Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Higher level relationships of Apiales (Apiaceae and Araliaceae) based on phylogenetic analysis of rbcL sequences

The two families of the order Apiales (Apiaceae and Araliaceae) represent a classic example of the difficulty in understanding evolutionary relationships between tropical-temperate family pairs. In Apiales, this problem is further compounded by phylogenetic confusion at almost every taxonomic level, including ordinal, interfamilial, and infrafamilial, due largely to difficulties in understanding trends in morphological evolution. Phylogenetic analyses of rbcL sequences were employed to resolve relationships at the ordinal and familial levels. The results of the ordinal analysis confirm the placement of Apiales in an expanded subclass Asteridae as the sister group to Pittosporaceae, and refute the traditional alliance of Apiales with Cornales and Rosidae. This study has also resolved relationships of a number of enigmatic genera, suggesting, for example, that Melanophylla, Aralidium, Griselinia, and Toricellia are close relatives of Apiales. Clarification of phylogenetic relationships has concomitantly provided insights into trends of morphological evolution, and suggests that the ancestral apialean taxon was probably bicarpellate, simple-leaved, woody, and paleotropical. Phylogenetic analysis at the family level suggests that apiaceous subfamily Hydrocotyloideae, often envisioned as an intermediate group between Apiaceae and Araliaceae, is polyphyletic, with some hydrocotyloids closely allied with Araliaceae rather than Apiaceae. With the exception of some hydrocotyloids, Apiaceae appear to be monophyletic. The relationship between Apiaceae and Araliaceae remains problematic. Although the shortest rbcL trees suggest that Apiaceae are derived from within a paraphyletic Araliaceae, this result is only weakly supported.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzyme-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

A model and lexicon for pollen fate

As pollination biology undergoes unprecedented growth as a discipline, confusion in the use of terms has become increasingly common. The need for a flexible yet unambiguous terminology has become urgent. As an example we discuss how the term “pollination efficiency” is used differently by 18 studies, and “pollinator effectiveness” by seven others. Here we present flowcharts of two general models of pollination systems (biotic and abiotic) that trace all the events from pollen production to development of seed or fruit, and we develop a lexicon for the quantities of pollen, processes of transfers (to a vector, to a stigma), and ratios of quantities that are of interest in studies of pollination and mating systems. An appendix includes a glossary of the definitions we suggest.