Metabolic diversity in epibiotic microflora associated with the Pompeii wormsAlvinella pompejana andA. caudata (Polychaetae: Annelida) from deep-sea hydrothermal vents

Specimens of alvinellid polychaetes (Alvinella pompejana Desbruyères and Laubier, 1980 andA. caudata Desbruyères and Laubier, 1986) and their tubes were sampled from deep-sea hydrothermal vents at 13°N from the manned submersible Nautile during the Hydronaut cruise (October to November 1987) on the East Pacific Rise. Samples were subjected to bacterial analysis aboard the mother ship Nadir to detect bacteria involved in the nitrogen and sulphur cycles, in non-specific heterotrophic processes, and displaying resistance to selected heavy metals. Cultures were incubated at different temperatures under atmospheric and in situ (250 atm) pressures. Bacterial growth was observed in enrichment cultures for most metabolic types screened. Heavy-metal-resistant bacteria were also detected in many samples. No filamentous bacterial form was observed in the cultures. The results demonstrate a high metabolic diversity in episymbiotic flora, indicating that the worm (A. pompejana orA. caudata), its tube and its epiflora represent a complex micro-ecosystem.     

Characterisation of HTO diffusion properties by an in situ tracer experiment in Opalinus clay at Mont Terri

A long-term single borehole diffusion experiment using tritiated water as tracer was carried out in Opalinus clay, an argillaceous rock formation that is accessible at the Mont Terri Underground Research Laboratory, situated in the Swiss Jura. The tracer was diluted in reconstituted formation water and introduced into a packed-off section of a borehole located in saturated rock. Pressure in this interval was maintained equal to the pore pressure of the surrounding rock in order to prevent any hydraulic gradient around the borehole and to avoid advective transport processes. The evolution of the tracer concentration in the injection system was monitored over time. After 1 year of diffusion, the claystone surrounding the interval was retrieved by overcoring the whole borehole and packer system, and by an adjacent oblique borehole. Compressed air was used as drilling fluid to reduce rock disturbances. The recovered overcore was sampled along profiles perpendicular to the borehole wall with a view to determining the tracer-concentration profiles in the rock. To avoid further evaporation of tritiated water, subsamples were immediately transferred into polyethylene bottles and disaggregated by adding a known amount of tracer-free water. Fifteen profiles were determined and showed a decreasing tracer concentration with distance into the rock. The pore-water contents were constant along those profiles, confirming that only very little water was lost during overcoring operations. The evolution of tritium-tracer concentration in the injection system over time and in situ profiles were interpreted with a 3-D numerical simulation of the experiment. That allowed for the identification of the transport parameters (orthotropic diffusion tensor and porosity) by minimising the relative quadratic error between the experimental and simulated data. The fitting is good and the results are consistent with data obtained on drill-core samples. The result of tritiated water is discussed regarding (1) the potential effect of mechanical and/or chemical disturbances around the injection borehole and (2) the specific behaviour of tritiated water.     

The occurrence and need for conservation of metallophytes on mine wastes in Europe

Metalliferous substrates are important in the distribution of various plants such asArmeria maritima, Minuartia verna, Thlaspi alpestre, Silene maritima andCochlearia officinalis, in parts of the United Kingdom. Metalliferous substrates are similarly important for a slightly different assemblage of metallophytes in the Aachen-Liège orefield in Belgium and Germany. The significance of metalliferous substrates in the distribution of the metallophytes varies from orefield to orefield and this is investigated. Certain sites, by virtue of their environmental and species diversity, are outstanding as habitats for the metallophytes and some of these are described. Observations on the importance and problems of the conservation of the metallophyte habitats are included.     

Chalk and limestone quarries as wildlife habitats

Natural recolonisation by volunteer species of chalk and limestone quarries has led to the development of attractive and species-rich communities in many parts of the United Kingdom. These communities have a basic similarity in floristic composition wherever they occur, but they also show a great deal of individual variation resulting from differences in the location, history and variability of the quarry itself and in the nature of the surrounding habitats. The interest of old quarries may be enhanced by the presence of individually rare or local species and especially by their refuge status in relation to the loss of semi-natural calcareous grassland in the district. It is suggested that some sites play an important role in wildlife conservation and that this factor should be considered in any programme of land reclamation. Natural regeneration is very slow compared with the rate of land use in modern quarrying but the development of ecological techniques may make it possible to rehabilitate derelict and barren sites for amenity and conservation purposes using older quarries as models.     

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

Prenatal diagnosis of tetrasomy 12p by in situ hybridization: Varying levels of mosaicism in different fetal tissues

Prenatal diagnosis of tetrasomy 12p is complicated by the discrimination of the 12p isochromosome from the duplication 21q as well as the level of mosaicism demonstrated in the particular tissue sampled. In this disease, a high percentage of chromosomally abnormal cells are generally found in fibroblastic cells, but lymphocyte karyotypes from the same individual may be normal. We report on the pregnancy of a 37-year-old female who presented to our centre at 16 weeks' gestation for genetic amniocentesis. Sonography of the fetus revealed dextrocardia and diaphragmatic hernia. Chromosome analysis of amniocytes demonstrated mosaicism of a 47,XY,+i(12p) line in 80 per cent of cells and a normal male line (20 per cent), consistent with the Pallister-Killian syndrome. Following termination, a 220 g male fetus of 18 weeks was examined. A flattened nose and low-set ears were noted. In situ hybridization with a chromosome 12 centromeric probe in lymphocytes and skin cells unequivocally confirmed the karyotype and showed the presence of a single centromere in the abnormal chromosome, suggesting a true isochromosome. Chromosome analysis of various fetal tissues was performed and the following percentages of abnormal cells were found: skin 100 per cent, chorion 50 per cent, placenta 30 per cent, and blood 80 per cent. The high frequency of tetrasomic cells in fetal blood at this early gestational age is noteworthy, since most reports of this syndrome show a very low percentage of abnormal cells postnatally.     

Trisomy 12 mosaicism in amniocytes and dysmorphic child despite normal chromosomes in fetal blood sample

Trisomy 12 mosaicism (44 per cent) was detected prenatally in cultured amniocytes. A cordocentesis was performed to confirm the result. Only normal cells were found in the fetal blood sample. The fetus was estimated to be at a low risk of having a chromosomal abnormality and the pregnancy continued. Eight days after birth, a congenital heart defect was detected in the child. Several dysmorphic features were also evident. Further karyotyping of different tissues revealed normal blood and urinary cells but trisomic cells in the placenta (100 per cent) and in skin fibroblasts (25 per cent). The child died at 5 weeks of age. In this case, the fetal blood sample failed to reveal the real chromosome constitution of the fetus.     

Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.     

Prenatal identification of i(YP) by molecular cytogenetic analysis

An i(Yp) is a rare marker chromosome. We present a case of de novo 46,X,i(Yp) detected prenatally in an amniotic fluid specimen. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes identified the marker chromosome as i(Yp). Comparative genomic hybridization (CGH) studies further confirmed the diagnosis. Upon pregnancy termination, external examination of the fetus revealed a generally well-developed male fetus with slight facial dysmorphism and prominent rocker-bottom feet. The molecular cytogenetic data in this case proved very useful in genetic counselling and served as a good example illustrating the important role of molecular techniques for accurate identification of marker chromosomes.     

Cytogenetic analysis of 1375 amniotic fluid specimens from pregnancies with gestational age less than 14 weeks

Our laboratory has received 1375 early amniotic fluid (EA) specimens during the past 5-year period for cytogenetics analysis. The gestational ages of the EA specimens were less than 14 weeks as estimated by ultrasound. The average volume of specimen received was 16 ml. Specimens were typically received in two collection tubes and cultured in Chang A and in supplemented MEM media using the in situ technique. Of the 1375 EA specimens received, 1356 were successfully cultured and yielded results. Abnormal results were found in 67 (4.9 per cent) of the cases. Nineteen specimens (1.4 per cent) failed to yield a result. The mean turn-around time (TAT) for all EA specimens was 8.28 days. In 1991, the average TAT for the EA specimens was 8.00 days compared with a TAT of 6.59 days for all specimens received over 14 weeks gestational age. The number of EA specimens received has increased from 1.5 per month in 1986 to 57 per month in 1991. In summary, our experience with EA specimens for cytogenetic analysis has demonstrated that the success rate is 98.6 per cent and that an increasing number of obstetricians are performing early amniocentesis as they seek to provide their patients with earlier results and an alternative to chorionic villus sampling.