Effect of Vitamin C and E supplementation on biochemical and ultrastructural indices of muscle damage after a 21 km run.

This study investigated whether 4 weeks of daily supplementation with 500 or 1000 mg of Vitamin C and 500 or 1000 IU of Vitamin E could modify biochemical and ultrastructural indices of muscle damage following a 21 km run. Fifteen experienced male distance runners were divided into two groups (vitamin or placebo) and received supplementation for four weeks before completing the first 21 km run in as fast a time as possible. A four-week "washout" period followed before the subjects crossed over and received the alternate supplement for the next four weeks. They then completed a second 21 km run. Before, immediately after and 24 h after each run venous blood samples were taken and analysed for serum creatine kinase, myoglobin, malondialdehyde and vitamin C and E (before-samples only) concentrations. A subgroup of six subjects also had muscle biopsy (gastrocnemius) samples taken 24 h before and 24 h after each 21 km run, which were later analysed by electron microscopy. The two dosages of supplementation produced similar results, so a single vitamin group was formed for further analysis of results. Significant increases (p < 0.05) in creatine kinase and myoglobin, but not in malondialdehyde, were found post-run in both groups. However, no significant differences were found between the vitamin and placebo groups for creatine kinase, myoglobin and malondialdehyde concentrations recorded after the 21 km runs. A qualitative ultrastructural examination of pre-run muscle samples revealed changes consistent with endurance training, but little further change was seen after the 21 km run in either the vitamin or placebo groups. It was concluded that vitamin C and E supplementation (500 or 1000 mg or IU per day) for four weeks does not reduce either biochemical or ultrastructural indices of muscle damage in experienced runners after a half marathon.

Biochemical and ultrastructural indices of muscle damage after a twenty-one kilometre run.

Increased serum concentrations of intracellular proteins are generally accepted as good indicators of muscle damage. The mechanism of this damage is, however, poorly understood. Twenty male runners completed a 21 km run in as fast a time as possible. Blood samples were obtained from each subject just prior to, immediately after, and 24 hr after the run. Samples were analysed for haemoglobin, haematocrit, creatine kinase (CK), myoglobin (Mb) and malondialdehyde (MDA) concentrations and corrected for percentage change in plasma volume (PV). Percutaneous muscle biopsies were taken from the lateral gastrocnemius muscle of 6 of the subjects 24 hr before and 24 hr after the run and examined by electron microscopy. Mb levels in the serum increased significantly (p < 0.001) immediately post-exercise, while CK levels increased significantly (p < 0.001) at 24 hours post-exercise. The PV corrected serum MDA levels were very close (p = 0.06) to a significant increase immediately post-exercise. Ultrastructural examination of pre-exercise samples revealed evidence of muscle changes consistent with endurance exercise training, but no further damage was evident at 24 hr post-exercise. It is thus suggested that the increased serum levels of CK and Mb after the 21 km run may be a result of free radical induced cell membrane damage and increased permeability, as evidenced by elevated serum MDA levels, and not due to mechanical muscle damage.

Molecular mechanisms of sepsis: molecular biology of the cell.

Complex and interrelated biological processes are at work in the expression of the host response to sepsis. To a large degree, these processes reflect drastic changes in the molecular workings of cells of the body. The protean nature of sepsis reflects this molecular adaptation. Studies are continuing to accrue that describe aspects of this process in tissue culture, animal models, and man. However, without an understanding of the basic mechanisms of molecular biology, the understanding of this important and expanding literature is limited. This review describes the basic molecular processes involved in replication of deoxyribonucleic acid (DNA) and transcription of DNA to ribonucleic acid (RNA) in the nucleus, translation of messenger RNA into proteins and the posttranslational modifications of these proteins in the cytoplasm. It uses the process of endotoxin-induced cellular activation as its model and highlights important aspects of DNA promoter and enhancer processes in this activation. Specific examples of known promoter genes and genomic translation are described. This review serves as a "primer" for the subsequent three review articles in this series that will follow it in preceding issues.

Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise.

Red blood cell (RBC) susceptibility to oxidative and osmotic stress in vitro was investigated in cells from trained and untrained men before and after submaximal exercise. Whilst no significant change in peroxidative haemolysis occurred immediately after 1 h of cycling at 60% of maximal aerobic capacity (VO2max), a 20% increase was found 6h later in both groups (P < 0.05). The RBC osmotic fragility decreased by 15% immediately after exercise (P < 0.001) and this was maintained for 6h (P < 0.001). There was an associated decrease in mean cell volume (P < 0.05). Training decreased RBC susceptibility to peroxidative haemolysis (P < 0.025) but it did not influence any other parameter. These exercise-induced changes were smaller in magnitude but qualitatively similar to those found in haemopathological states involving haem-iron incorporation into membrane lipids and the short-circuiting of antioxidant protection. To explore this similarity, a more strenuous and mechanically stressful exercise test was used. Running at 75% VO2max for 45 min reduced the induction time of O2 uptake (peroxidation), consistent with reduced antioxidation capacity, and increased the maximal rate of O2 uptake in RBC challenged with cumene hydroperoxide (P < 0.001). The proportion of high-density RBC increased by 10% immediately after running (P < 0.001) but no change in membrane-incorporated haem-iron occurred. In contrast, treatment of RBC with oxidants (20-50 mumol.l-1) in vitro increased cell density and membrane incorporation of haem-iron substantially. These results showed that single episodes of submaximal exercise caused significant changes in RBC susceptibility to oxidative and osmotic stress. Such responses may account for the increase in RBC turnover found in athletes undertaking strenuous endurance training.

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons.

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.

N4-(6-aminohexyl)cytidine and -deoxycytidine nucleotides can be used to label DNA.

Bisulfite is known to catalyze transamination between cytidine derivatives and amines. Using 1,6-diaminohexane we describe the synthesis and recovery of the 5'-triphosphates of N4-(6-aminohexyl)cytidine and -deoxycytidine (dahCTP). Both may be incorporated into DNA by nick translation with DNA polymerase I of Escherichia coli to provide reactive sites for the attachment of immunological or other labels. Biotinyl dahCTP is actively incorporated into DNA by the same system and can be detected by the binding of streptavidin complexed to an indicator enzyme such as acid phosphatase. Such labeled DNA is a suitable nonradioactive probe for detection of related sequences by hydridization.

The nucleotide sequence of tRNAVal3a and tRNAVal3b from Drosophila melanogaster.

The nucleotide sequences of two valine tRNA's of Drosophila melanogaster are the following: tRNAVal3a, (sequence in text) tRNAVal3b, (sequence in text) tRNAVal3a shows greater similarity to prokaryotic and eukaryotic organelle tRNAVal's than does tRNAVal3b.

1980 Melbourne marathon study.

Most of the 5423 entrants in the Melbourne 1980 Big M Marathon were non-elite athletes. A study of a stratified random sample of 459 entrants (which represented a 42% response rate) found that, while entrants reflected the community standards of disease, they pursued healthier lifestyles. Preparation for the marathon led to a number of positive changes in the health standard of runners. The principal negative consequence of marathon training was the high rate of musculoskeletal problems (30%). Before the race, only 4% of participants had an adequate fluid intake; 33% had pre-existing problems, mainly involving muscles and joints (63%) and viral or gastrointestinal illnesses (41%). These entrants had a 60% less chance of finishing the race. Symptoms during the race were reported by 92% of entrants, but most of these were not serious; only 6% of entrants were unable to finish the race. The pattern of symptoms after the race was similar to that during the race; 50% of these resolved within three hours. Ninety-seven entrants (2%) required medical attention during the race. Serious problems were rare (only in three entrants), and no runner required admission to hospital for longer than 24 hours. Entrants were at greater risk of requiring medical attention or experiencing problems during and after the race if they had a shorter preparation (less than two months), ran fewer kilometres per week (less than 60 km/week) in the last two or three months before the race, and had performed fewer long training runs (more than 24 km).

The structure of tRNA 5 Lys from Drosophila melanogaster.

The nucleotide sequence of Drosophila melanogaster tRNA 5 Lys is pGCCCGGAUAm2GCUCAGDCGGDAGAGCA psi psi GGACUsU*UUt6A*A psi CCAAGGm7GDm5CCAGGGTm psi CAm1AGUCCCUGUUCGGGCGCCA. The sU* is probably 5-methylcarboxymethyl-2-thiouridine and t6A* is a mixture of modified derivatives of t6A including t6A itself and a component sensitive to treatment with cyanogen bromide. This tRNA 5 Lys is 95% homologous to the rabbit liver tRNA 5 Lys.

The structures of genes hybridizing with tRNA4Val from Drosophila melanogaster.

Segments of cloned Drosophila DNA from four recombinant plasmids that hybridize with tRNA4Val have been sequenced. The segments from pDt92R and pDt120R that hybridize to 90C on the third polytene chromosome appear to be either repeats or alleles. They contain one structural gene each of identical sequence but differ at eight sites in 506 base pairs. The structural genes differ at four sites from the sequence expected from that of tRNA4Val. A third plasmid, pDt14, which hybridizes to 89BC on the third chromosome, also contains a structural gene with the same sequence as those in pDt92R and pDt120R. In addition, pDt14 has a gene for tRNA2Phe 214 base pairs upstream and with the same polarity as the tRNA4Val gene. The tRNA2Phe gene contains a 23-base pair segment identical with the corresponding segment in the tRNA4Val genes except for one base pair. The fourth plasmid investigated, pDt55, hybridizes to 70BC. It contains two tRNA4Val genes 525 base pairs apart with opposite polarity. These genes have identical sequences, which corresponds to that expected from the sequence of tRNA4Val. There is no evidence that the first three tRNA4Val genes are expressed at any stage during the development of Drosophila.

The nucleotide sequence of tRNA4Val of Drosophila melanogaster. Chloroacetaldehyde modification as an aid to RNA sequencing.

The nucleotide sequence of tRNA4Val from Drosophila melanogaster was determined to be pGUUUmCCGUm1GGUG psi AGCGGDU(acp3U)AUCACA psi CUGCCmUIACAm5CGCAGAAGm7GCCCCCGGT psi CGm1AUCCCGGGCGGAAACACCA. It is probable that residue C 49 is modified to m5C. The use of tRNA modified with chloroacetaldehyde to overcome secondary structure problems in sequencing is described.

Localization of tRNA genes of Drosophila melanogaster by in situ hybridization.

Six purified tRNAs labeled with 125I by chemical or enzymatic methods were hybridized to polytene chromosomes of Drosophila melanogaster. The main chromosomal regions of hybridization wer: tRNAGly(GGA), 58A, 84C, and 90E; tRNALeu(2), 44E, 66B5-8, and 79F; tRNASer(2b), 86A, 88A9-12, and 94A6-8; tRNAThr(3), 47F and 87B; tRNAThr(4), 93A1-2; and tRNATyr(1 gamma), 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNATyr(1 gamma) was polymorphic in the giant strains. When the hybridization of three valine isoacceptors studied previously was re-investigated, it was found that only one hybridization site, 90BC, was shared between tRNAVal(3b) and tRNAVal(4). tRNAVal(3a) did not have any sites in common with the other two.

Hybridization of tRNAs of Drosophila melanogaster to the region of the 5S RNA genes of the polytene chromosomes.

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled 125I-tRNA Asp2 gamma occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNAAsp2 gamma labeled by introducing 125I-5-iodocytidylyl residues into the 3'-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to other sites. The hybridization of tRNALys2, tRNA3Gly and tRNAMet3 at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNALys2 no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNAAsp2 gamma in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila silivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC, tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5-7, 72F1-2, 83F-84A; tRNA3Met, 46A1-2, 61D1-2, 70F1-2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84d3-4, 92b1-9; tRNA4Val, 56D3-7, 70BC.

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes.

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.

The nucleotide sequence of the initiator tRNA from Drosophila melanogaster.

The nucleotide sequence of Drosophila melanogaster methionine tRNAi was determined to be: pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-m2G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-C-G-A-G-G-A-U-C-G-m1A-A-A-C-C-U-U-G-C-U-C-U-G-C-U-A-C-C-A(OH). It differs from vertebrate initiator tRNAs in only 6 out of 75 positions.