Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein.

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands.

Nuclear expressed sequence Tag (NEST) analysis: a novel means to study transcription through amplification of nuclear RNA.

We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing.

Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome p450 gene superfamily.

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics.

Adapting the Biomek 2000 Laboratory Automation Workstation for printing DNA microarrays.

The Biomek 2000 Laboratory Automation Workstation is used for liquid handling and other repetitive operations in many laboratories. Since it has very good spatial positioning capabilities, we have modified this workstation to deliver samples at high densities onto microscope slides to produce DNA microarrays. The workstation tool, originally designed for bacterial colony replication, was adapted to carry special printing pins and was further modified to improve its positional accuracy.

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants.

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GALA regulation, the Z.

Genetic studies of the mouse mutations mahogany and mahoganoid.

The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain.

Green-fluorescent protein fusions for efficient characterization of nuclear targeting.

The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the beta-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments.

Evidence for enteroviral persistence in humans.

We have sought evidence of enteroviral persistence in humans. Eight individuals with chronic fatigue syndrome (CFS) were positive for enteroviral sequences, detected by PCR in two serum samples taken at least 5 months apart. The nucleotide sequence of the 5' non-translated region (bases 174-423) was determined for each amplicon.

Enteroviral polymerase chain reaction in the investigation of aseptic meningitis.

We used a nested polymerase chain reaction (nPCR) to seek evidence for enteroviruses in clinical samples from patients with symptoms of aseptic meningitis. When compared with conventional virus isolation methods on a total of 366 samples collected during 1994-1995, an increase in positivity from 6% to 27% was shown. The results indicate that nPCR would be a valuable aid to the laboratory diagnosis of enteroviral infections as it can detect those enteroviruses that cannot be identified by current isolation methods.

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.

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.

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.

Depth dose flattening of electron beams using a wire mesh bolus.

A common problem with low-energy electron beams (< 15 MeV) is their low surface dose when the incident electrons are monodirectional. This makes it difficult to deliver a uniform dose to tumor with any precision, limiting the clinical usefulness of such beams. A practical method is presented for greatly increasing the tissue depth enclosed by the 95% isodose region, while delivering the entire dose in a single uninterrupted treatment.

Comparison of coxsackie B neutralisation and enteroviral PCR in chronic fatigue patients.

Coxsackie B enteroviruses have been implicated repeatedly as agents associated with chronic fatigue syndrome (CFS). The objective of this study was to compare the serological evidence for the presence of Coxsackie B virus neutralising antibody, with the polymerase chain reaction (PCR) detecting a portion of the 5' nontranslated region (NTR) of the enterovirus genome. Serum samples from 100 chronic fatigue patients and from 100 healthy comparison patients were used in this study.

Coxsackie B virus infection and onset of childhood diabetes.

Infection with Coxsackie B viruses has been linked to insulin-dependent diabetes mellitus. Nine of 14 serum samples (64%) taken from children at the onset of diabetes were positive for enterovirus RNA by PCR. All of the children were under age six, and five were under age three.

Phylogenetic analysis of short enteroviral sequences from patients with chronic fatigue syndrome.

This study used phylogenetic analysis based on a region of the 5' non-translated region (5'NTR) of a variety of enteroviral sequences to compare sequences associated with chronic fatigue syndrome (CFS) and those from enteroviruses causing acute infections. Direct sequencing of PCR products was used to obtain the nucleic acid sequences from CFS patients. The inferred phylogenetic tree identified three groupings, one correlating with the diagnosis of CFS.

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.

Detection of enterovirus-specific RNA in serum: the relationship to chronic fatigue.

The serum of 88 chronic fatigue patients was screened for enteroviral specific sequences by polymerase chain reaction (PCR) assay. The PCR method used was "nested" PCR targetting the 5' nontranslated region of the enteroviral genome which yielded a final fragment length of 264 base pairs. Samples were obtained from patients during 1990-1991.