Biomedical subjects

Lee Smith

Publications and source records attributed to Lee Smith.

10 recordsLinked to original sources

PubMed2006

Spreading of neutrophils: from activation to migration.

Neutrophils rely on rapid changes in morphology to ward off invaders. Time-resolved dynamics of spreading human neutrophils after activation by the chemoattractant fMLF (formyl methionyl leucyl phenylalanine) was observed by RICM (reflection interference contrast microscopy). An image-processing algorithm was developed to identify the changes in the overall cell shape and the zones of close contact with the substrate. We show that in the case of neutrophils, cell spreading immediately after exposure of fMLF is anisotropic and directional. The dependence of spreading area, A, of the cell as a function of time, t, shows several distinct regimes, each of which can be fitted as power laws (A ~ t(b)). The different spreading regimes correspond to distinct values of the exponent b and are related to the adhesion state of the cell. Treatment with cytochalasin-B eliminated the anisotropy in the spreading.

Actins↗

PubMed2006

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct.

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.

Adaptor Proteins, Signal Transducing↗

PubMed2005

SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data.

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.

Algorithms↗

PubMed2003

DNA microarrays and development.

Gene expression is a central concept in molecular biology: its control, frequently exquisite in terms of cell specificity and timing, forms part of our explanation of most biological processes. The importance of the control of gene expression for developmental biologists is made obvious by just considering the nature of their discipline. Development is the term we use to describe the coordination in time and space of numerous cellular activities such as mitosis, migration, differentiation and apoptosis. Understanding the role of genes in these processes thus necessitates the use of methods to determine patterns of transcription during development with a high degree of sensitivity and specificity, conventionally by in situ hybridization. However, there is a widespread conviction amongst biologists that the description of gene expression patterns is of no immediate functional relevance: definitive functional data are the exclusive prerogative of biochemistry and genetics. In this review of recent applications of DNA microarray technology by developmental biologists, we suggest that genome-wide expression profiling has met with some resistance owing to such preconceived ideas about the status of gene expression pattern descriptions and, in particular, the format in which these are delivered by microarrays.

Animals↗

PubMed2003

Single primer amplification (SPA) of cDNA for microarray expression analysis.

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.

Animals↗

PubMed2003

Candidate testis-determining gene, Maestro (Mro), encodes a novel HEAT repeat protein.

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell-dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data.

Amino Acid Sequence↗

PubMed2003

Acute lower gastroenteric bleeding retrospective analysis (the ALGEBRA study): an analysis of the triage, management and outcomes of patients with acute lower gastrointestinal bleeding.

Many algorithms have been developed for patients with acute lower gastrointestinal hemorrhage (ALGIH). Their clinical usefulness is not readily apparent. It is important first to observe patterns in admission, triage, and management to formulate hypotheses as to how outcomes might be affected. We reviewed patient charts with the diagnosis of gastrointestinal hemorrhage from June 1998 to January 2001. Patients with ALGIH were entered into a database. We defined patients as having ALGIH if presentation included melena or hematochezia. Patients with hematemesis, bloody nasogastric aspirate, or occult fecal blood were excluded. Observations were made on 420 patients. Seventy-six per cent of patients were admitted to the medical service. Lower endoscopy was the first diagnostic method in 33 per cent. Medical management comprised 52 per cent of first management strategies. Surgeons used angiography (3% vs 1%) or surgery (25% vs 5%) more than other services. Fourteen per cent of patients managed with endoscopy, 16 per cent medically, 17 per cent with surgery, and 67 per cent with interventional radiology required two or more subsequent packed red blood cell transfusions. Mean admission Acute Physiology and Chronic Health Evaluation II score was 9.2 whereas that for those with mortality was 13.5. We conclude that the construction of a database will allow for formation and testing of hypotheses in managing ALGIH.

APACHE↗

PubMed2002

Fibroblast growth factors (FGFs) in the cochlear nucleus of the adult mouse following acoustic overstimulation.

To see if fibroblast growth factors (FGFs) might function in the central changes following auditory overstimulation we tracked immunostaining in the cochlear nucleus of adult mice with monoclonal antibodies to FGFs (FGF-1, FGF-2) and FGF receptor. After exposure nearly all outer hair cells died, while inner hair cell and fiber loss were restricted to a region midway along the cochlear spiral. FGFs staining in the cochlear nucleus appeared in hypertrophied astrocytes in the regions of nerve fiber degeneration only. For normal-sized astrocytes there was an increase in the number stained and the intensity of staining across all frequency domains, but not in neurons. The increases were modest at 3-7 days, pronounced at 14 days, modest again by 30 days, and back to control levels by 60 days. FGF receptor staining of neurons occurred equally in all mice, exposed or not. The findings suggest that astrocytes play a role in the central responses to acoustic overstimulation and cochlear damage, involving FGFs, possibly regulating the activity of intrinsic neurons or signaling axonal growth. Not limited to regions of cochlear nerve fiber and inner hair cell loss, the changes in FGFs may represent a reaction to outer hair cell damage which spreads broadly across the central pathways.

Animals↗

PubMed2002

Sexually dimorphic expression of Gata-2 during mouse gonad development.

We report that Gata-2 is expressed in a sexually dimorphic fashion during mouse gonadogenesis. Gata-2 transcripts accumulate rapidly in the fetal ovary from 11.5 days post coitum (dpc) onwards, but are not detected in the fetal testis throughout the period studied (10.5-15.5 dpc). Ovarian expression of Gata-2 ceases by 15.5 dpc. Examination of ovaries from embryos homozygous for the extreme allele of c-kit(W(e)) (Nature, 335, 88; Cell, 55, 185) demonstrates that ovarian Gata-2 expression is dependent upon the presence of germ cells. Comparative in situ hybridisation using the germ cell marker Oct4 (EMBO J., 8, 2543) indicates that Gata-2 transcripts are restricted to the germ cell lineage at 13.5 dpc.

Animals↗

PubMed2002

Targeting microparticles to select tissue via radiation-induced upregulation of endothelial cell adhesion molecules.

PURPOSE: Certain endothelial cell adhesion molecules are up regulated in tissue that has been irradiated for therapeutic purposes. This up-regulation of adhesion molecules provides a potential avenue for targeting drugs to select tissues. METHODS: Microspheres were coated with a mAb to ICAM-1 and the level of adhesion of the anti-ICAM-1 microspheres to irradiated tissue in vitro and in vivo was quantified. RESULTS: Under in vitro flow conditions, the number of adherent microspheres on irradiated HUVEC was 4.8 +/- 0.9 times that of control; the adhesion of anti-ICAM-1 microspheres on irradiated HUVEC could be enhanced by more than 170% in the presence of RBC (20% hematocrit) in the medium. In vivo in a rat cranial window model, the number of adherent anti-ICAM-1 microspheres in locally irradiated cerebral tissue was 8 and 13 times that of IgG microspheres at 24 h and 48 h post-irradiation, respectively and returned to baseline 7 days post-irradiation. In locally irradiated animals, the number of adhering microspheres in unirradiated tissue remained at the basal level. CONCLUSIONS: Radiation-induced up-regulation of endothelial cell adhesion molecules may be exploited to target drugs and/or genes to select segments of the endothelium.

Animals↗