A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

Ammonia production and kinetic properties of glutamate dehydrogenase in the sipinculid Phascolosoma arcuatum exposed to anoxia

The amounts of total NH ₄ ⁺ detected in the external media in which Phascolosoma arcuatum had been exposed to various periods of anoxia were significantly greater than those in which the worms were exposed to normoxia for a similar period. The increased NH ₄ ⁺ production by P. arcuatum during anoxic exposure was unlikely to be due to an increased catabolism of adenine nucleotides or urea. In contrast, there were significant decreases in the concentrations of several free amino acids in the coelomic plasma and body tissues of individuals during the 48 h of anoxic exposure. The amount of NH ₄ ⁺ produced by the anoxic P. arcuatum could be accounted for by the decreases in the concentrations of aspartate or glycine. Increases in the catabolism of free amino acids (FAA), leading to the increased production of NH ₄ ⁺ , in P. arcuatum during anoxia were supported by the detection of significant changes in the kinetic properties of glutamate dehydrogenase (GDH), in the deaminating direction, from worms exposed to anoxia for 48 h. The apparent increase in the affinity of GDH from the anoxic worm to glutamate would bring about a greater deaminating activity at physiological concentrations of ths substrate. P. arcuatum used in these experiments were collected from the mangrove swamp at Mandai, Singapore between 1990 and 1993.     

THE INFLUENCE OF SETTINGS AND ENVIRONMENTAL EVENTS ON METAMOTIVATIONAL STATE AND AROUSAL: FURTHER TESTS OF REVERSAL THEORY ON ASIAN SAMPLES

The two studies reported further test reversal theory hypotheses concerning human psychological responses to different types of settings and environmental events on an Asian sample. In study 1, individuals in four settings representing different combinations of metamotivational state and arousal level (telic and paratelic/low arousal; telic and paratelic/high arousal) completed the telic state measure (TSM; Svebak & Murgatroyd, 1985). Significant differences between participants' mean scores across the four settings were obtained for TSM items serious–playful, planning–spontaneous, felt arousal, preferred arousal, effort and arousal discrepancy. In study 2 different environmental events in the same setting (changes in reading tasks), used in a crossover design, were used to induce reversals in individuals' operative metamotivational states. The results obtained from TSM scores pretask 1, between tasks and posttask 2. indicated significant time-of-testing interaction effects on the TSM serious–playful, planning–spontaneous and effort items. In addition, a main effect for TSM felt arousal was found. These results showed that reversals in metamotivational states were induced by the environmental event and that felt arousal scores increased across the three administrations of the TSM.     

New birds on the block: new neighbors increase defensive costs for territorial male willow ptarmigan

Previous researchers have hypothesized that site-faithful animals may benefit from the presence of familiar neighbors. This study compares the relative costs of territorial defense against new and former neighbors by male willow ptarmigan (Lagopus lagopus). Territorial defense against new neighbors appeared to require a greater expenditure of both time and effort than did defense against former neighbors. Territorial males that had several new neighbors spent a higher proportion of time fighting than did males with fewer new neighbors, and males with both new and former neighbors spent a greater amount of time fighting with their new neighbors, on average, than with their former neighbors. In addition, fights with new neighbors occurred relatively more frequently and were longer than fights with former neighbors. Finally, fights involving new neighbors tended to escalate to higher levels than fights between former neighbors. Reduced defensive costs for site-faithful, territorial males may provide one explanation for the tendency of males to be more site-faithful than females in many species.     

The State of the Mammal Community of Boreal Forest Ecosystems in the Vicinity of a Nickel-Smelting Plant

The effect of industrial air pollution on natural small mammal populations has been studied in the northern taiga subzone of the boreal forest zone. The results of long-term monitoring have been used to demonstrate the possibility of predicting changes in the main population and community characteristics of the animal species studied as dependent on the degree of anthropogenic impact.     

Structure of a Mediterranean cryptobenthic fish community and its relationships with habitat characteristics

In the last 10 years, several studies have been carried out on the fish fauna of the Ustica Island marine reserve, yet no investigation was specifically addressed to the cryptobenthic fish assemblage. The first task of this study, conducted along the shallow rocky reefs of Ustica, was to determine the species composition, diversity and relative density of the resident cryptobenthic fishes. Furthermore, we aimed to assess the effects of some macro- and microscale habitat characteristics on the distribution pattern of fishes. In particular, the effect of predator density was indirectly evaluated by comparing density data collected within and outside the integral reserve zone. Overall, 20 species belonging to Blenniidae, Gobiidae, Tripterygiidae, Scorpaenidae and Gobiesocidae were recorded. Gobius bucchichi, Scorpaena maderensis, Tripterygion delaisi, T. melanurus and T. tripteronotus were the numerically dominant and most common species. The effects of zone (i.e. of predator density), bottom type and depth on species richness, diversity and evenness were not significant. A greater total fish density was observed on stones compared with rocky cliff and plateau, but only in the shallowest depth range. At level of single species, G. bucchichi was more abundant inside than outside the integral reserve, but only on stones and at 0–2 m depth range. Density of G. bucchichi was generally higher on stones than on rocky cliffs or plateau and between 0 and 5 m depth, although these differences were not always significant. T. delaisi was conversely more abundant in the deepest stratum (7–10 m). Canonical analyses demonstrated that bottom type and depth influenced significantly the fish assemblage structure. The observed differences in the assemblage structure relied mainly upon the dominant species. T. tripteronotus was mainly associated with rocky plateau and the intermediate depth range (3–5 m), whereas S. maderensis, T. melanurus and Lipophrys trigloides inhabited preferentially the rocky cliffs. At microscale level, the habitat choice of the investigated species was almost entirely based on whether the substrate was either vegetated or composed of bare rock. T. delaisi and T. tripteronotus were associated with substrata covered by algae, whilst G. bucchichi, S. maderensis and T. melanurus preferred bare rock bottoms. In some species, the electivity indices for the less abundant type of cover, measured at different spatial scale, changed accordingly. For instance, the smaller the size of the sampled area, the higher was the intensity of the association between G. bucchichi and Anemonia viridis.     

Toxicological Assessment of Wastewater and Runoff from the Baikal Pulp and Paper Mill

Russian Journal of Ecology -