Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

First-trimester diagnosis of an unusual case of α-mannosidosis

Chorionic villi obtained in the first trimester from a pregnancy at risk for α-mannosidosis were shown to have reduced α-mannosidase (EC 3.2.1.24) activity. The pregnancy was terminated and subsequent enzyme studies of the fetal tissues were consistent with the diagnosis of α-mannosidosis. Like the enzyme in the child's fibroblast, α-mannosidase of the chorionic villi from a pregnancy at risk for α-mannosidosis was activated by high substrate concentration and by Zn^{2 +}, and displayed a K_m value two-fold higher than normal. Our results confirm that chorionic villi can be used for early prenatal diagnosis of α-mannosidosis.     

Prenatal diagnosis for dystrophia myotonica using the polymerase chain reaction

The polymerase chain reaction has been used to detect an abundant class of short repeat DNA families of the form (dC-dA)n.(dG-dT)n, known as microsatellites. These units are found throughout the human genome and have been characterized for several loci including APOC2 on chromosome 19ql2-ql3.2. The locus APOC2 is linked to the gene for dystrophia myotonica and a microsatellite within this locus was used to derive polymorphisms in a family to predict the inheritance of the disease. Chorionic villus sampling (CVS) was performed at 151/2 weeks' gestation. Following DNA extraction from the CVS material and parental blood samples, microsatellite analysis was carried out by the polymerase chain reaction.     

An approach to preimplantation diagnosis of β-thalassaemia

Using the polymerase chain reaction (PCR), it was possible to amplify a single copy fragment of the β-globin gene from 2–32 human embryonic cells obtained from arrested preimplantation embryos. For the detection of β-thalassaemia mutations, allele specific priming of the PCR using nested primers was employed using approximately 10 pg of DN A from individuals known to carry these mutations. This approach was successful in detecting the presence or absence of five Asian Indian β-thalassaemia mutations that were selected for this study. In spite of meticulous precautions against contamination, false-positive amplification was observed, a problem that will have to be overcome before this approach can be used in clinical practice.     

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: A multiparametric study involving transmission electron microscopy and fetal dna amplification

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free inter-villous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.     

镉诱导长江华溪蟹肝胰腺细胞凋亡研究

应用显微与亚显微技术和琼脂糖凝胶电泳方法,研究了不同浓度Cd2+(0、7.25、14.50、29.00、58.00和116.00mg·L-1)处理不同时间(24、48、72和96h)对长江华溪蟹(Sinopotamon yangtsekiense)肝胰腺细胞凋亡的影响.结果显示,29.00mg·L-1Cd2+处理48h,光镜下肝胰腺细胞凋亡主要表现为染色质不规则凝聚、固缩和边集;58.00mg·L-1Cd2+处理72h,细胞核碎裂,形成凋亡小体;116mg·L-1Cd2+处理96h,出现坏死细胞;电镜下48h和72h处理组肝胰腺细胞呈现典型的细胞凋亡特征,与光镜检测结果一致,具体表现为核边集、核膜折叠、核裂解形成凋亡小体.琼脂糖凝胶电泳图谱中48和72h处理组出现凋亡细胞所特有的DNA梯状条带,58.00mg·L-1Cd2+处理72h后条带尤为清晰.随着Cd2+浓度的增加和处理时间的延长,肝胰腺中凋亡细胞所占比例呈现先升后降的趋势,凋亡指数的变化表现出显著的剂量和时间效应关系.研究表明,镉能够诱导长江华溪蟹肝胰腺出现细胞凋亡.细胞凋亡的检测及凋亡指数的变化能够灵敏地反映出镉对水生动物的胁迫程度及毒性大小,可作为水生态环境镉污染的生物评估指标.     

Challenges to achieving a successful transition to a low carbon economy in South Africa: examples from poor urban communities

South Africa faces several development challenges including those linked to climate change. Energy usage in South Africa, for example, is already constrained because of a range of development challenges (the dependence on cheap coal as a heating source; energy availability; access; affordability of alternative energy sources; and a range of health impacts, including air pollution). Notwithstanding calls for a transition to a low carbon economy, there have been few, recent assessments in South Africa of the costs associated with such a transition, particularly the social and economic costs for the poor who use energy in a variety of ways. In this paper we focus on trying to unravel some of the complex energy-use behaviour including understanding what drives consumers in resource-poor areas to choose and use persistently risky energy options. Analysis of qualitative data in two township areas, Doornkop and Kwaguqa, shows that township households, whether electrified or not, continue to burn coal. In both study areas, an estimated 80% of electrified households burn coal for space heating and cooking and also make use of multiple fuel sources for a range of activities. Although the major obstacles preventing people from discontinuing domestic coal combustion are poverty, the ready availability and social acceptability of coal together with other social customs cannot be underrated. This paper therefore highlights some of the persistent challenges associated with sustainable energy transitions in South Africa including implications for improved mitigation and adaptation for the energy sector in wider climate change efforts.     

Vegetation changes in Conservation Reserve Program lands in interior Alaska

Over 14 million hectares of erosion prone cropland in the United States has been converted into grasslands through the Conservation Reserve Program (CRP) administered by the United States Department of Agriculture, however, studies of the effects of CRP enrollment on plant communities and subsequent plant succession are largely lacking. In Delta Junction, Alaska plant communities in CRP fields are transitioning from grasslands to shrub dominated plant communities, which are resulting in compliance problems with program regulations that state “fields must be maintained in a condition that permits easy conversion to cropland”. To determine plant succession and how previous land management and soils might influence the transition, we measured plant populations in 20 CRP fields throughout Delta Junction using modified-Whittaker plots. These data were combined with data on current management practices, previous farming history, soils, soil properties, diversity indices, and time since land was cleared and analyzed with nonmetric multidimensional scaling ordination to determine factors that influence plant succession. Time in the CRP was the only factor consistently influencing plant succession. As time in the CRP increased, the planted introduced grasses brome grass (Bromus inermis) and red fescue (Festuca rubra) and the native pteridophyte (Equisetum arvense) decreased, whereas a native grass (Calamigrostis canadensis), five native forb, two native shrub, and three native tree species increased. Plant diversity increased at a rate of more than 2 species per 1000 m² per year. Regression analyses of plant species and plant groups using time in the CRP as the dependent variable resulted in the identification of outlier CRP fields with significantly more or less than expected covers of vegetation. All fields with these outliers had reasonable explanations for the differences in cover that were unrelated to the overall rate of plant succession. Current management practices will result in incompliant fields and different management practices that result in woody vegetation control is key to maintaining CRP fields in compliance.