Long-term enhancement of pulmonary gas exchange after high-altitude residence during maturation.

In a previous study, our laboratory showed that young dogs born at sea level (SL) and raised from 2.5 mo of age to beyond somatic maturity at a high altitude (HA) of 3,100 m show enhanced resting lung function (Johnson RL Jr, Cassidy SS, Grover RF, Schutte JE, and Epstein RH. J Appl Physiol 59: 1773-1782, 1985). To examine whether HA-induced adaptation improves pulmonary gas exchange during exercise and whether adaptation is reversible when animals return to SL before somatic maturity, we raised 2.5-mo-old foxhounds at HA (3,800 m) for 5 mo (to age 7.5 mo) before returning them to SL. Lung function was measured under anesthesia 1 mo and 2 yr after return to SL and during exercise approximately 1 yr after return. In animals exposed to HA relative to simultaneous litter-matched SL controls, resting circulating blood and erythrocyte volumes, lung volumes, septal volume estimated by a rebreathing technique, and lung tissue volume estimated by high-resolution computed tomography scan were persistently higher. Lung diffusing capacity, membrane diffusing capacity, and pulmonary capillary blood volume estimated at a given cardiac output were significantly higher in animals exposed to HA, whereas maximal oxygen uptake and hematocrit were similar between groups. We conclude that relatively short exposure to HA during somatic maturation improves long-term lung function into adulthood.     

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.     

Insulin-stimulated hexose transport and glucose oxidation in rat adipocytes is inhibited by sphingosine at a step after insulin binding

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.     

A model of the role of natural killer cells in immune surveillance — II

The functioning of natural killer (NK) cells as immune surveillance effector cells against tumors is explored. In part I (J. Math. Biol. 12, 363-373 (1981], it was predicted that susceptible tumors would be eliminated if they have parameter lambda 0 value negative. They would not be eliminated if lambda 0 greater than 0. As the lambda 0 less than 0 result was local, one expected either that tumors of all sizes with lambda 0 less than 0 will be eliminated (global stability) or that tumor population will go to zero if in a domain of attraction of the critical point which is not all of the positive orthant. In this paper, the second is shown to be true. The general results are illustrated by a specific model.     

Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum

Long-chain (sphingoid) bases may serve as another category of "lipid second messenger" because they inhibit protein kinase C and affect multiple cellular functions. Free sphingosine has been found in rat liver (Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171, 373-381); hence, this study determined if liver plasma membranes contain free long-chain bases and have the ability to form them from endogenous enzymes and substrates. Isolated plasma membranes contained 0.45 nmol of sphingosine/mg of protein which, based on the recovery of the membranes, was equivalent to 3.5 +/- 1.2 nmol/g of liver and at least half of the total free sphingosine in liver. When the membranes were incubated at 37 degrees C, the amount increased at an initial rate of 5-25 pmol/min/mg, resulting in a 2-3-fold increase over an hour. Sphingosine formation required divalent cations, was optimal at neutral to alkaline pH, and was temperature-dependent. Activities with these characteristics were not identified in microsomes or lysosomes (lysosomal activities with acidic pH optima were detected, however); hence, they appear to reflect a separate plasma membrane system. Sphingosine formation was stimulated by ceramides either added exogenously or formed endogenously by treating the membranes with sphingomyelinase (but not endoglycoceramidase). Sphingomyelin hydrolysis to ceramide was also observed during incubation of the plasma membranes alone. Some of the properties of this system resembled the neutral sphingomyelinase and ceramidase activities of liver. While the physiological significance of this endogenous sphingosine is not known, this system has the appropriate subcellular location to provide sphingosine as a participant in signal transduction.