Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN^t) gene. An oligonucleotide was designed to be specific to the SMN^t nucleotidic sequence with exonic mismatch G (for SMN^t)→A (for SMN^c) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN^t gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.     

Anreicherung atmosphärischer Depositionen von Metallen und Stickstoff in Moosen Mecklenburg-Vorpommerns von 1990 bis 2005

Background, aim and scope Since 1990 the UN ECE Heavy Metals in Mosses Surveys provide data inventories of the atmospheric heavy metal bioaccumulation across Europe. In the survey 2005 the nitrogen accumulation was measured for the first time in most of the participating countries. In Germany, the surveys were conducted in close cooperation of the relevant authorities of both the Federal Republic and the sixteen states. Therefore, statistical evaluations of the moss survey data with regard to the whole German territory and single federal states are of interest. This article concentrates on Mecklenburg-Western Pomerania, dealing with the mapping of the spatiotemporal trends of metal accumulation from 1990 to 2005, the spatial patterns of nitrogen accumulation in 2005, and the spatial variability of bioaccumulation due to characteristics of the sampling sites and their surroundings. Materials and methods The bioaccumulation of up to 40 trace elements in mosses was determined according to a Europe-wide harmonised methodology. The according experimental protocol regulates the selection of sampling sites and moss species, the chemical analysis and quality control and the classification of the measured values for mapping spatial patterns. In Mecklenburg-Western Pomerania all sampling sites were described with regard to topographical and ecological characteristics and several criteria to be fulfilled according to the guideline. Together with the measurements this metadata was combined with other information regarding land use in the surroundings of the sampling sites in the WebGIS MossMet. The spatial structure of the metal bioaccumulation was analysed and modelled by variogram analyses and then mapped by applying different kriging techniques. Furthermore, multi metal indices (MMI) were derived for both the sampling sites and raster maps with help of percentile statistics: The MMI_1990–2005 was calculated for As, Cd, Cr, Cu, Fe, Ni, Pb, Ti, V and Zn. The statistical association of the metal bioaccumulation, site specific characteristics as well as information on land use and emissions was analysed by bivariate nonparametric correlation analysis, contingency tables and Chi-square Automatic Interaction Detection (CHAID). Results The results of the quality controlled chemical analyses show a significant decrease of the metal bioaccumulation in Germany from 1990 to 2000 for all elements. However, in Mecklenburg-Western Pomerania the concentrations of Cr and Zn are even significantly higher than those found in 1990. From 2000 to 2005?a further non-significant increase can be stated for As, Cu, Ni and Ti. The concentrations of Cd and Pb decreased significantly throughout all four surveys. The MMI illustrates the temporal trend of the metal bioaccumulation as a whole: After a significant decrease from 1990 to 2000 it increased significantly till 2005. The N concentration in mosses in Mecklenburg-Western Pomerania reaches from 1.3 to 2.3?% in dry mass and is negatively correlated with the forest ratio in the surroundings of the moss sampling sites and to the same degree positively correlated with the area ratio of agricultural land uses. Except for Cd, Pb and Sb all metal concentrations in the mosses are negatively correlated with the forest ratio around the sampling sites. With the exception of Cr all metal concentrations are further negatively correlated with the precipitation sums of the accumulation periods. Only the Cu and Zn concentrations show no or rather a negative correlation with the tree height whereas all other elements exhibit positive correlations. Furthermore, all elements except Cr are significantly associated to the sampled moss species, the growth pattern and the frequency of occurrence of the mosses at the respective sampling sites. Exemplified for Cu multivariate correlations were furthermore detected by CHAID. It could be shown that the frequency of the mosses, the sampled moss species, the distance to motorways and the distance to the Baltic Sea are the statistically most significant boundary conditions of the Cu concentrations in the mosses sampled in Mecklenburg-Western Pomerania in 2005. No correlations were found between the modelled total depositions and the concentrations of Cd, Hg and Pb in the mosses at p < 0.1. For Pb in 1995?r is 0.52 at p = 0.012, for the other surveys no correlations at p < 0.05 could be found. Discussion The increase of the Cr bioaccumulation from 2000 till 2005 is particularly pronounced in Mecklenburg-Western Pomerania. This trend is confirmed with regional differences in the national average as well as in other participating countries like in Switzerland. Deposition measurements did not register this trend. In contrast to the UNECE area, the federal territory and several federal states no correlations were found between the modelled total depositions and the metal concentrations in the mosses. Conclusions The fact that no correlations were found between the modelled total depositions and the element concentrations in the mosses may be caused by the low spatial resolution (50?×?50?km) of the EMEP data. The moss surveys contribute to the heavy metal and the multi-component-model of CLRTAP because they prove on different spatial scales how air pollution control influences the accumulation of emitted substances in environmental subjects of protection like vegetation. In contrast to deposition measurement networks the moss monitoring identified a trend reversal in Mecklenburg-Western Pomerania: The continuous decrease of the metal bioaccumulation in mosses from 1990 till 2000 has changed to an increase of several metals between 2000 and 2005. This increase is significant for Cr and Zn. Recommendations and perspectives The spatial resolution of the EMEP deposition data should be enhanced based on the Europe-wide regression relationship between the element concentrations in the deposition and in the mosses. For regional studies the existing but so far not useable deposition measurement data of the federal states should be made available. It should further be investigated what caused the increase of the Cr concentrations above the level of 1990 – perhaps emissions or biogenic effects as a consequence of simultaneously increased nitrogen loads? The Heavy Metals in Mosses Surveys are a positive example for environmental monitoring activities reaching across three spatial and administrative levels: regional (e.?g. federal state or natural landscape), nation wide (e.?g. Germany) and continental (e.?g. Europe). In Germany the harmonised and quality controlled moss data are made available via a WebGIS portal. Therefore the moss data may easily be accessed for environmental monitoring purposes and the control of environmental political actions. Hence, the continuous task of environmental monitoring can be met and carried on in the future. It should further be considered to expand the moss monitoring on the survey of persistent organic pollutants and apply it in human-biomonitoring. This would facilitate the acquisition of indoor and outdoor pollution with the same receptor.     

Bacteriolytic Bacillus species isolated from brackish waters of the Southern Baltic Sea

The present study examines antagonistic relationships between different microorganisms inhabiting brackish water and includes a systematic screening for bacteriolytic prokaryotes in the brackish waters of the Southern Baltic Sea (Nordruegenscher Bodden) sampled in July/August 2009. Ten of the 35 marine bacteriolytic isolates belong to the genus Bacillus. Five isolates (B. pumilus, B. subtilis, B. megaterium, B. licheniformis) lysed living microbial cells such as the bacteria Arthrobacter citreus, Arthrobacter protophormiae, Micrococcus luteus, Pseudomonas putida and the yeast Trichosporon mucoides. These and other bacteria (Aeromonas sp., Bacillus subtilis, Chromobacterium violaceum, Citrobacter freundii, Enterobacter aerogenes, Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens) were also lysed as autoclaved and pasteurized cells on agar plates. One isolate of Bacillus pumilus showed a distinct bacteriolysis activity against pasteurized cells of A. citreus in liquid culture. Our results suggest that Bacillus species may play a role as opportunistic predators in the marine microbial food web.     

Area-based assessment of extinction risk

Underpinning the International Union for Conservation of Nature (IUCN) Red List is the assessment of extinction risk as determined by the size and degree of loss of populations. The IUCN system lists a species as Critically Endangered, Endangered, or Vulnerable if its population size declines 80%, 50%, or 30% within a given time frame. However, effective implementation of the system faces substantial challenges and uncertainty because geographic scale data on population size and long-term dynamics are scarce. I develop a model to quantify extinction risk using a measure based on a species' distribution, a much more readily obtained quantity. The model calculates the loss of the area of occupancy that is equivalent to the loss of a given proportion of a population. It is a very simple yet general model that has no free parameters and is independent of scale. The model predicted well the distributions of 302 tree species at a local scale and the distributions of 348 species of North American land birds. This area-based model provides a solution to the long-standing problem for IUCN assessments of lack of data on population sizes, and thus it will contribute to facilitating the quantification of extinction risk worldwide.     

利用酵母菌处理高浓度味精废水的连续小试

利用批量实验对从高浓度味精废水中筛选出的一组酵母菌混合菌群进行了脱氢酶活性(DHA)测试.结果表明用该菌群对COD、硫酸根和氨氮均接近20000mg/L的味精离交尾液进行处理时,其DHA值在前36h高于对离交尾液稀释液的处理,说明高浓度氨氮和硫酸盐对酵母菌活性的影响不显著.利用接种了该酵母菌群的生物接触氧化反应器对味精废水进行的连续小试处理结果表明,COD容积负荷2.0～14.3kg/(m³·d)时,COD去除率稳定在80%以上;适当地补充磷源有利于维持稳定的处理效果对出水pH值和C     

The political significance of local agenda 21: The early stages of some European experience

Sustainable development, as it emerged in Agenda 21 from the Rio conference in 1992, will only be meaningful when it touches the lives of ordinary people; then it becomes a reality. Local Agenda 21 (LA21) seeks to achieve that objective. This article assesses the origins of LA21, reviews its social and political significance, and considers its prospects in the light of case study experience emerging from the UK, Germany and Norway, focusing on the role of local government as a major stakeholder in Agenda 21 (A21). The range of response to LA21 has proved to be varied. A successful transformation to a more sustainable world will require visionary political leadership, supportive administrations, networks of experience sharing, alliances with non-governmental organisations and local industry, and effective community mobilisation. All of that, in turn, requires equally supportive economic and social policy backing from national governments. This article will indicate that, not surprisingly, it is the domestic political context, nationally and locally, which in the main determines the speed and nature of response to LA21, now and in the future. By understanding and being aware of these contexts, factors impeding progress towards LA21 may be addressed, whilst at the same time retaining the diversity of response which is an essential part of local sustainability.     

Physiologische Anpassungen eines marinen insekts. II. Die Eigenschaften von schwimmenden und absinkenden Eigelegen

The gelatinous egg-masses of Clunio marinus (Diptera; Chironomidae) consist of a tube of jelly containing up to 175 eggs. The swimming egg-mass of the Atlantic populations (Race A) and the sinking egg-mass of the Baltic populations (Race B) have a higher density than the sea water, on which the female spawns; salinity is not important to the swimming or sinking of the egg-masses. Density and sinking velocity of gelatinous egg-masses from Atlantic midges are significantly higher than those from Baltic midges if the eggs are laid on water of the same salinity. The density of the eggs and the quantitative relation of eggs to jelly are the same in both races. The jelly of the egg-masses from the Baltic Sea race swells more than that from the Atlantic Sea race (increase of volume by absorption of water = 3-fold and 2.4-fold, respectively). During swelling the jelly alters from a trough-like shape into a tube-like from which closes around the eggs. If the surface tension of the spawning medium is lowered by use of detergents, the A-spawn will sink. A- and B-spawns occupy different positions at an oil-water interface. Interference contrast microscopy revealed that the A-jelly forms a well-defined outer line in relationship to the surrounding aqueous medium; this line is missing in the B-jelly. The morphological fine surface structures, as seen by the scanning electron microscope, do not explain the different properties of the A- and B-spawn. Extracts and hydrolysates of jelly material were separated by thin-layer chromatography. The results indicate that the spawn jelly of C. marinus consists of a polysaccharide-protein-complex, similar to the jellies of other invertebrates. It is rather probable that the chemical structure or the components of the jellies from A- and B-populations are not exactly the same; this would explain the different properties described. The ecological significance of swimming and sinking gelatinous egg-masses and the taxonomical position of both midge populations are discussed.     

Chemisch-analytische Charakterisierung technischen tierischen Fettes aus einer Tierkörperbeseitigungsanstalt

Aims and Scope

German rendering plants produce 300,000 tons of animal fat per year. Until recently, these raw products have been in wide use as nutritional additive for cattle and other animals fodder but now they have been banned due to their assumed role in BSE infection. Therefore, alternative fields of usage are required. In this context the Deutsche Bundesstiftung Umwelt (DBU) is sponsoring a joint research project which deals with the production and testing of cooling lubricants based on animal fat esters.

Methods

In a first step, characteristics and quality of the animal fat were analyzed and monitored for a whole year in order to identify e.g. seasonal variations. The investigations covered the following fat specific and trace analytical parameters: total contamination, sulfates ash, water content, peroxide number, iodine value, kinematical viscosity, neutralisation number (free fatty acids), fatty acid spectra, elements / heavy metals (Al, B, Ca, Cd, Cr, Cu, Fe, K, Mo, Mn, Na, Ni, P, Pb, S, Sn, V, W and Zn), polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans.

Results

Valuable hints on the design of the technical process of fatty acid methylesters production were gained by regarding the fat specific parameters. For example, filtration and dewatering of the fats proved necessary. Small variations of iodine value, viscosity and fatty acid spectrum pointed at a quite constant composition of the raw material in the course of the year. Clues to the degree of hydrolysis and decay of the fat were given by the share of free fatty acids. The saturated fatty acids, most interesting for the production of the cooling lubricants, were present in the fatty acid spectra in the rage of 36.4% to 54.6%. Trace analytical investigations concerning inorganic and organic pollutants proved a low basic contamination of the technical animal fats.

Outlook

Next step will be to present the pilot plant for the production of the cooling lubricants and further analytical results in context with the production process.     

Role of nitric oxide in the genotoxic response to chronic microcystin-LR exposure in human-hamster hybrid cells

Microcystin-LR (MC-LR) is the most abundant and toxic microcystin congener and has been classified as a potential human carcinogen (Group 2B) by the International Agency for Research on Cancer. However, the mechanisms underlying the genotoxic effects of MC-LR during chronic exposure are still poorly understood. In the present study, human-hamster hybrid (A_L) cells were exposed to MC-LR for varying lengths of time to investigate the role of nitrogen radicals in MC-LR-induced genotoxicity. The mutagenic potential at the CD59 locus was more than 2-fold higher (p < 0.01) in A_L cells exposed to a cytotoxic concentration (1 μmol/L) of MC-LR for 30 days than in untreated control cells, which was consistent with the formation of micronucleus. MC-LR caused a dose-dependent increase in nitric oxide (NO) production in treated cells. Moreover, this was blocked by concurrent treatment with the NO synthase inhibitor N^G-methyl-_L-arginine (L-NMMA), which suppressed MC-LRinduced mutations as well. The survival of mitochondrial DNA-depleted (ρ⁰) A_L cells was markedly decreased by MC-LR treatment compared to that in A_L cells, while the CD59 mutant fraction was unaltered. These results provided clear evidence that the genotoxicity associated with chronic MC-LR exposure in mammalian cells was mediated by NO and might be considered as a basis for the development of therapeutics that prevent carcinogenesis.     

Schnelle Elementanalytik zur Abschätzung von Dioxinbelastungen: Ein neues Werkzeug zur Untersuchung von Brandschäden?

A car and a railway coach with an InterCityExpress interior were combusted in a tunnel. The deposition of pollutants could be detected by passive sampling devices over the distance of several hundred meters. Cotton passive collectors were analyzed for the elements Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sb, Sn, V and Zn. A comparison with analytical results of PCDD/F and PAH (US EPA) showed a close similarity between the concentration profiles of some elements and the organic pollutants. As one outcome, Cd, Cu, Pb, Sb, Sn and Zn could be used as indicators for the estimation of surface contamination with PCDD/F or PAH in the event of such fires, thereby providing the advantages of faster results and lower costs.