Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest™

Abstract

Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest^?. The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.     

Highly Sensitive Flow Injection Analysis of Lipid Hydroperoxides in Foodstuffs

Lipid hydroperoxides in oils and foods were measured by a flow injection analysis system with high sensitivity and selectivity. After sample injection, lipid hydroperoxides were reacted with diphenyl-1-pyrenylphosphine (DPPP) in a stainless steel coil, then the fluorescence intensity of DPPP oxide, that was produced by the reaction, was monitored. By this method, trilinolein hydroperoxide showed good linearity between 0.4 and 79pmol and their detection limits were 0.2pmol (signal-to-noise ratio = 3). The method made it possible to inject samples at 2-min intervals. There was a good agreement of the amounts of lipid hydroperoxides in oils and foods between by the batch method with DPPP and by the proposed method (coefficient of correlation: r = 0.999; n = 21; peroxide value = 0.09–167 meq/g). With this method, the calibration graph of trilinolein hydroperoxide was useful for all samples tested.     

Implications of climate change for potamodromous fishes

There is little understanding of how climate change will impact potamodromous freshwater fishes. Since the mid 1970s, a decline in annual rainfall in south‐western Australia (a globally recognized biodiversity hotspot) has resulted in the rivers of the region undergoing severe reductions in surface flows (ca. 50%). There is universal agreement amongst Global Climate Models that rainfall will continue to decline in this region. Limited data are available on the movement patterns of the endemic freshwater fishes of south‐western Australia or on the relationship between their life histories and hydrology. We used this region as a model to determine how dramatic hydrological change may impact potamodromous freshwater fishes. Migration patterns of fishes in the largest river in south‐western Australia were quantified over a 4 year period and were related to a number of key environmental variables including discharge, temperature, pH, conductivity and dissolved oxygen. Most of the endemic freshwater fishes were potamodromous, displaying lateral seasonal spawning migrations from the main channel into tributaries, and there were significant temporal differences in movement patterns between species. Using a model averaging approach, amount of discharge was clearly the best predictor of upstream and downstream movement for most species. Given past and projected reductions in surface flow and groundwater, the findings have major implications for future recruitment rates and population viabilities of potamodromous fishes. Freshwater ecosystems in drying climatic regions can only be managed effectively if such hydro‐ecological relationships are considered. Proactive management and addressing existing anthropogenic stressors on aquatic ecosystems associated with the development of surface and groundwater resources and land use is required to increase the resistance and resilience of potamodromous fishes to ongoing flow reductions.     

Nuclear DNA content variation in seeds from 22 half-sib families of jack pine (Pinus banksiana,Pinaceae)

In order to understand the amount of DNA content variation and its potential roles, both absolute DNA amount and cell cycle phases in 22 half-sib families of jack pine were examined using flow cytometry. When the variability due to differences in speed of germination was taken into account, embryos from superior families (classified on the basis of height growth during field trials) had significantly higher levels of all nuclei classes greater than 4C. Mean DNA contents per nucleus were significantly lower in embryos from superior families compared to inferior ones. Analysis of megagametophyte tissue showed that the mother trees of these embryos expressed a similar pattern. Absolute DNA values were also established on the emerging radicle and the hypocotyl + cotyledons region (HC) separately in five of the families. Nuclei isolated from the emerging radicles had significantly lower levels of DNA than those isolated from the HC region. For three of these families, absolute DNA values from nuclei of the hypocotyl + cotyledons region were established on individual embryos with varying cotyledon numbers. In all three families total DNA amount per nucleus decreased with increasing cotyledon number. A better understanding of differences observed in DNA content during germination, as well as in total DNA content per nuclei among different half-sib families of jack pine, may help in the identification of factors that influence growth and adaptation of this species.     

Cytogeography of the Phleum pratense group (Poaceae) in the Carpathians and Pannonia

Cytogeographical variability within the Phleum pratense group in the Carpathians and adjacent part of Pannonian lowland, based on 132 populations analysed by flow cytometry, is described. Only diploid and hexaploid plants were detected among 635 samples from the studied area. Diploids were found to be less frequent (127 plants, 20%) than hexaploids (508, 80%). With the exception of the single pure diploid population, diploids always co-occured with hexaploids (30 localities, 22.7%). The majority of populations (101, 76.5%) consisted of hexaploid plants. Most mixed populations occur in the Western Carpathians (26). In the Eastern Carpathians, mixed populations are much rarer, with three populations in Ukraine and one in Romania. In the Southern Carpathians, only hexaploids occur. The conventional taxonomic concept of the two species, diploid P. bertolonii and hexaploid P. pratense , was followed in spite of their sympatric occurence. Distribution maps based on chromosome number data from previous studies and on ploidy level estimates are given for both species in the studied area. The pattern of different distribution of the two taxa within the Carpathians is discussed.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 475–485.     

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β₂ integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β₂ integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.     

Desert ants Cataglyphis fortis use self-induced optic flow to measure distances travelled

While foraging, desert ants of the genus Cataglyphis use a vector navigation (route integration) system for homing. Any vector navigation system requires that the animal is able to evaluate the angles steered and the distances travelled. Here we investigate whether the ants acquire the latter information by monitoring self-induced optic flow. To answer this question, the animals were trained and tested within perspex channels in which patterns were presented underneath a transparent walking platform. The patterns could be moved at different velocities (up to > 0.5 the ant's walking speed) in the same or in the opposite direction relative to the direction in which the animal walked. Experimental manipulations of the optic flow influenced the ant's homing distances (Figs. 2 and 4). Distance estimation depends on the speed of self-induced image motion rather than on the contrast frequency, indicating that the motion sensitive mechanism involved is different from mechanisms mediating the optomotor response. Experiments in which the ants walked on a featureless floor, or in which they wore eye covers (Fig. 6), show that they are able also to use additional (probably kinesthetic) cues for assessing their travel distance. Hence, even though optic flow cues are not the only ones used by the ants, the experiments show that ants are obviously able to exploit such cues for estimation of travel distance.     

Is embryo abortion a post‐zygotic barrier to gene flow between Littorina ecotypes?

Genetic incompatibilities contribute to reproductive isolation between many diverging populations, but it is still unclear to what extent they play a role if divergence happens with gene flow. In contact zones between the "Crab" and "Wave" ecotypes of the snail Littorina saxatilis, divergent selection forms strong barriers to gene flow, while the role of post‐zygotic barriers due to selection against hybrids remains unclear. High embryo abortion rates in this species could indicate the presence of such barriers. Post‐zygotic barriers might include genetic incompatibilities (e.g. Dobzhansky–Muller incompatibilities) but also maladaptation, both expected to be most pronounced in contact zones. In addition, embryo abortion might reflect physiological stress on females and embryos independent of any genetic stress. We examined all embryos of >500 females sampled outside and inside contact zones of three populations in Sweden. Females' clutch size ranged from 0 to 1,011 embryos (mean 130 ± 123), and abortion rates varied between 0% and 100% (mean 12%). We described female genotypes by using a hybrid index based on hundreds of SNPs differentiated between ecotypes with which we characterized female genotypes. We also calculated female SNP heterozygosity and inversion karyotype. Clutch size did not vary with female hybrid index, and abortion rates were only weakly related to hybrid index in two sites but not at all in a third site. No additional variation in abortion rate was explained by female SNP heterozygosity, but increased female inversion heterozygosity added slightly to increased abortion. Our results show only weak and probably biologically insignificant post‐zygotic barriers contributing to ecotype divergence, and the high and variable abortion rates were marginally, if at all, explained by hybrid index of females.