End-sequencing and characterization of silkworm () bacterial artificial chromosome libraries-1

<p><b>Copyright information:</b></p><p>Taken from "End-sequencing and characterization of silkworm () bacterial artificial chromosome libraries"</p><p>http://www.biomedcentral.com/1471-2164/8/314</p><p>BMC Genomics 2007;8():314-314.</p><p>Published online 7 Sep 2007</p><p>PMCID:PMC2014780.</p><p></p>io of alignment length to BES length, as a match. BES+ denotes a BES with a single match, and BES++ a BES with multiple matches. BES- denotes a BES without a match, and BES-- a BES without a "raw BLAST hit.

3D endothelial network formation in the depth direction in EC and EC+EPC models.

<p>Representative fluorescent images of a confluent EC layer on the surface of collagen gel (<b>A</b>, <b>C</b>) and EC networks in the collagen gel (<b>B</b>, <b>D</b>) are shown. (<b>E</b>) The depth of the EC and EC+EPC models was quantitatively analyzed. The depth of 3D endothelial networks in the EC+EPC model was deeper than that in the EC model. However, the difference was not remarkable according to the concentration of bFGF. Data are presented as the means ± SD (n = 6–26; *<i>p</i> < 0.001 vs. EC model with 10 ng/mL bFGF; ^†<i>p</i> < 0.001 vs. EC model with 30 ng/mL bFGF). Scale bar, 100 μm.</p

EC network formation and growth of EPCs in EC and EC+EPC models.

<p>In the presence of bFGF, ECs formed a confluent monolayer on the gel (<b>A</b>) and invaded into the underlying gel (<b>B</b>). After 5 days in culture, ECs formed longer EC networks (<b>C</b>, arrowheads). EPCs embedded between double collagen gel layers gradually proliferated in culture (<b>D</b>: day 1, E: day 3, F: day 5). Scale bar, 100 μm.</p

EC network formation in the EC and EC+EPC models.

<p>After 5 days in culture, bright-field images indicate EC network formation in the EC (<b>A</b>) and EC+EPC models (D) with 10 ng/mL bFGF. Using these images, EC networks were marked in red (<b>B</b>, <b>E</b>) and skeletonized (<b>C</b>, <b>F</b>), and the length and number of the EC networks were measured. The EC+EPC model significantly increased both the length (<b>G</b>) and number (<b>H</b>) of EC networks compared to the EC model at days 3–5. Data are presented as the means ± SD (n = 27–51; *<i>p</i> < 0.05 vs. EC model). Scale bar, 300 μm.</p

VEGF concentration in 3D endothelial network models.

<p>CM from the EC and EC+EPC models and collagen gel from the EC+EPC model were collected and analyzed by ELISA to compare VEGF concentrations. The concentration of VEGF in collagen gel was significantly more than that in CM from the EC and EC+EPC models. Data are presented as the means ± SD (n = 3–8; *<i>p</i> < 0.03 versus CM from EC and EC+EPC models).</p

Three-dimensional endothelial network models.

<p>(<b>A</b>) In the EC model, ECs were seeded onto collagen gel. The EC monoculture served as a control. (<b>B</b>) In the EC+EPC model, EPCs were sandwiched with double layers of collagen gel. ECs were then cultured on the top of the upper collagen gel layer. In each model, some ECs in a confluent monolayer invaded the underlying collagen gel with the addition of bFGF (Sprout) and formed 3D endothelial networks in culture (3D network).</p

Effect of supplemented bFGF on EC network formation.

<p>The EC and EC+EPC models with 30 ng/mL bFGF significantly increased both the length (<b>A</b>) and number (<b>B</b>) of EC networks compared to 10 ng/mL bFGF at day 5. Furthermore, the EC+EPC model with 30 ng/mL bFGF enhanced the length (<b>A</b>) and number (<b>B</b>) of EC networks to a greater extent than the EC model with 30 ng/mL bFGF. Data are presented as the means ± SD (n = 8–51; *<i>p</i> < 0.03 vs. EC model with 10 ng/mL bFGF; ^†<i>p</i> < 0.001 vs. EC+EPC model with 10 ng/mL bFGF; **<i>p</i> < 0.001 vs. EC model with 30 ng/mL bFGF).</p

Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

<div><p>We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.</p></div

Identification of Key Uric Acid Synthesis Pathway in a Unique Mutant Silkworm <i>Bombyx mori</i> Model of Parkinson’s Disease

<div><p>Plasma uric acid (UA) levels decrease following clinical progression and stage development of Parkinson’s disease (PD). However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm <i>Bombyx mori</i> (<i>B.mori</i>) <i>op</i>. Initially, we investigated the causality of the phenotypic “<i>op”</i> by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in <i>B. mori</i>. We found that the mRNA levels of genes in this pathway were significantly lower in <i>B. mori op</i> mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of <i>B.mori</i> tyrosine hydroxylase (TH) and DJ-1 mRNA were significantly lower in the brain of <i>B. mori op</i> mutants. UA content was significantly lower in the <i>B. mori op</i> mutant tissues and hemolymph. The possibility that the <i>B. mori op</i> mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the <i>B. mori op</i> mutant. In the case of <i>B. mori op</i> mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.</p></div

Schematic diagram of the DNA decontamination of Phi29 DNA polymerase.

<p>GST-fused Phi29 DNA polymerase (GST-ø29pol) is expressed in <i>E. coli</i> and affinity purified by glutathione sepharose 4B resin (GS4B) of the cell lysate after polyethylenimine (PEI) precipitation to remove the host DNA. Then, GST-ø29pol is treated with ethidium monoazide (EMA) and irradiated with visible light (VL) to reduce both ssDNA and dsDNA, which may be captured in GST-ø29pol. After washing away the free EMA, the remaining oligonucleotides are reduced by the endogenous 3′-5′ exonuclease activity of the polymerase. Finally, DNA-free Phi29 DNA polymerase is separated from GS4B by digestion with PreScission protease and collected.</p