Dynamic response of the peripheral chemoreflex loop to changes in end-tidal O2.

We studied the peripheral ventilatory response dynamics to changes in end-tidal O2 tension (PETO2) in 13 cats anesthetized with alpha-chloralose-urethan. The arterial O2 tension in the medulla oblongata was kept constant using the technique of artificial perfusion of the brain stem. At constant end-tidal CO2 tension, 72 ventilatory on-responses due to stepwise changes in PETO2 from hyperoxia (45-55 kPa) to hypoxia (4.7-9.0 kPa) and 62 ventilatory off-responses due to changes from hypoxia to hyperoxia were assessed. We fitted two exponential functions with the same time delay to the breath-by-breath ventilation and found a fast and a slow component in 85% of the ventilatory on-responses and in 76% of the off-responses. The time constant of the fast component of the ventilatory on-response was 1.6 +/- 1.5 (SD) s, and that of the off-response was 2.4 +/- 1.3 s; the gain of the on-response was smaller than that of the off-response (P = 0.020). For the slow component, the time constant of the on-response (72.6 +/- 36.4 s) was larger (P = 0.028) than that of the off-response (43.7 +/- 28.3 s), whereas the gain of the on-response exceeded that of the off-response (P = 0.031). We conclude that the ventilatory response of the peripheral chemoreflex loop to stepwise changes in PETO2 contains a fast and a slow component.     

Kinetics of ammonia oxidation by a marine nitrifying bacterium: Methane as a substrate analogue

In pure culture, the marine ammonia oxidizer,Nitrosococcus oceanus, exhibits normal Michaelis Menten kinetics with respect to its primary substrate, ammonia.N. oceanus also exhibits a kinetic response to methane. In the absence of methane, oxidation of ammonia is first order with respect to ammonia concentration under atmospheric oxygen concentrations at seawater pH. In the presence of methane, ammonia oxidation is inhibited, and the amount of inhibition is related to the relative concentrations of methane and ammonia. Using semicontinuous batch cultures as a source of organisms for short-term kinetic experiments, I investigated the relationship between ammonia and methane oxidation inN. oceanus by varying the absolute and relative concentration of both substrates. Methane appeared to act as a substrate analogue, and its effect on ammonia oxidation was modeled as a permutation of competitive inhibition involving a cooperative enzyme system. Methane was oxidized byN. oceanus, even in the absence of measurable ammonia oxidation, but the process was inhibited at increasing methane concentrations. Of the two product pools analyzed, an average of 37% of methane oxidized was detected in particulate (cell) material and the remainder was detected in¹⁴CO₂. The contribution of methane to total carbon assimilation varied with the ratio [CH₄]/[NH₃] and may be significant under substrate concentrations typical of a dilute aquatic environment.     

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.     

Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1, a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene.

Saccharomyces cerevisiae cyclic AMP-dependent protein kinase (A kinase) activity is essential for growth and cell cycle progression. Dependence on A kinase function can be partially relieved by the inactivation of a second kinase encoded by the gene YAK1. We have isolated two new genes, SOK1 and SOK2 (suppressor of kinase), as gene dosage suppressors of the conditional growth defect of several temperature-sensitive A kinase mutants. Overexpression of SOK1, like lesions in YAK1, also restores growth to a strain (tpk1 tpk2 tpk3) lacking all A kinase activity. The SOK1 gene is not essential, but a sok1::HIS3 disruption abrogates suppression of an A kinase defect by yak1. These results suggest that Yak1 and Sok1 define a linear pathway that is partially redundant with that of the A kinase. Activation of Sok1, by SOK1 overexpression or by inactivation of the negative regulator Yak1, renders a cell independent of A kinase function. The implications of such a model are particularly intriguing in light of the nuclear localization pattern of the overexpressed Sok1 protein and the primary sequence homology between SOK1 and a recently described, developmentally regulated mouse gene.     

Kinetics of cell detachment: peeling of discrete receptor clusters.

Clustering of cell surface adhesion receptors is an essential step in the development of focal contacts, specialized cell-substrate attachment sites where receptors are simultaneously linked to extracellular ligand and cytoskeletal proteins. Previously, we examined the effect of receptor clustering on attachment strength. Here, we employ the numerical methodology developed by Dembo and colleagues (Dembo, M., D.C. Torney, K. Saxman, and D. Hammer. 1988. Proc. R. Soc. Lond. B. 234:55-83) to investigate the kinetics of cell detachment when receptors are clustered into discrete patches. We show that the membrane peeling velocity decreases if receptors are clustered within a patch located inside the contact region. Peeling of clusters is influenced by the chemistry and mechanics of receptor-ligand bonds within the patch. Detachment is also prohibited if the applied tension equals the critical tension of the patch, unless the patch length is small compared with the boundary length over which membrane bending occurs, in which case the patch will peel. Peeling of these short patches only occurs when the mechanical stiffness of clustered bonds is within an optimal range. We compare our model predictions with experimental measurements of T lymphocyte detachment from ICAM-1 substrates. We demonstrate that if discrete patches of receptors are present, detachment occurs through intervals of slow and fast peeling, similar to the dynamics of T lymphocyte peeling, indicating that clustering of LFA-1 receptors is one possible explanation for the observed detachment kinetics in this system.     

Intermediates of adeno-associated virus DNA replication in vitro.

Intermediates of adeno-associated virus type 2 (AAV) DNA replication in an in vitro assay have been characterized. The assay involves rescue and replication of an AAV insert in pBR322. Intermediates were shown to be duplex molecules in which at least one terminus was in the extended configuration, in contrast to the hairpinned ends seen after rescue in the absence of AAV DNA replication. Also present were linear double-stranded dimers, which were characterized as either head-to-head or tail-to-tail tandems; no head-to-tail dimers were detected. The results are in accord with the current model of AAV DNA replication.