Induction of the catalytic protein of (Na+ plus K+)-ATPase in the salt gland of the duck.

The (Na+ plus K+)-ATPase activities in salt gland homogenates increased 3- to 4-fold after saline treatment of ducks for 3 weeks. The ATPase was purified to a specific activity of 460 and 1015 mumol Pi/mg protein per h, respectively, in control and saline-treated ducks. The catalytic protein was identified on polyacrylamide electrophoresis gels by phosphorylating the enzyme with (32P)ATP. The molecular weight of the protein was estimated to be 98 000. The amount of catalytic unit increased commensurately with the enzyme activity after saline treatment. It is therefore concluded that the increased enzyme activity is due to a de novo enzyme synthesis and is not an activation effect. Phospholipid concentration in the salt gland tissue increased 1.7-fold after the saline treatment. Significant increases occurred in the percentage of the total phospholipids as phosphatidylserine and sphingomyelin. In the partially purified (Na+ plus K+)-ATPase preparation, the percentage composition of phosphatidylserine and phosphatidylethanolamine increased after saline treatment.     

A unique automation platform for measuring low level radioactivity in metabolite identification studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.     

Increased hepatic fibrosis and JNK2-dependent liver injury in mice exhibiting hepatocyte-specific deletion of cFLIP

Chronic liver disease promotes hepatocellular injury involving apoptosis and triggers compensatory regeneration that leads to the activation of quiescent stellate cells in the liver. The deposition of extracellular matrix from activated myofibroblasts promotes hepatic fibrosis and the progression to cirrhosis with deleterious effects on liver physiology. The role of apoptosis signaling pathways in the development of fibrosis remains undefined. The aim of the current study was to determine the involvement of the caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) during the initiation and progression of fibrosis. Liver injury and fibrosis from carbon tetrachloride (CCl(4)) and thioacetamide (TAA) were examined in mice exhibiting a hepatocyte-specific deletion of cFLIP (flip(-/-)). Acute liver injury from CCl(4) and TAA were enhanced in flip(-/-) mice. This was accompanied by increased activation of caspase-3 and -9, pronounced phosphorylation of JNK, and decreased phosphorylation of Erk. Deletion of the cJun NH(2)-terminal kinase 2 (JNK2) in flip(-/-) mice protected from injury. Hepatic fibrosis was increased at baseline in 12-wk-old flip(-/-) mice, and progression of fibrosis from TAA was accelerated compared with the wild type. In conclusion, deletion of cFLIP in hepatocytes leads to increased fibrosis and accelerated fibrosis progression. This is accompanied by increased injury involving the activation of caspases and JNK2. Thus predisposition to liver injury involving increased hepatocellular apoptosis is a critical mediator of accelerated fibrogenesis, and prevention of liver injury will be a most important measure for patients with chronic liver disease.     

An alternative theoretical approach to escape decision-making: the role of visual cues

Escape enables prey to avoid an approaching predator. The escape decision-making process has traditionally been interpreted using theoretical models that consider ultimate explanations based on the cost/benefit paradigm. Ultimate approaches, however, suffer from inseparable extra-assumptions due to an inability to accurately parameterize the model's variables and their interactive relationships. In this study, we propose a mathematical model that uses intensity of predator-mediated visual stimuli as a basic cue for the escape response. We consider looming stimuli (i.e. expanding retinal image of the moving predator) as a cue to flight initiation distance (FID; distance at which escape begins) of incubating Mallards (Anas platyrhynchos). We then examine the relationship between FID, vegetation cover and directness of predator trajectory, and fit the resultant model to experimental data. As predicted by the model, vegetation concealment and directness of predator trajectory interact, with FID decreasing with increased concealment during a direct approach toward prey, but not during a tangential approach. Thus, we show that a simple proximate expectation, which involves only visual processing of a moving predator, may explain interactive effects of environmental and predator-induced variables on an escape response. We assume that our proximate approach, which offers a plausible and parsimonious explanation for variation in FID, may serve as an evolutionary background for traditional, ultimate explanations and should be incorporated into interpretation of escape behavior.     

Immunolocalization of hypochlorite-induced, catalase-bound free radical formation in mouse hepatocytes

The establishment of oxidants as mediators of signal transduction has renewed the interest of investigators in oxidant production and metabolism. In particular, H(2)O(2) has been demonstrated to play pivotal roles in mediating cell differentiation, proliferation, and death. Intracellular concentrations of H(2)O(2) are modulated by its rate of production and its rate of decomposition by catalase and peroxidases. In inflammation and infection, some of the H(2)O(2) is converted to hypochlorous acid, a key mediator of the host immune response against pathogens. In vivo HOCl production is mediated by myeloperoxidase, which uses excess H(2)O(2) to oxidize Cl(-). Mashino and Fridovich (Biochim. Biophys. Acta 956:63-69; 1988) observed that a high excess of HOCl over catalase inactivated the enzyme by mechanisms that remain unclear. The potential relevance of this as an alternative mechanism for catalase activity control and its potential impact on H(2)O(2)-mediated signaling and HOCl production compelled us to explore in depth the HOCl-mediated catalase inactivation pathways. Here, we demonstrate that HOCl induces formation of catalase protein radicals and carbonyls, which are temporally correlated with catalase aggregation. Hypochlorite-induced catalase aggregation and free radical formation that paralleled the enzyme loss of function in vitro were also detected in mouse hepatocytes treated with the oxidant. Interestingly, the novel immuno-spin-trapping technique was applied to image radical production in the cells. Indeed, in HOCl-treated hepatocytes, catalase and protein-DMPO nitrone adducts were colocalized in the cells' peroxisomes. In contrast, when hepatocytes from catalase-knockout mice were treated with hypochlorous acid, there was extensive production of free radicals in the plasma membrane. Because free radicals are short-lived species with fundamental roles in biology, the possibility of their detection and localization to cell compartments is expected to open new and stimulating research venues in the interface of chemistry, biology, and medicine.     

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.