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Biomedical subjects

D W Galbraith

Publications and source records attributed to D W Galbraith.

13 recordsLinked to original sources
PubMed1996

Automated particle classification based on digital acquisition and analysis of flow cytometric pulse waveforms.

In flow cytometry, the typical use of front-end analog processing limits the pulse waveform features that can be measured to pulse integral, height, and width. Direct digitizing of the waveforms provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, and Fourier properties. In this work, we have first demonstrated that the Fourier properties of the pulse can be employed usefully for discrimination between different types of cells that otherwise cannot be classified by using only time-domain features of the pulse. We then implemented and evaluated automatic procedures for cell classification based on neural networks. We established that neural networks could provide an efficient means of classification of cell types without the need for user interaction. The neural networks were also employed in an innovative manner for analysis of the digital flow cytometric data without feature extraction. The performance of the neural networks was compared with that of a more conventional means of classification, the K-means clustering algorithm. Neural networks can be realized in hardware, and this, in addition to their highly parallel architecture, makes them an important potential part of real-time analysis systems. These results are discussed in terms of the design of a real-time digital data acquisition system for flow cytometry.

Animals↗
PubMed1996

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

Base Sequence↗
PubMed1995

Flow cytometric analysis using digital signal processing.

Current commercial flow cytometers employ analog circuits to produce the feature values of the pulse waveforms that result from particle analysis. The use of analog pulse processing limits the features that can be measured to pulse integral, pulse height, and pulse width, and a large amount of potentially relevant information about the shape of the pulse waveform is lost. Direct digitizing of the waveform provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, as well as the Fourier properties of the pulse. Here we describe a digital pulse waveform processing system that is compatible both with a commercial flow cytometer, and with a readily available computational platform. The performance of the digital and analog systems were compared through analysis of synthetic waveforms, and the waveforms produced by standard fluorescence microspheres and biological particles. The digital waveform processing system was found to be accurate and flexible, and the value of several of its unique attributes was demonstrated using biological cells. A protocol was designed in which digital pulse processing provided a means for the quantitative monitoring of the optical alignment of the flow cytometer. It was shown that digital pulse processing could be used to discriminate between particle classes which produce feature values indistinguishable through analog pulse processing, and to discriminate accurately single cells from doublets and larger aggregates.

Animals↗
PubMed1995

Green-fluorescent protein as a new vital marker in plant cells.

The green-fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat-shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single-cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co-factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence-activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co-transfection, transformation, protein trafficking and localization, protein-protein interaction, cell separation and purification, and cell lineage in higher plants.

Animals↗
PubMed1990

Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

Base Sequence↗
PubMed1988

Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts.

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.

Cell Separation↗
PubMed1988

Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

Antibodies, Monoclonal↗
PubMed1987

Factors governing the flow cytometric analysis and sorting of large biological particles.

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.

Cell Separation↗
PubMed1977

The isolation of plasma membrane from protoplasts of soybean suspension cultures.

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Cell Fractionation↗