Exposure of bovine embryos to trypsin during washing does not decrease embryonic survival

The objective of this study was to assess whether the exposure of zona pellucida-intact bovine embryos to the proteolytic enzyme, trypsin, during embryo washing has a detrimental effect on their subsequent survival and development. Embryos were collected nonsurgically from superovulated cows (n = 19) 7.5 d after insemination. Grade 1 and Grade 2 embryos were washed 12 times in modified Dulbecco's phosphate buffered saline (PBS) containing 0.4% bovine serum albumin (BSA), or in a series of five washes in BSA-PBS (without Ca++ and Mg++), two in 0.25% trypsin in Hank's solution (without Ca++ and Mg++) and five in PBS-BSA medium. Within 30 min after washing, embryos were either transferred nonsurgically into recipient cows, 7 to 8 d post estrus, or cryopreserved and transferred later. Frozen-thawed embryos from five of the donors were cultured for 72 h in vitro and their development was evaluated. Pregnancy rates did not differ (P>0.1) between recipient cows receiving control-washed and trypsin-washed embryos transferred fresh (51.0 vs 56.3%). However, pregnancy rates were higher (P<0.05) for frozen-thawed embryos treated with trypsin before cryopreservation than for frozen-thawed, control-washed embryos (68.2 vs 38.5%). Survival and development of embryos in vitro after cryopreservation did not differ between embryos subjected to the control- and trypsin-wash procedures. These results suggest that exposure of bovine embryos to trypsin for 2 to 3 min during washing did not have a detrimental effect on embryonic development, but may have enhanced cryopreservation of the embryos.     

Lack of alveolar O2 during lung reperfusion does not decrease edema formation

We previously reported that pulmonary arterial occlusion for 48 h followed by 4 h of reperfusion in awake dogs results in marked edema and inflammatory infiltrates in both reperfused and contralateral lungs (Am. Rev. Respir. Dis. 134: 752-756, 1986; J. Appl. Physiol. 63: 942-950, 1987). In this experiment we study the effects of alveolar hypoxia on this injury. Anesthetized dogs underwent thoracotomy and occlusion of the left pulmonary artery. Twenty-four hours later the dogs were reanesthetized, and a double-lumen endotracheal tube was placed. The right lung was continuously ventilated with an inspiratory O2 fraction (FIO2) of 0.35. In seven study animals the left lung was ventilated with an FIO2 of 0 for 3 h after the left pulmonary artery occluder was removed. In six control animals the left lung was ventilated with an FIO2 of 0.35 during the same reperfusion period. Postmortem bloodless wet-to-dry weight ratios were 5.87 +/- 0.20 for the left lower lobe and 5.32 +/- 0.12 for the right lower lobe in the dogs with hypoxic ventilation (P less than 0.05 for right vs. left lobes). These values were not significantly different from the control dog lung values of 5.94 +/- 0.22 for the left lower lobe and 5.11 +/- 0.07 for the right lower lobe (P less than 0.05 for right vs. left lobes). All values were significantly higher than our laboratory normal of 4.71 +/- 0.06. We conclude that reperfusion injury is unaffected by alveolar hypoxia during the reperfusion phase.     

Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis

The present study was designed to assess the impact of coingestion of various amounts of carbohydrate combined with an ample amount of protein intake on postexercise muscle protein synthesis rates. Ten healthy, fit men (20 +/- 0.3 yr) were randomly assigned to three crossover experiments. After 60 min of resistance exercise, subjects consumed 0.3 g x kg(-1) x h(-1) protein hydrolysate with 0, 0.15, or 0.6 g x kg(-1) x h(-1) carbohydrate during a 6-h recovery period (PRO, PRO + LCHO, and PRO + HCHO, respectively). Primed, continuous infusions with L-[ring-(13)C(6)]phenylalanine, L-[ring-(2)H(2)]tyrosine, and [6,6-(2)H(2)]glucose were applied, and blood and muscle samples were collected to assess whole body protein turnover and glucose kinetics as well as protein fractional synthesis rate (FSR) in the vastus lateralis muscle over 6 h of postexercise recovery. Plasma insulin responses were significantly greater in PRO + HCHO compared with PRO + LCHO and PRO (18.4 +/- 2.9 vs. 3.7 +/- 0.5 and 1.5 +/- 0.2 U.6 h(-1) x l(-1), respectively, P < 0.001). Plasma glucose rate of appearance (R(a)) and disappearance (R(d)) increased over time in PRO + HCHO and PRO + LCHO, but not in PRO. Plasma glucose R(a) and R(d) were substantially greater in PRO + HCHO vs. both PRO and PRO + LCHO (P < 0.01). Whole body protein breakdown, synthesis, and oxidation rates, as well as whole body protein balance, did not differ between experiments. Mixed muscle protein FSR did not differ between treatments and averaged 0.10 +/- 0.01, 0.10 +/- 0.01, and 0.11 +/- 0.01%/h in the PRO, PRO + LCHO, and PRO + HCHO experiments, respectively. In conclusion, coingestion of carbohydrate during recovery does not further stimulate postexercise muscle protein synthesis when ample protein is ingested.     

Flowering does not decrease vegetative competitiveness of Lolium perenne

The theory of life-history evolution commonly assumes a trade-off between sexual and vegetative reproduction. Hence, production of flowers and fruits should have measurable costs in terms of reduced vegetative growth. This trade-off may be meaningful for breeding of forage and turf grasses as reduced flowering could free resources and increase productivity. But if so, less-flowering cultivars might be more competitive and invade natural swards. We tested for costs of sexual reproduction on vegetative propagation and competitiveness of the perennial grass Lolium perenne, one of the most important forage and turf grasses worldwide. We used the differences in vernalisation requirement between northern and southern European provenances to manipulate the degree of flowering. Over three growing seasons, we counted the number of flower stems and measured the clone diameter. The vernalisation treatments were successful in producing clones with largely differing degrees of flowering. However, we found no negative correlation between flowering and vegetative propagation and competitiveness. Early and strongly flowering southern provenances showed less clonal growth and higher mortality, but within provenances the response of clone diameter to flowering was positive or neutral. We conclude that investment of resources into flowering has no measurable costs on vegetative propagation and competitiveness of L. perenne. The apparent lack of costs of sexual reproduction could be explained by bet-hedging strategy that is focused on survival and growth rather than reproductive effort in order to maximise the life-time fitness.     

Nitric oxide availability is increased in contracting skeletal muscle from aged mice,but does not differentially decrease muscle superoxide

Reactive oxygen and nitrogen species have been implicated in the loss of skeletal muscle mass and function that occurs during aging. Nitric oxide (NO) and superoxide are generated by skeletal muscle and where these are generated in proximity their chemical reaction to form peroxynitrite can compete with the superoxide dismutation to hydrogen peroxide. Changes in NO availability may therefore theoretically modify superoxide and peroxynitrite activities in tissues, but published data are contradictory regarding aging effects on muscle NO availability. We hypothesised that an age-related increase in NO generation might increase peroxynitrite generation in muscles from old mice, leading to an increased nitration of muscle proteins and decreased superoxide availability. This was examined using fluorescent probes and an isolated fiber preparation to examine NO content and superoxide in the cytosol and mitochondria of muscle fibers from adult and old mice both at rest and following contractile activity. We also examined the 3-nitrotyrosine (3-NT) and peroxiredoxin 5 (Prx5) content of muscles from mice as markers of peroxynitrite activity. Data indicate that a substantial age-related increase in NO levels occurred in muscle fibers during contractile activity and this was associated with an increase in muscle eNOS. Muscle proteins from old mice also showed an increased 3-NT content. Inhibition of NOS indicated that NO decreased superoxide bioavailability in muscle mitochondria, although this effect was not age related. Thus increased NO in muscles of old mice was associated with an increased 3-NT content that may potentially contribute to age-related degenerative changes in skeletal muscle.     

Amino acid ingestion improves muscle protein synthesis in the young and elderly

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 +/- 4 yr) and elderly (67 +/- 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol.kg(-1).min(-1)) of L-[ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of 15 g of essential amino acids (EAA) in young (n = 6) and elderly (n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by approximately 0.04%/h (P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: 115.6 +/- 5.4 nmol/ml; elderly: 150.2 +/- 19.4 nmol/ml). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.1 +/- 1.2 to 21.3 +/- 3.1 microIU/ml) but not in elderly subjects (3.0 +/- 0.6 to 4.3 +/- 0.4 microIU/ml). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.     

Alpha-linolenic acid supplementation during human pregnancy does not effect cognitive functioning

Increasing evidence suggests a positive association between docosahexaenoic acid (DHA, 22:6n-3) and cognitive performance. In addition, pregnancy is associated with a reduction of the DHA status and cognitive deficits. In the current study, cognition was assessed in pregnant women receiving a margarine enriched with alpha-linolenic acid (ALA, 18:3n-3, the ultimate dietary precursor of DHA) and some linoleic acid (LA, 18:2n-6, to prevent a possible reduction in n-6 fatty acids). A control group received a margarine enriched with LA only. ALA supplementation hardly affected the maternal DHA status and no significant differences were found in cognitive performance between the two groups. This indicates that ALA supplementation during pregnancy does not affect cognitive performance during and 32 weeks after gestation. At week 14 of pregnancy and 32 weeks after delivery, higher plasma DHA levels were associated with lower cognitive performance as indicated by longer reaction times on the finger precuing task (partial correlation coefficients 0.3705 and 0.4633, respectively, P<0.01). Since this could imply an unexpected adverse association between DHA and certain aspects of cognitive functioning this certainly needs further investigation.     

Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans

There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKalpha2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKalpha2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 +/- 1% V(O2 peak). In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKalpha2 activity, AMPKalpha2 Thr(172) phosphorylation and acetyl-CoA Ser(222) phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.     

Preexercise medium-chain triglyceride ingestion does not alter muscle glycogen use during exercise.

This investigation determined whether ingestion of a tolerable amount of medium-chain triglycerides (MCT; approximately 25 g) reduces the rate of muscle glycogen use during high-intensity exercise. On two occasions, seven well-trained men cycled for 30 min at 84% maximal O(2) uptake. Exactly 1 h before exercise, they ingested either 1) carbohydrate (CHO; 0.72 g sucrose/kg) or 2) MCT+CHO [0.36 g tricaprin (C10:0)/kg plus 0.72 g sucrose/kg]. The change in glycogen concentration was measured in biopsies taken from the vastus lateralis before and after exercise. Additionally, glycogen oxidation was calculated as the difference between total carbohydrate oxidation and the rate of glucose disappearance from plasma (R(d) glucose), as measured by stable isotope dilution techniques. The change in muscle glycogen concentration was not different during MCT+CHO and CHO (42.0 +/- 4.6 vs. 38.8 +/- 4.0 micromol glucosyl units/g wet wt). Furthermore, calculated glycogen oxidation was also similar (331 +/- 18 vs. 329 +/- 15 micromol. kg(-1). min(-1)). The coingestion of MCT+CHO did increase (P < 0.05) R(d) glucose at rest compared with CHO (26.9 +/- 1.5 vs. 20.7 +/- 0. 7 micromol.kg(-1). min(-1)), yet during exercise R(d) glucose was not different during the two trials. Therefore, the addition of a small amount of MCT to a preexercise CHO meal did not reduce muscle glycogen oxidation during high-intensity exercise, but it did increase glucose uptake at rest.     

Amino acid pools in cultured muscle cells

Compartmentalization of cellular amino acid pools occurs in cultures of cardiac and skeletal muscle cells, but the factors involved in this are not clear. We have further defined this problem by analyzing the intracellular free leucine and the transfer-RNA-(tRNA)-bound leucine pool in cultures of skeletal and cardiac muscle incubated with ³H-leucine in the presence and absence of serum and amino acids. Withdrawal of nitrogen substrates caused substantial changes in leucine pool relationships–in particular, a change in the degree to which intracellular free leucine and tRNA-leucine were derived from the culture medium. In separate experiments, the validity of our tRNA measurements was confirmed by measurements of the specific activity of newly synthesized ferritin after iron induction. We discuss the implications of these findings with regard-to factors involved in the control of amino acid flux through the cell, as well as with regard to design of experiments using isotopic amino acids to measure rates of amino acid utilization.     

Amino acid composition and protein dimension

There is indirect evidence that the amino acid composition of proteins depends on their dimension. The amino acid composition of a nonredundant set of about 550,000 proteins was determined and it was observed that, in the range of 50-200 residues, the percentage of occurrence of most of the residue types significantly depends on protein dimension. This result should prove useful in analyzing protein sequences and genomics.