Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Microhabitat distribution of protostelids in a Tropical Wet Forest in Costa Rica

A microhabitat study of protostelids was carried out in a Tropical Wet Forest at the La Selva Biological Station in Costa Rica. Nine species were recorded from sterile wheat straws placed out and then re-collected over a period of six weeks from two different litter microhabitats in an area of primary forest. All nine species were present on straws placed in the aerial litter microhabitat, but only six species were present on straws placed in the forest floor litter microhabitat. Total colonies, percent of straws colonized, and mean number of species per straw increased significantly over time. One species (Schizoplasmodiopsis pseudoendospora) typical of temperate litter was the overwhelming dominant on the forest floor litter, while Echinostelium bisporum, a species rare in temperate litter microhabitats, was the single most abundant species in the aerial litter microhabitat. Both of these species had significantly increased frequencies over time. Two species abundant in temperate aerial litter microhabitats and one species abundant in temperate forest floor litter were rare at La Selva. Our data conform to those obtained in an earlier study carried out in tropical forests in the mountains of Puerto Rico and provide additional support towards developing a model of microhabitat distribution of protostelids in terrestrial ecosystems.     

Estimating the latitudinal origins of migratory birds using hydrogen and sulfur stable isotopes in feathers: influence of marine prey base

Hydrogen stable isotope analysis of feathers is an important tool for estimating the natal or breeding latitudes of nearctic-neotropical migratory birds. This method is based on the latitudinal variation of hydrogen stable isotope ratios in precipitation in North America (deltaD(p)) and the inheritance of this variation in newly formed feathers (deltaD(f)). We hypothesized that the typically strong relationship between deltaD(p) and deltaD(f) would be decoupled in birds that forage in marine food webs because marine waters have relatively high deltaD values compared to deltaD values for local precipitation. Birds that forage on marine prey bases should also have feathers with high delta(34)S values, since delta(34)S values for marine sulfate are generally higher than delta(34)S values in terrestrial systems. To examine this potential marine effect on feather stable isotope ratios, we measured deltaD and delta(34)S in the feathers of nine different species of raptors from both inland and coastal locations across North America. Feathers from coastal bird-eating raptors had consistently higher deltaD and delta(34)S values than feathers from inland birds. Birds that had high delta(34)S values also deviated strongly from the typical relationship between deltaD(p) and deltaD(f). We recommend measuring both sulfur and hydrogen stable isotope ratios in feathers when some members of a migrant population could potentially forage in marine habitats. We suggest using a practical cutoff of delta(34)S >10 per thousand to remove marine-foraging birds from a migrant sample when using stable isotopes of hydrogen to estimate the latitudinal origins of migrants because high deltaD(f) values for marine-foraging birds could potentially distort estimates of origins.     

Purification and preliminary characterization of brain aspartoacylase

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.     

The development of enantiostyly

Enantiostyly, the deflection of the style either to the left (left-styled) or right (right-styled) side of the floral axis, has evolved in at least ten angiosperm families. Two types of enantiostyly occur: monomorphic enantiostyly, in which individuals exhibit both stylar orientations, and dimorphic enantiostyly, in which the two stylar orientations occur on separate plants. To evaluate architectural or developmental constraints on the evolution of both forms of enantiostyly, we examined inflorescence structure and floral development among unrelated enantiostylous species. We investigated relations between the position of left- and right-styled flowers and inflorescence architecture in four monomorphic enantiostylous species, and we examined the development of enantiostyly in nine monomorphic and dimorphic enantiostylous species from five unrelated lineages. The location of left- and right-styled flowers within inflorescences ranged from highly predictable (in Solanum rostratum) to random (in Heteranthera mexicana). There were striking differences among taxa in the timing of stylar bending. In Wachendorfia paniculata, Dilatris corymbosa, and Philydrum lanuginosum, the style deflected in the bud, whereas in Heteranthera spp., Monochoria australasica, Cyanella lutea, and Solanum rostratum, stylar bending occurred at the beginning of anthesis. Comparisons of organ initiation and development indicated that asymmetries along the left-right axis were expressed very late in development, despite the early initiation of a dorsiventral asymmetry. We suggest that the evolution of dimorphic enantiostyly from monomorphic enantiostyly may be constrained by a lack of left-right positional information in the bud.     

Neonatal hypothyroidism alters the localization of gap junctional protein connexin 43 in the testis and messenger RNA levels in the epididymis of the rat

The objectives of this study were to determine the effects of propylthiouracil (PTU)-induced neonatal hypothyroidism on the gap junctional protein Cx43 in rat testis and epididymis. PTU (0.02%) was administered via lactation from birth to Day 30, and the rats were sampled at 14, 18, 22, 26, 30, and 91 days of age. Testicular Cx43 was localized along the plasma membranes and cytoplasm of Sertoli cells until Day 22. At Day 30, the immunostaining was localized exclusively along the plasma membrane of Sertoli cells. In PTU-treated rats, Cx43 did not localize to the plasma membrane and was still cytoplasmic at 30 days of age. Occludin was present in tubules of treated rats, but was not localized to the blood-testis barrier in 30-day-old rats, as in controls. There were no differences in Cx43 immunostaining in the adult testis. In the proximal epididymis (initial segment, caput, corpus), Cx43 mRNA levels were lower in PTU-treated rats at 14, 18, and 22 days of age, but no differences were observed in the distal (cauda) epididymis at these ages. In 22- and 30-day-old rats, Cx43 was localized along the plasma membrane between principal and basal cells throughout the epididymis. In PTU-treated rats, Cx43 was not detectable in initial segment, caput, or corpus epididymidis. In the cauda epididymidis, however, Cx43 immunostaining in PTU-treated rats was similar to controls. These data suggest that thyroid hormones regulate Cx43-dependent gap junctional communication in the testis and epididymis.     

Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases

This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract.Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.