Inter-rater agreement on chromosome 5 breakage in FISH-based mutagen sensitivity assays (MSAs)

In chromosome breakage assays, validated, universal criteria for selection of cells and classification of chromosome aberrations may enhance their utility for cancer susceptibility screening. To standardize a fluorescence in situ hybridization (FISH) modification of the mutagen sensitivity assay (MSA), scoring criteria were evaluated by web-based validation. Two hundred digital FISH images were assigned random identification numbers. With this set of images, criteria for inclusion of cells and measurement of the frequency of abnormal cells were evaluated by eight observers, all of whom had five or more years of experience. Observers included doctoral and MS/BS level cytogeneticists, and were drawn from a randomized pool of 54 volunteers. Questions addressed were: (1) how uniformly were criteria applied to analysis of a standard digital FISH image set and (2) did concordance vary with educational level? These data suggest inter-rater agreement within a factor of 2 for average breakage frequency, but revealed greater variability in cell selection. These results aid in estimating the components of assay variance due to definitions, technical parameters and biological variables.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

Effects of peroxidase substrates on the Amplex red/peroxidase assay: antioxidant properties of anthracyclines

Oxidation of Amplex red (AR) by H(2)O(2) in the presence of horseradish peroxidase (HRP) gives rise to an intensely colored product, resorufin. This reaction has been frequently employed for measurements of low concentrations of H(2)O(2) in biological samples. In the current study, we show that alternative peroxidase substrates, such as p-hydroquinone, acetaminophen, anticancer mitoxantrone, and ametantrone, inhibit AR oxidation by consuming H(2)O(2) in a competitive process. In contrast, the anthracycline agents doxorubicin, daunorubicin, and 5-iminodaunorubicin are markedly less efficient as competitors in these reactions, as is salicylic acid. When [H(2)O(2)]>[AR], the generated resorufin was oxidized by HRP and H(2)O(2). In the presence of anthracyclines, this process was inhibited and occurred with a lag time, the duration of which depended on the concentration of anthracycline. We propose that the mechanism of this inhibition is due to the antioxidant activity of anthracyclines involving the reduction of the resorufin-derived phenoxyl radical by the drugs' hydroquinone moiety back to resorufin. In addition to HRP, lactoperoxidase, myeloperoxidase, and HL-60 cells, naturally rich in myeloperoxidase, also supported these reactions. Results of this study suggest that extra caution is needed when using AR to measure cellular H(2)O(2) in the presence of alternative peroxidase substrates. They also demonstrate that the anticancer anthracyclines may function as antioxidants.     

The biogeographical assignment of a west Kenyan rain forest remnant: further evidence from analysis of its reptile fauna

Aim The Kakamega Forest, western Kenya, has been biogeographically assigned to both lowland and montane forest biomes, or has even been considered to be unique. Most frequently it has been linked with the Guineo‐Congolian rain forest block. The present paper aims to test six alternative hypotheses of the zoogeographical relationships between this forest remnant and other African forests using reptiles as a model group. Reptiles are relatively slow dispersers, compared with flying organisms (Aves and Odonata) on which former hypotheses have been based, and may thus result in a more conservative biogeographical analysis. Location Kakamega Forest, Kenya, Sub‐Saharan Africa. Methods The reptile diversity of Kakamega Forest was evaluated by field surveys and data from literature resources. Faunal comparisons of Kakamega Forest with 16 other African forests were conducted by the use of the ‘coefficient of biogeographic resemblance’ using the reptile communities as zoogeographic indicators. Parsimony Analysis of Endemism and Neighbour Joining Analysis of Endemism were used to generate relationship trees based on an occurrence matrix with paup *. Results The analysis clearly supports the hypothesis that the Kakamega Forest is the easternmost fragment of the Guineo‐Congolian rain forest belt, and thus more closely related to the forests of that Central–West African complex than to any forest further east, such as the Kenyan coastal forests. Many Kenyan reptile species occur exclusively in the Kakamega Forest and its associated forest fragments. Main conclusions The Kakamega Forest is the only remnant of the Guineo‐Congolian rain forest in the general area. We assume that the low degree of resemblance identified for the Guineo‐Congolian forest and the East African coastal forest reflect the long history of isolation of the two forest types from each other. Kenyan coastal forests may have been historically connected through forest ‘bridges’ of the southern highlands with the Congo forest belt, allowing reptile species to migrate between them. The probability of a second ‘bridge’ located in the region of southern Tanzanian inselbergs is discussed. Although not particularly rich in reptile species, the area should be considered of high national priority for conservation measures.