during the four 16-contraction periods were 6.12, 2.56, 2.17, and 0.64 mmol X kg dry muscle-1 X s-1 contraction time. Glycolysis provided 58%, phosphocreatine 40% and a decreased ATP store 2% of the consumed energy during the initial 16 contractions. Glycolysis was responsible for 90% of the total ATP production beyond contraction 16. Absolute glycolytic ATP production decreased to 60, 55, and 17% of the amount in the initial 16 contractions during the final three periods, respectively. In conclusion glycolysis produced approximately 195 mmol ATP/kg dry muscle during the initial 48 contractions (76.8 s) and only approximately 15 mmol ATP/kg dry muscle during the final 16 contractions. Equivalent values for total ATP turnover were 278 and 16.5 mmol/kg dry muscle.     

Regulation of glycogenolysis in human skeletal muscle

The role of inorganic phosphate on the regulation of glycogenolysis in resting and contracting muscle was studied in human quadriceps muscle. Increased Pi content was achieved by intermittent electrical stimulation of the muscle followed by occlusion of the blood flow. Occlusion resulted in the maintenance of a high Pi content over a 60-s observation period during which the muscle was either at rest or was stimulated electrically. The study was performed with and without infusion of epinephrine (EPI). In the absence of EPI the phosphorylase a fraction was 50% immediately at the end of the initial stimulation period, declining to 22% after 60 s. With EPI corresponding values for phosphorylase a were 91% initially, 56% after 30 s, and 33% after 60 s, respectively. In both cases the Pi content was increased by approximately 35 mmol/kg dry muscle during the stimulation and remained constant during the occlusion. In neither of these situations was significant degradation of glycogen observed during the occlusion. In the study performed with electrical stimulation during the occlusion period, muscle glycogen degradation was observed both with and without EPI. Phosphorylase a fractions and Pi contents in this study were similar to those observed when muscle was rested over the 60-s occlusion period. The paradox of a high Pi content and extensive transformation of phosphorylase to the a form but low glycogenolytic activity points to additional factors in the regulation of glycogen breakdown.     

Seed transmission of turnip yellow mosaic virus in winter turnip and winter oilseed rapes

This is the first record of seed transmission of turnip yellow mosaic virus (TYMV) in oilseed and turnip rapes. The seed transmission of TYMV in a naturally infected winter turnip rape (Brassica napus var. silvestris) cultivar Perko PVH was investigated. By ELISA 1.6%, 3.2% and 8.3% seed transmission of the virus was found in seed of plants from three localities. The proportion of infected seeds produced by artificially infected plants of winter oilseed rape (Brassica napus ssp. oleifera) and winter turnip rape cultivars was determined. The virus transmission rate, expressed as the proportion of virus-infected plants which germinated from the seed was for the oilseed rape cvs Jet Neuf 0.1%, Solida 0.4%, Silesia 0.8%, Darmor 1.2%, SL-507 0.2%, SL-509 0.0% and for the winter turnip rape cv. Perko 1.5%. ELISA cannot be used in direct tests on bulk seed lots to estimate proportion of infected seed, but must be used on germinated seedlings.     

Aspects of hadrosaurian cranial anatomy

Supraorbital bones in Saurolophus angustirostris are described, and their presence in all hadrosaurs is suggested. Frontal-nasal and premaxillar-nasal fontanellae are distinguished in hadrosaurs; their presence is explained as connected with growth and considered to he responsible for the variability of crest structures. New data indicating the presence of a cartilaginous diverticulum nasi within the circumnarial depression in Saurobphus ongustirostris are presented. A physiological (respiratory and/or thermoregulatory) function of the nasal diverticulum is proposed.     

Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing

A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.     

Adaptation of mitochondrial ATP production in human skeletal muscle to endurance training and detraining.

The adaptation of mitochondrial ATP production rate (MAPR) to training and detraining was evaluated in nine healthy men. Muscle samples (approximately 60 mg) were obtained before and after 6 wk of endurance training and after 3 wk of detraining. MAPR was measured in isolated mitochondria by a bioluminometric method. In addition, the activities of mitochondrial and glycolytic enzymes were determined in skeletal muscle. In response to training, MAPR increased by 70%, with a substrate combination of pyruvate + palmitoyl-L-carnitine + alpha-ketoglutarate + malate, by 50% with only pyruvate + malate, and by 92% with palmitoyl-L-carnitine + malate. With detraining MAPR decreased by 12-28% from the posttraining rate (although not significantly for all substrates). No differences were found when MAPR was related to the protein content in the mitochondrial fraction. The largest increase in mitochondrial enzyme activities induced by training was observed for cytochrome-c oxidase (78%), whereas succinate cytochrome c reductase showed only an 18% increase. The activity of citrate synthase increased by 40% and of glutamate dehydrogenase by 45%. Corresponding changes in maximal O2 uptake were a 9.6% increase by training and a 6.0% reversion after detraining. In conclusion, both MAPR and mitochondrial enzyme activities are shown to increase with endurance training and to decrease with detraining.     

Social Class,Social Mobility and Risk of Psychiatric Disorder - A Population-Based Longitudinal Study

Objectives

This study explored how adult social class and social mobility between parental and own adult social class is related to psychiatric disorder.

Material and Methods

In this prospective cohort study, over 1 million employed Swedes born in 1949-1959 were included. Information on parental class (1960) and own mid-life social class (1980 and 1990) was retrieved from the censuses and categorised as High Non-manual, Low Non-manual, High Manual, Low Manual and Self-employed. After identifying adult class, individuals were followed for psychiatric disorder by first admission of schizophrenia, alcoholism and drug dependency, affective psychosis and neurosis or personality disorder (N=24 659) from the Swedish Patient Register. We used Poisson regression analysis to estimate first admission rates of psychiatric disorder per 100 000 person-years and relative risks (RR) by adult social class (treated as a time-varying covariate). The RRs of psychiatric disorder among the Non-manual and Manual classes were also estimated by magnitude of social mobility.

Results

The rate of psychiatric disorder was significantly higher among individuals belonging to the Low manual class as compared with the High Non-manual class. Compared to High Non-manual class, the risk for psychiatric disorder ranged from 2.07 (Low Manual class) to 1.38 (Low Non-manual class). Parental class had a minor impact on these estimates. Among the Non-manual and Manual classes, downward mobility was associated with increased risk and upward mobility with decreased risk of psychiatric disorder. In addition, downward mobility was inversely associated with the magnitude of social mobility, independent of parental class.

Conclusions

Independently of parental social class, the risk of psychiatric disorder increases with increased downward social mobility and decreases with increased upward mobility.