Digital extractor: analysis of digital differential display output

Digital Extractor is a program for the high-throughput processing of data sets derived from digital differential display-based comparisons of EST libraries. These comparisons can be utilized to identify discrete subsets of genes whose expression is restricted to distinct tissue types. The program facilitates these investigations by permitting parallel annotation of genes identified as being differentially expressed.     

Rostral Ventrolateral Medulla C1 Neurons and Cardiovascular Regulation

1. The rostral ventrolateral medulla (RVLM) is essential for the normal control of arterial pressure. This region of the brainstem is heterogeneous, and contains spinally projecting neurons that contain phenylethanolamine-N-methyltransferase (C1 neurons) and non-C1 neurons.2. The availability of a toxin, a dopamine--hydroxylase antibody conjugated to saporin, that can selectively destroy C1 neurons within the RVLM allows for the study of cardiovascular regulation in rats following depletion of the C1 neuronal population.3. C1 neurons are not essential for the maintenance of resting blood pressure in anesthetized rats, but do contribute to many cardiovascular responses mediated through the RVLM.4. The depressor response elicited by clonidine injected into the RVLM is dependent upon C1 neurons.5. Studies in rats with selective toxin-induced destruction of RVLM C1 neurons demonstrate that C1 neurons contribute importantly to cardiovascular regulation.     

Database resources of the National Center for Biotechnology Information

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap'99, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri-tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov.     

Mediators of alkalosis-induced relaxation of piglet pulmonary veins

Pulmonary venous constriction leads to significant pulmonary hypertension and increased edema formation in several models using newborns. Although alkalosis is widely used in treating neonatal and pediatric pulmonary hypertension, its effects on pulmonary venous tone have not previously been directly measured. This study sought to determine whether alkalosis caused pulmonary venous relaxation and, if so, to identify the mediator(s) involved. Pulmonary venous rings (500-microm external diameter) were isolated from 1-wk-old piglets and precontracted with the thromboxane mimetic U-46619. Responses to hypocapnic alkalosis were then measured under control conditions after inhibition of endothelium-derived modulator activity or K(+) channels. In control rings, alkalosis caused a 34.4 +/- 4.8% decrease in the U-46619-induced contraction. This relaxation was significantly blunted in rings without functional endothelium and in rings treated with nitric oxide synthase or guanylate cyclase inhibitors. However, neither cyclooxygenase inhibition nor voltage-dependent, calcium-dependent, or ATP-dependent K(+)-channel inhibitors altered alkalosis-induced relaxation. These data suggest that alkalosis caused significant dilation of piglet pulmonary veins that was mediated by the nitric oxide-cGMP pathway.     

DMPO-Alkyl radical spin trapping: an in situ radiolysis steady-state ESR study

Short-lived free radicals formed in the reaction of 11 substrates and radiolytically produced hydroxyl radicals were trapped successfully with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in dilute aqueous solution. The in situ radiolysis steady-state ESR spectra of the spin adducts were analyzed to determine accurate ESR parameters for these spin adducts in a uniform environment. Parent alkyl radicals include methyl, ethyl, 1-propyl and 2-propyl (1-methylethyl). Hydroxyalkyl parent radicals were hydroxymethyl, hydroxyethyl, 2-hydroxy-2-propyl (1-methyl-1-hydroxyethyl), 1-hydroxypropyl and 2-hydroxy-2-methylpropyl. Carboxyl radical (carbon dioxide anion, formate radical) and sulfite anion radical were the sigma radicals studied. The DMPO spin adduct of 1-propyl was identified for the first time. For most spin adducts, g factors were also determined for the first time. In DMPO spin adducts of hydroxyalkyl radicals, nitrogen and C(2)-proton hyperfine coupling constants are smaller than those of alkyl radical adducts; the hydroxyalkyl spin adducts possess larger g values than their unsubstituted counterparts. These changes are ascribed to the spread of pi conjugation to include the hydroxyl group. Strong evidence of spin addend-aminoxyl group interaction can be seen in the asymmetrical line shapes in the hydroxyethyl and the hydroxypropyl spin adducts.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells

Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis.     

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.