Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke: a pilot investigation.

BACKGROUND: Direct brain biopsy is rarely indicated during acute stroke. This study uses peripheral blood mononuclear cells (PBMCs) to determine whether a systemic gene expression profile could be demonstrated in patients with acute ischemic stroke. METHODS AND RESULTS: Using oligonucleotide microarrays, we compared the gene expression profile of an index cohort of 20 patients with confirmed ischemic stroke on neuroimaging studies with that of 20 referent subjects. Validation studies used quantitative real-time polymerase chain reaction to measure the levels of 9 upregulated genes in the index cohort, and an independent cohort of 9 patients and 10 referent subjects was prospectively studied to determine the accuracy of the Prediction Analysis for Microarrays list to classify stroke. After correction for multiple comparisons with the Bonferroni technique, 190 genes were significantly different between the stroke and referent groups. Broad classes of genes included white blood cell activation and differentiation (approximately 60%), genes associated with hypoxia and vascular repair, and genes potentially associated with an altered cerebral microenvironment. Real-time polymerase chain reaction confirmed increased mRNA expression in 9 of 9 upregulated stroke-associated genes in the index cohort. A panel of 22 genes derived from the Prediction Analysis for Microarrays algorithm in the index cohort classified stroke in the validation cohort with a sensitivity of 78% and a specificity of 80%. Control for the Framingham stroke risk score revealed only a partial dependence of the stroke gene expression profile in PBMCs on vascular risk. CONCLUSIONS: This study demonstrated an altered gene expression profile in PBMCs during acute ischemic stroke. Some genes with altered expression were consistent with an adaptive response to central nervous system ischemia.

Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector.

We describe the in vitro isolation and expansion of cells capable of forming neurosphere-like aggregates from human adult bone marrow. Cells within these passaged spheroids can differentiate into astrocytes, specific neuronal subtypes, and oligodendrocytes and have gene expression profiles similar to human fetal brain-derived neural stem cells. Genetically modified neural-competent bone marrow-derived cells efficiently migrate toward distant sites of brain injury and tumor in vivo, where they differentiate and express therapeutic transgenes when transplanted into the brains of mice. These studies suggest that adult bone marrow may serve as a large reservoir for autologous neural stem-like cells for future therapeutic strategies.

Development of a Position Sensitive Neutron Detector with High Efficiency and Energy Resolution for Use at High-Flux Beam Sources.

We are developing a high-efficiency neutron detector with 1 cm position resolution and coarse energy resolution for use at high-flux neutron source facilities currently proposed or under construction. The detector concept integrates a segmented (3)He ionization chamber with the position sensitive, charged particle collection methods of a MicroMegas detector. Neutron absorption on the helium produces protons and tritons that ionize the fill gas. The charge is amplified in the field region around a wire mesh and subsequently detected in current mode by wire strips mounted on a substrate. One module consisting of a high-voltage plate, a field-shaping high-voltage plate, a grid and wire strips defines a detection region. For 100 % efficiency, detector modules are consecutively placed along the beam axis. Analysis over several regions with alternating wire strip orientation provides a two-dimensional beam profile. By using (3)He, a 1/v absorption gas, each axial region captures neutrons of a different energy range, providing an energy-sensitive detection scheme especially useful at continuous beam sources.

Catabolism of sulfamate by Mycobacterium sp. CF1.

A bacterium able to utilize sulfamate as N-source for growth was isolated from soil and identified as a Mycobacterium sp. An apparently previously unrecorded enzyme, sulfamate hydrolase (EC 3.10.1.-), converts sulfamate to equimolar amounts of ammonia and sulfate. This enzyme was purified to homogeneity and had a Km for sulfamate of 26.36 +/- 4.01 mM. Its Specificity Constant value, 74 M(-1) s(-1), was low, indicating that it was not a particularly good catalyst for this reaction and it may be a hydrolase recruited to this role from some other reaction sequence. However, under equivalent conditions it showed no detectable action on the other sulfamates, cyclamate and sulfamoylbenzoate, or on urea or methylamine.

Expression, purification, crystallization and preliminary characterization of an HHED aldolase homologue from Escherichia coli K12.

An ORF designated b2245 (yfaU) in the Escherichia coli K12 genome sequence, identified as an HHED aldolase homologue, was cloned into the high-expression plasmid pT7-7 and overexpressed in E. coli B835(DE3). The enzyme was purified in three steps to 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Crystals suitable for structural studies were grown from solutions containing 0.4 M ammonium dihydrogen phosphate and grew to a maximum dimension of approximately 0.5 mm. Diffraction data to 1.7 A were collected using an in-house Cu Kalpha radiation source at 100 K. The crystals belong to space group C222(1), with unit-cell parameters a = 105.1, b = 136.6, c = 123.1 A. A 90% complete data set was collected to 1.78 A from a single native crystal using in-house facilities.

2-phenylethylamine catabolism by Escherichia coli K-12: gene organization and expression.

A gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map. It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction. The purified PAD acted almost equally well on phenylacetaldehyde, 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde. It had a subunit size of 54 kDa and its deduced amino acid sequence was approximately 40% identical to various eukaryotic and prokaryotic aldehyde dehydrogenases. A third gene encoding a positive regulatory protein required for expression of the amine oxidase and PAD genes was located next to the PAD gene. A gene previously located in this position was reported to encode a second amine oxidase but this was not confirmed. The nucleotide sequence from 1447 to 1450 kb on the E. coli K-12 physical map has been determined.