Orthophosphate determination in the presence of high concentrations of ATP

A method to measure orthophosphate which contaminates samples of ATP was developed. Concentrations of orthophosphate as low as 0.4% of the ATP concentration were determined using a zinc-molybdate reagent [D. A. Bencini, J. R. Wild, and G. A. O'Donovan, Anal. Biochem. 132, 254-258 (1983)] and continuous spectrophotometric monitoring of chromophore formation. Since the rate of ATP hydrolysis was pseudo-first order and was slow compared to the rate of chromophore formation, the initial concentration of phosphate could be readily determined by extrapolation to zero time. The method is rapid and reproducible, and requires a single, stable reagent.     

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.     

The preferred route of kynurenine metabolism in the rat

It has been suggested (Ueda, T., Otsuka, H. and Goda, K. (1978) J. Biochem. 84, 687–696) that direct cleavage of kynurenine, catalysed by kynureninase, followed by microsomal hydroxylation of the resultant anthranilic acid, may provide an alternative to the established pathway of kynurenine metabolism that involves direct hydroxylation followed by cleavage to 3-hydroxyanthranilic acid. To test this suggestion, anthranilic acid was administered to rats; there was no increase in either the concentration of nicotinamide nucleotides in the liver or the urinary excretion of N¹-methyl nicotinamide. However, injection of either kynurenine or 3-hydroxyanthranilic acid did increase the concentration of nicotinamide nucleotides in the liver. The kinetics of kynurenine hydroxylase (K_m = 1.8±0.6·10^?5 mol/l) and kynureninase (K_m = 2.5±0.8·10^?4 mol/l, liver steady-state kynurenine = 4.9±0.9 μmol/kg) are such that the preferred route of kynurenine metabolism is probably by way of hydroxylation rather than cleavage.     

Evaluation of the dermal carcinogenic potential of liquids produced from the Cold Lake heavy oil deposits of northeast Alberta

This study assessed the dermal carcinogenic potential of raw bitumen derived from the Cold Lake Oil Sands deposit (located in Northeast Alberta, Canada) and two liquids which were under evaluation as part of a process to refine the crude bitumen at the Cold Lake site. The crude bitumen was dermally carcinogenic, inducing tumors in 26% of the treated animals with a median latency of 106 weeks. This response was significantly greater than the tumor yield previously reported for a raw bitumen derived from Athabasca tar sands by the Syncrude process, but was not substantially different from the carcinogenic potential of two crude petroleum oils. The GO-FINING product, a high boiling (259-519 degrees C), catalytically cracked gas oil was a relatively potent dermal carcinogen, inducing tumors in 86% of the treated animals with a median latency of 46 weeks. This result is consistent with the fact that the GO-FINING product contained appreciable levels of high boiling aromatic compounds. The HYCRACKING product, a high boiling (102-498 degrees C), severely hydroprocessed liquid was noncarcinogenic. This result was also consistent with the compositional data; the high boiling components were predominantly saturated species. Thus the carcinogenic properties of the liquid products prepared by these two processes were as predicted from the compositional information.     

Regulatory effects of protein S-acylation on insulin secretion and insulin action

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.