Pulmonary and circulatory adjustments determining the limits of depths in breathhold diving.

Data on pulmonary gas exchange were collected in breathhold dives to 90 feet in a tank and in open-sea breathhold dives to depths of 217.5 and 225 feet. Thoracic blood volume displacements were measured at depths of 25, 50, 90, and 130 feet, by use of the impedance plethysmograph. The open-sea dives were carried out with an average speed of descent of 3.95 feet per second and an average rate of ascent of 3.50 feet per second. End-dive alveolar oxygen tensions did not fall below 36 millimeters of mercury, while alveolar carbon dioxide tension did not rise above 40 millimeters of mercury except in one case. These findings indicate that for diver Croft, who has unusual lung capacity, neither hypoxia nor hypercapnia determined the depth limits under those conditions. At depths of 90 and 130 feet blood was forced into the thorax, amounting to 1047 and 850 milliliters respectively.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

Effect of vitamin E supplementation on the health and fertility of dairy cows: a review.

The currently recommended intake of vitamin E for dairy cows is based on the prevention of nutritional myopathy, a calf disease. However, it is likely that the vitamin E requirement of the modern dairy cow is very different from that of a calf. This review of the literature investigates the effect of vitamin E supplementation on the health and fertility of the dairy cow. Supplementation of high levels of vitamin E (at least 1000 iu per day) during the dry period and early lactation can reduce the incidence of mastitis, possibly because of an increase in immune system activity and function, but there appears to be little benefit of supplementation on infectious diseases other than mastitis. The evidence for a response in the reproductive system is more equivocal. In herds with a history of selenium deficiency and a high incidence of retained fetal membranes, supplementation, in conjunction with selenium, can reduce retention, but the evidence for an effect of supplementation on other reproductive diseases, such as cystic ovarian disease and metritis, is based on a very limited number of cases. The literature suggests that the current recommendations for vitamin E are inadequate. In particular, it suggests that the current linking of requirement to dry matter intake is incorrect, because vitamin E requirement is probably at its highest when intake is at its lowest However, the majority of the data on which this conclusion is based, come from North America where cows will encounter significantly different levels of oxidative stress from cows in the EU.

Kinetic assay methods.

The purpose of this appendix is to provide a brief review of issues important in the design of initial-rate assay methods.

Kinetic assay methods.

The purpose of this unit is to provide a brief review of issues important in the design of initial-rate assay methods. General aspects of kinetic assay design are discussed, including enzyme and substrate purity, concentration and stability. Also covered are issues such as continuous versus stop-time assay formats, coupled enzyme assays, binding studies, and presentation of initial-rate data.

Concerning the equilibrium exchange kinetics and mechanism of the sheep brain glutamine synthetase reaction.

Isotope exchange measurements of sheep brain glutamine synthetase have yielded conflicting experimental findings and interpretation, leaving in doubt the question of whether the enzyme operates via ordered or random pathways of enzyme-substrate interactions. We now report new experimental evidence that demonstrates the earlier discrepant results may be attributed to the choice of reaction conditions used to achieve equilibration of the chemical reaction prior to addition of isotopic tracer. A random kinetic mechanism, perhaps not of the rapid-equilibrium type, is most compatible with the exchange data. We also discuss other potential time-dependent processes that may compromise the equilibration of reaction systems and affect the outcome of exchange experiments, and criteria for equilibration are suggested for the aid of other workers.

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.

Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase.

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.