Improved cardiovascular health following a progressive walking and dietary intervention for type 2 diabetes.

AIM: To examine the impact of two different lifestyle programmes on cardiovascular health and glycaemic control among people with type 2 diabetes. METHODS: A two-phase 24-week randomized trial. During the first phase, participants were to increase daily steps using a pedometer. At week 12, participants were randomly allocated to either an enhanced lifestyle programme (ELP) targeting walking speed or a basic lifestyle programme (BLP) targeting total daily steps. Both programmes focused on increasing the intake of low glycaemic index foods but utilized different goal setting strategies. Clinical measurements were completed at baseline, week 12 and week 24. Principal outcomes were change in resting pulse rate (PR) and glycated haemoglobin A1c (A1c) between week 12 and week 24 compared between groups using analysis of covariance. RESULTS: Forty-one participants [mean +/- s.d. : age = 56.5 +/- 7.2 years, body mass index (BMI) = 32.7 +/- 6.1 kg/m(2)] were randomized. After 12 weeks, we observed an increase in average total daily steps of 1688 (95% confidence interval: 330-3040, [corrected] p = 0.02). Weight, BMI and systolic and diastolic blood pressure improved (p < 0.01 for all). No changes were observed for energy intake. At week 24, those in the ELP had a lower resting PR (71 +/- 12 b.p.m.) compared with those in the BLP (78 +/- 12 b.p.m.) (adjusted p = 0.03), while no group differences for total daily steps or glycaemic control were observed. CONCLUSIONS: Improvements in cardiovascular health can be expected following a pedometer-based lifestyle modification programme that progresses from walking more to walking faster.

Serotonin and central nervous system fatigue: nutritional considerations.

Fatigue from voluntary muscular effort is a complex phenomenon involving the central nervous system (CNS) and muscle. An understanding of the mechanisms within muscle that cause fatigue has led to the development of nutritional strategies to enhance performance. Until recently, little was known about CNS mechanisms of fatigue, even though the inability or unwillingness to generate and maintain central activation of muscle is the most likely explanation of fatigue for most people during normal daily activities. A possible role of nutrition in central fatigue is receiving more attention with the development of theories that provide a clue to its biological mechanisms. The focus is on the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] because of its role in depression, sensory perception, sleepiness, and mood. Nutritional strategies have been designed to alter the metabolism of brain 5-HT by affecting the availability of its amino acid precursor. Increases in brain 5-HT concentration and overall activity have been associated with increased physical and perhaps mental fatigue during endurance exercise. Carbohydrate (CHO) or branched-chain amino acid (BCAA) feedings may attenuate increases in 5-HT and improve performance. However, it is difficult to distinguish between the effects of CHO on the brain and those on the muscles themselves, and most studies involving BCAA show no performance benefits. It appears that important relations exist between brain 5-HT and central fatigue. Good theoretical rationale and data exist to support a beneficial role of CHO and BCAA on brain 5-HT and central fatigue, but the strength of evidence is presently weak.

Effects of carbohydrate and chromium ingestion during intermittent high-intensity exercise to fatigue.

PURPOSE: This study was designed to test the hypothesis that addition of chromium (Cr) to a carbohydrate-electrolyte drink would enhance the reported benefits of carbohydrate on exercise capacity during intermittent high-intensity shuttle running. METHODS: Eight physically active men performed 3 exercise trials while ingesting 6% carbohydrate-electrolyte (CHO), CHO plus chromium picolinate (400 mg) (CHO + Cr+3), or placebo (P) using a double-blind, counterbalanced design. Each trial consisted of 5 3 15 min bouts of shuttle running (walk, sprint, and run at 95 and 55% of estimated VáO2max, separated by 3-min rest). This was followed by a fatigue test (running alternating 20-m lengths at 55 and 95% of estimated VáO2 until fatigue). RESULTS: During the standardized shuttle running, blood glucose was higher with both CHO and CHO + Cr+3 than P. Plasma free fatty acid was higher in P than both CHO and CHO + Cr+3 at 75 min of exercise and at fatigue. In the fatigue test, subjects ran longer with both CHO and CHO + Cr+3 than P. CONCLUSIONS: The data confirm an ergogenic benefit of ingesting CHO during exercise designed to imitate sports like basketball, soccer, and hockey, but do not support the hypothesis that the addition of Cr would enhance this effect.

Effects of branched-chain amino acids and carbohydrate on fatigue during intermittent, high-intensity running.

Experimental support for the hypothesized benefits of BCAA supplements on endurance performance is limited. However, it is theorized that the benefits may be enhanced if 1) BCAA are taken along with a pre-event carbohydrate meal as well as during exercise, and 2) the exercise is intermittent in nature. This study tested the effects of ingesting carbohydrate beverages with and without BCAA before and during intermittent high-intensity running to fatigue. Eight subjects performed 3 exercise trials consisting of intermittent shuttle running (walking, sprinting, and running) to fatigue. Subjects drank either carbohydrate drinks given 1 h before (5 mL/kg, 18% carbohydrate) and during exercise (2 mL/kg, 6% carbohydrate) (CHO), carbohydrate drinks with BCAA (7 g) added to the portions consumed 1 h before and immediately before exercise (CHO+BCAA), or flavored water placebos (P). Subjects ran longer when fed either CHO or CHO+BCAA as compared to P, with no differences between CHO and CHO+BCAA. CHO and CHO+BCAA also had higher plasma glucose and insulin, and lower FFA (p < 0.05). These findings confirm a beneficial effect of carbohydrate feedings on fatigue during exercise designed to mimic the activity pattern that occurs in sports like soccer, basketball, and hockey. They do not, however, support the hypothesis of an added benefit of BCAA supplements.

Preparation and characterization of two phosphopeptide fractions from calf thymus nuclei; similarity to corresponding fractions in the DNA prepared from the nuclei.

In this study two phosphopeptide (PP) fractions from purified, calf thymus nuclei, prepared under protective conditions were characterized. The nuclei were lysed and dialyzed, and the material in the dialysates was fractionated by anion exchange column chromatography. This revealed the presence of two main phosphopeptide (PP) fractions, numbers 1 and 5. Amino acid analysis of the fraction 1, occurring in the greatest amount, showed that, after deionization, it contained the metal ion complexes of phosphoserine, aspartic and glutamic acids in large amounts, and nine other amino acids. In the same way the two main PP fractions, P1 and P5, were obtained during the dialysis of the EDTA-reacted, highly purified DNA (N-DNA) prepared from calf thymus nuclei. Fraction 1 from nuclei had practically the same amino acid composition as fraction P1 from EDTA-reacted N-DNA. In both cases, the second main fraction obtained (fraction 5) had nearly the same amino acid composition as fraction 1, but the total Pser molar ratio, was about twice as high as for fraction 1. In contrast to the low molecular weights of 900-1400 daltons observed for PPs isolated directly from the dialysates in water without exposure to HCOOH, the high molecular weight of 10,260 daltons was obtained for the PP fraction 1, after exposure to HCOOH used in the gradient for the column chromatography.

A method for the preparation of DNA from calf thymus nuclei in maximum purity and yield, analysis of this DNA compared with that obtained by other methods.

In the preparation of calf thymus DNA, a method (1) was developed to yield DNA in maximum yield and purity, with a minimum of shear degradation, which differed in three respects from those used in standard preparations: 1) use of calf thymus nuclei rather than whole tissue as a source of the DNA; 2) use of initial Pronase, then on the next cycle RNAase + Pronase digestions; 3) use of optimal concentrations of reagents to precipitate denatured proteins in the purification cycles. The optimal reagent concentrations were determined in tests by changing each variable one at a time to be 3M NaCl, 3% sarcosyl. The purification cycles consisted of adjustment of DNA solution in buffer to 3M NaCl-3% sarcosyl, centrifugation, alcohol precipitation, and redissolving DNA fibers in buffer. The DNA from nuclei, digested with Pronase then with RNAase + Pronase was then subjected to five additional purification cycles. The purified DNA thus obtained (N-DNA) had a protein content of 0.64%. When purified calf thymus nuclei are subjected directly to salt-sarcosyl purification cycles, the initial protein content is about 21%, but after the second cycle the percentages of protein fell off in subsequent cycles in a parallel manner as compared to the DNA obtained by the Blin and Stafford method (2) and then subjected to NaCl-sarcosyl purification. The degree of purity obtained by these purification methods was appreciably greater than obtained for a modified method of Gross-Bellard et al. (3). Two portions of DNA purified by this method were analyzed: 1) dialyzed 11 times after the Proteinase K digestion, and 2) dialyzed 6 times. Portion 1) gave a protein content of 0.96% and a yield of 0.135% (g/100 g tissue). Portion 2) gave a protein content of 1.34% and a yield of 0.133%.

Amino acid analysis of proteins and tightly bound phosphopeptides in calf thymus DNA: indications of a novel regulatory mechanism.

Purification of DNA prepared from calf thymus nuclei, which were isolated at temperatures of -10 degrees to -15 degrees C, termed N-DNA, reduced the protein content from 17% to 0.7%. Amino acid analysis of the crude N-DNA revealed that the protein material removed in this process contained lys and phe, whereas in purified N-DNA neither of these amino acids was present. The crude N-DNA had a lower total Pser molar ratio (5.9) than that of the purified N-DNA (22.6). The purification also effected a large decrease of molecular weight, from 170 to 15 million daltons, roughly corresponding to the size of replicons. The amino acid composition of the N-DNA was compared with those of the two PP fractions, a) the PPs released into the dialysates, and b) the core material bound in the DNA, probably covalently, after reaction of N-DNA with EDTA and subsequent dialysis. N-DNA had an intermediate total Pser molar ratio between that of a) (35.3) and b) (11.9). Analysis of a standard preparation of DNA (S-DNA), which involved initial rupture of nuclei in the presence of cytoplasmic components, gave elevated values of asp and glu, a total Pser average value of 8.7 before, and 9.0, after the purification, and again phe and lys in the crude DNA.

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA.

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.

Characterization of phosphopeptides released during dialysis of EDTA-reacted highly purified DNA prepared from calf thymus nuclei.

The treatment of highly purified DNA obtained from calf thymus nuclei (N-DNA) with a chelating agent and subsequent repeated dialyses led to release of phosphopeptides (PPs) into the dialysates. By means of anion exchange column chromatography, the PPs were separated into 9 main fractions. Two of them (P1 and P5) contained the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by deionization with nitrilotriacetate + Chelex. The proportion of phosphoserine was about twice as great in P5 as in P1. Whereas P1 and P5 contained essentially no nucleotide material, the other fractions contained ribonucleotides and deoxynucleotides. The deoxynucleotide content was less than 10% of that of total nucleotides. After a deionizing treatment, the amounts of nucleotides in these fractions were reduced to a level corresponding to 1 nucleotide per peptide of 5-15 amino acids.

Phosphopeptides in highly purified calf thymus DNA.

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.

Evidence for covalent binding between phosphopeptides and terminal deoxynucleotides in highly purified calf thymus DNA.

Highly purified DNA from calf thymus nuclei (N-DNA) was found to cleave after reaction with a chelating agent and subsequent dialysis. During the cleavage phosphopeptides (PPs) were released into the dialysates. At the end of the cleavage, approximately one half of the PP material remained with the DNA. Since it was so strongly bound, it was considered to be retained in the DNA structure by covalent bonding. In order to confirm this, a commercial DNA (S-DNA) was ultrasonicated and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. DEAE Sephacel chromatography of the digested material yielded 5 fractions. The fraction 2, having the highest proportion of proteinaceous material, was digested with Pronase. Amino acid analysis of the hydrolysis mixture yielded phosphoserine (Pser), asp, thr, ser, glu, gly, ala, val, ile, leu, and arg. The mixture was chromatographed again on DEAE Sephacel. From this a single fraction, number 5, was found to contain both deoxynucleotides and the amino acids, Pser, asp, ser, glu, and gly in a molar ratio of greater than 7:3:2:2:5. The mixture obtained by hydrolysis of this fraction with snake venom diesterase was again chromatographed on DEAE Sephacel. This fractionation gave two main peaks, one corresponding to the same 5 amino acids and the other to deoxynucleotide material. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between the deoxynucleotide and amino acid portions.

Organization of highly purified calf thymus DNA. I. Cleavage into subunits and release of phosphopeptides.

DNA (N-DNA) prepared under conditions eliminating the exposure of chromatin to cytoplasmic components exhibits some special properties not observed for DNA prepared by standard methods (S-DNA).N-DNA, having a sedimentation coefficient of 24.7 S and a firmly bound protein content of 0.7%, can be cleaved (in contrast to S-DNA) by treatment with chelating agents, into stable subunits having a mean molecular weight of about 500 000. This cleavage was shown to be an ordered process which involved no enzymatic or shear degradation. It was accompanied by the release of phosphopeptides. The analyses of these phosphopeptides revealed the presence of two main fractions. One contained phosphoserine and glycine (Mr about 1400), and the other contained phosphoserine, glycine, alanine, glutamic and aspartic acids (Mr about 900). The amount of released phosphopeptides could be correlated to the extent of cleavage.

Relationship between the purity and molecular weight of calf thymus DNA.

The molecular weight and purity of calf thymus DNA, prepared by different procedures of isolation, were determined. The highest molecular weight was obtained by the method of Blin and Stafford (12 x 10(7)). This DNA contained 17% protein. The further purification of this DNA led both to increase of purity and to reduction of the molecular weight. The relationship between molecular weight and achieved purity was found to be a monotonic sigmoid-shape function ranging between the molecular weights of 12 x 10(7) and 12 x 10(6) and between the purities of 17 and 0.7% protein. After achieving this highest level of purity (0.7% protein content), despite repetition of the purification cycles, no further increase of purity or reduction of the molecular weight was achieved. The use of [14C]T4 bacteriophage DNA as internal standard demonstrated that the reduction of the molecular weight does not result from an artifact, such as shear or enzymatic degradation, but that the high-molecular-weight calf thymus DNA preparation must be considered to consist of either aggregates of small subunits stabilized by laterally attached proteins or of tandemly joined units connected by protein or peptide linkers.