Notes: Genomic DNA from Arabidopsis thaliana was isolated using the Wizard® Genomic DNA Purification Kit. The extracted DNA was used to amplify the 3´ UTR of AteIF5A-2, A. thaliana translation initiation factor 5A, or the entire gene for creating transgenic plants. Leaf discs from wild-type Arabidopsis were infected with Pseudomonas syringae pv tomato DC3000, a virulent strain regulated by AteIF5A-2, using a syringe. After 24 and 72 hours, 0.4cm leaf discs were fixed, labeled using the DeadEnd™ Fluorometric TUNEL System and stained for AteIF5A-2 protein. (3979)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: The authors designed a high-throughput gene-expression screen to identify compounds that induce a neuroblastoma gene signature in BE(2)-C cells. The screen used Valproic Acid (VPA), an inhibitor of histone deacetylase, as an enhancer. The top hit from the screen was all-trans retinoic acid (ATRA). The authors proposed that ATRA + VPA would reduce cell viability and might induce apoptosis by inducing genes associated with terminal differentiation. They used the CellTiter-Glo^® Luminescent Cell Viability Assay in a 96-well format to assess the effects of ATRA and a variety of inhibitors of histone deacetylase on BE(2)-C cell viability. (3931)

Notes: Lynch syndrome (LS) is due to mutations in at least four DNA mismatch repair (MMR) genes. There are two tumor characteristics that can be screened for in an attempt to identify patients with CRC who are most likely to have LS. These characteristics are microsatellite instability (MSI) and loss of one or two of the MMR proteins in the tumor compared to normal tissue. MSI testing and IHC for the four mismatch repair proteins was performed on 500 tumors from unselected patients with CRC. If either MSI or IHC was abnormal, complete mutation analysis for the mismatch repair genes was performed. GoTaq Master Mix (Cat.# M7122, Cat.# M7132) (4111)

Notes: The authors of this study describe a reporter gene system that allows researchers to create one genetic construct that can be used for a variety of studies including imaging and protein immobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters. (3925)

Notes: The authors examined the heritability of primary angle closure using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4042)

Notes: The authors examined the heritability of certain eye characteristics that may be associated with glaucoma using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4043)

Notes: The authors examined strains of Salmonella enterica serovar Typhi isolated from typhoid cases originating in or around Indonesia or from travelers returning from Indonesia to examine if serovar Typhi from this area has a greater level of genetic diversity compared to other countries. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit, diluted to 4 ng/µl and used in locus-specific PCR genotyping. (3980)

Notes: Thees authors searched for small-molecule inhibitors of galactokinase (GALK). They developed an HTS assay using the Kinase-Glo^® Assay System. The HTS assay used 15 μM ATP and α-D-galactose as the substrate and was performed in 384-well plates against 50,000 small molecules. Two hundred compounds were identified from the primary screen as GALK inhibitors. (3934)

Notes: The authors describe an HTS assay to screen for inhibitors of measles virus infection. The CytoTox 96^® Non-Radioactive Cytotoxicity Assay was used to assess toxicity of all confirmed hit compounds from the primary screen. A luminescent assay was used to assess cell-to-cell fusion in the presence candidate compounds that appeared to inhibit entry. Effector cells that expressed the T7 polymerase and measles virus H and F envelope proteins were overlaid on target cells expressing firefly luciferase under the control of a T7 polymerase promoter. Inhibition of fusion should reduce luciferase expression compared to a positive fusion control. Ten of the eleven compounds tested caused a dose-dependent reduction in luciferase expression, suggesting they block viral entry into cells. Luminescence was detected using the Bright-Glo™ Assay System. (3933)

Notes: In this study of the frequency of human papilloma virus (HPV) in patients with oral squamous cell carcinoma (OSCC), the MagneSil^® Genomic, Fixed Tissue System was used to isolate DNA from formalin-fixed paraffin-embedded tissue samples from primary tumors and matched samples. Five microliters of genomic DNA was amplified using PCR Master Mix and primers for both a 110 bp fragment of human ß-globin gene and HPV genotype. After PCR, the product was analyzed on an 8% nondenaturing polyacrylamide gel and stained with AgNO₃. (3938)

Notes: This paper demonstrates use of a calpain assay in a cell-based format. (Calpain-Glo™ Assay). (3941)

Notes: RBCK1 is a RING-IBR (ring in between ring fingers) protein previously shown to have transcriptional activity and to bind protein Kinase Cβ. This paper demonstrates that RBCK1 also possesses ubiquitin ligase E3 activity. Both FLAG-tag and HaloTag® labeled proteins were used to demonstrate this activity. To demonstrate the interaction between RBCK1 and ubiquitinated proteins, HaloTag®-ubiquitin and FLAG-RBCK1 were coexpressed in HEK293 cells in the presence or absence of a proteasome inhibitor. The anti-FLAG immunoprecipitates isolated from these cells were analyzed by SDS-PAGE and Western blotting using anti-HaloTag® and anti-FLAG antibodies and self-ubiquitination of RBCK1 was demonstrated. RBCK2, a splice variant of RBCK1 lacking the RING domain showed no self ubiquitination activity but was demonstrated to interact with RBCK1 in vivo and in vitro, and to inhibit the self-ubiquitination activity of RBCK1 in a FLAG-tag assay. Pulse-chase experiments, using HEK293 cells expressing HaloTag®-RBCK1 with or without RBCK2 and treated with HaloTag®-TMR ligand, were used to show that the half-life of RBCK1 was extended by overexpression of RBCK2. (3874)

Notes: In this review, the author discusses the advantages of the Promega MSI Analysis System, including the use of mononucleotide markers that are monomorphic (i.e., almost all individuals are homozygous for the same common allele) to simplify data analysis. In addition, the inclusion of pentanucleotide repeat markers ensures that the normal and tumor tissue are from the same individual. The author shows representative data generated using MSI Analysis System, Version 1.1. (4118)

Notes: The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures. (3953)

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4^oC but slides stored at -20^oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70^oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

Notes: Trivalent arsenic (As³⁺) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As³⁺-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR. (3766)

Notes: In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting. (3986)

Notes: The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75^NTR) using GoTaq® Green Master Mix. RT-PCR results were confirmed by Western blot analysis. (3884)

Notes: In this study, the Maxwell^® 16 Instrument was used to purify genomic DNA from blood samples. The extracted DNA was amplified by PCR for subsequent genotype analysis. (3902)

Notes: The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors. (3886)

Notes: The authors characterized five open-reading frames flanking the alcohol-inducible alkane monooxygenase (BMO) structural gene of Pseudomonas butanovora. Strains with mutated bmoR, which encodes a putative transcriptional regulator, or bmoG, which encodes a putative chaperonin, were created by gene inactivation. The bmoR gene was amplified and cloned into the pGEM^®-T Vector for disruption with a kanamycin cassette. The two termini of the bmoG gene were amplified separately, ligated to the kanamycin cassette and cloned into the pGEM^®-T Easy Vector. Plasmids encoding the disrupted genes were transformed into Pseudomonas butanovora by electroporation. (3893)

Notes: The authors examined the role of specific amino acids within the second extracellular loop (ECL2) of C-C chemokine receptor 5 (CCR5), which is a coreceptor for human immunodeficiency virus type 1, in HIV-1-mediated cell fusion. The authors substituted single and multiple amino acids in ECL2 by site-directed mutagenesis, transfected MAGI cells with these CCR5 mutations, then monitored the effect of the mutations on the magnitude of cell-cell fusion. Their cell fusion assay used the pLTR-LucE plasmid, which encodes firefly luciferase and is transcriptionally activated by the HIV-1 tat protein. When tat⁺ cells fuse with pLTR-LucE⁺ cells, transcription of the luciferase gene is activated, and the level of luminescence becomes a measure of cell fusion. To perform the cell fusion assay, the researchers combined tat⁺, env⁺ 293T cells and pLTR-LucE⁺, CCR5⁺ MAGI cells for 6 hours, then measured luciferase activity using the Bright-Glo™ Luciferase Assay System. This assay allowed the researchers to identify amino acids within ECL2 that are involved in HIV-1-mediated cell fusion. (3971)

Notes: The asparagine-glycine-arginine (NGR) sequence can be converted to isoaspartate-glycine-arginine (isoDGR) by asparagine deamidation, and isoDRG can interact with αvβ3, an integrin involved in angiogenesis. The authors tested the hypothesis that a cyclic isoDGR peptide (CisoDGRC) can be used to target delivery of drugs or nanoparticles to tumor neovasculature. The authors produced tumor necrosis factor α tagged with NGR (NGR-TNF) and subjected it to conditions favoring asparagine deamidation to produce CisoDGRC-TNF. The isoaspartate content was determined using the IsoQuant® Isoaspartate Detection Kit. (3900)

Notes: The ORFs of Japanese eel follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) were ligated into the pSI Mammalian Expression Vector. Fifty nanograms of this construct or the pSI Vector was cotransfected with 0.5ng of pRL-null Vector and 1µg or a cAMP-responsive firefly luciferase reporter vector into COS cells seeded in 60mm dishes. The next day, the cells were trypsinized, replated into 96-well plates and allowed to recover for 1 day. The transfected cells were serum-starved then treated with hormones six hours before being lysed and enzymatic activity measured with the Dual-Luciferase® Reporter Assay System. (3988)