Aldehyde dehydrogenase, aldose reductase, and free radical scavengers in cataract

Human lens was found to contain aldehyde dehydrogenase at a level of activity similar to that of bovine lens, namely 1.76 +/- 0.51 IU/g. The enzyme, which appears to be a tetramer of 229 kD, was less susceptible to inhibition by cataractogenic agents than the bovine enzyme. The lipid peroxidation product malondialdehyde was a good substrate of the human lens enzyme. The in vitro aldose reductase reaction, which we have shown is caused by glyceraldehyde-stimulated free-radical NADPH oxidation, is inhibited by the potential anti-cataract agents, bendazac acid and bendazac lysine; these compounds also inhibit ferricytochrome c reduction in the presence of DL-glyceraldehyde and scavenge superoxide radicals. These results are consistent with the hypotheses that aldehyde dehydrogenase is a protective enzyme in the human lens, and that the peroxy radical scavenging effects of bendazac acid and bendazac lysine contribute to their anti-cataract activity.     

Relationship of membrane physical properties to alcohol dependence in mice selected for genetic differences in alcohol withdrawal

Genetic selection based on severity of withdrawal seizures following inhalation of ethanol vapor has produced two lines of mice, WSR (withdrawal seizure resistant) and WSP (withdrawal seizure prone), that differ markedly in withdrawal signs. In the present study, we report that these mice also differed in the severity of withdrawal seizures following consumption of an ethanol-containing liquid diet but did not differ in ethanol intake. In contrast to ethanol withdrawal seizures, the lines displayed similar sensitivity to electrical- or pentylenetetrazole-induced seizures. These results suggest that the lines differ in the development of physical dependence on ethanol rather than seizure sensitivity per se. Because decreased synaptic membrane fluidity has been associated with ethanol dependence, we used fluorescence polarization of diphenylhexatriene and trimethylammonium-diphenylhexatriene to evaluate membrane fluidity in WSP and WSR mice fed lab chow, an ethanol-containing liquid diet, or an isocaloric sucrose-containing liquid diet. Fluidity of brain synaptic membranes was identical for WSP and WSR mice fed lab chow. The control liquid diet did not alter membrane fluidity, and the ethanol diet decreased fluidity equally for WSP and WSR mice. Thus, the genetic difference in development of ethanol dependence found in these lines was not reflected in the physical properties of brain membranes.     

Bovine lens aldehyde reductase (aldose reductase). Purification, kinetics and mechanism.

Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones , including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

Oxidation of p-dimethylaminomethylbenzylamine by pig kidney diamine oxidase. A new method for spectrophotometric assay

1. The oxidation of some para-substituted benzylamines by diamine oxidase produces the corresponding aldehydes. This was studied to develop a spectrophotometric method for following the enzyme reaction, as the aldehydes produced absorb strongly at 250nm where the substrates are almost optically transparent. 2. p-Dimethylaminomethylbenzylamine was the most useful substrate and full details of its preparation are given. The synthesis of its related oxidation product, p-dimethylaminomethylbenzaldehyde, is also described. 3. The effects of variations in pH, ionic strength, temperature and oxygenation on the reaction are described and the usefulness of the method is illustrated by several applications and assessed by comparison with the standard spectrophotometric assay.     

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Three murine anxiety models: results from multiple inbred strain comparisons

The literature surrounding rodent models of human anxiety disorders is discrepant concerning which models reflect anxiety-like behavior distinct from general activity and whether different models are measuring the same underlying constructs. This experiment compared the responses of 15 inbred mouse strains (129S1/SvlmJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/B1NJ, PL/J, SJL/J and SWR/J) in three anxiety-like behavioral tasks (light/dark test, elevated zero-maze and open field) to examine whether responses were phenotypically and/or genetically correlated across tasks. Significant strain differences were found for all variables examined. Principal components analyses showed that variables associated with both activity and anxiety-like behaviors loaded onto one factor, while urination and defecation loaded onto another factor. Our findings differ from previous research by suggesting that general activity and anxiety-related behaviors are linked, negatively correlated and cannot easily be dissociated in these assays. However, these findings may not necessarily generalize to other unconditioned anxiety-like behavioral tests.     

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

Background  

Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections.     

Different data from different labs: lessons from studies of gene-environment interaction

It is sometimes supposed that standardizing tests of mouse behavior will ensure similar results in different laboratories. We evaluated this supposition by conducting behavioral tests with identical apparatus and test protocols in independent laboratories. Eight genetic groups of mice, including equal numbers of males and females, were either bred locally or shipped from the supplier and then tested on six behaviors simultaneously in three laboratories (Albany, NY; Edmonton, AB; Portland, OR). The behaviors included locomotor activity in a small box, the elevated plus maze, accelerating rotarod, visible platform water escape, cocaine activation of locomotor activity, and ethanol preference in a two-bottle test. A preliminary report of this study presented a conventional analysis of conventional measures that revealed strong effects of both genotype and laboratory as well as noteworthy interactions between genotype and laboratory. We now report a more detailed analysis of additional measures and view the data for each test in different ways. Whether mice were shipped from a supplier or bred locally had negligible effects for almost every measure in the six tests, and sex differences were also absent or very small for most behaviors, whereas genetic effects were almost always large. For locomotor activity, cocaine activation, and elevated plus maze, the analysis demonstrated the strong dependence of genetic differences in behavior on the laboratory giving the tests. For ethanol preference and water escape learning, on the other hand, the three labs obtained essentially the same results for key indicators of behavior. Thus, it is clear that the strong dependence of results on the specific laboratory is itself dependent on the task in question. Our results suggest that there may be advantages of test standardization, but laboratory environments probably can never be made sufficiently similar to guarantee identical results on a wide range of tests in a wide range of labs. Interpretations of our results by colleagues in neuroscience as well as the mass media are reviewed. Pessimistic views, prevalent in the media but relatively uncommon among neuroscientists, of mouse behavioral tests as being highly unreliable are contradicted by our data. Despite the presence of noteworthy interactions between genotype and lab environment, most of the larger differences between inbred strains were replicated across the three labs. Strain differences of moderate effects size, on the other hand, often differed markedly among labs, especially those involving three 129-derived strains. Implications for behavioral screening of targeted and induced mutations in mice are discussed.