Notes: A key component in the pathogenesis of Alzheimer's disease is cerebral accumulation of amyloid-beta protein (Aβ). Aβ is produced by proteolysis of amyloid-β-protein precursor (APP) by ß- and gamma-secretases, thus these enzymes are considered important drug targets for Alzheimer's disease. Existing assays for assessing inhibition of alpha-, beta- and gamma-secretases include HPLC or ELISA assays that are cumbersome, expensive and not well-suited to high-throughput screening. The authors developed a luciferase reporter system to identify new molecules that inhibit APP processing. They then successfully interfaced this sensitive, specific and quantitative assay with a high-throughput screen, useful for identifying both inhibitors and stimulators of APP processing. (3775)

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM^®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM^®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

Notes: HepG2 cells were plated in 96-well microdilution plates in triplicate. Twelve hours after plating, the cells were treated with serial dilutions of various microtubule-stabilizing drugs. Following a 48 hour incubation, the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay was used to determine cell survival. The cells were incubated for 4 hours with the CellTiter 96^® and absorbance was read using an ELISA plate reader. Survival curves were generated based on comparing the absorbance ratios of the drug-treated cells to control cells. (3303)

Notes: Mouse adenovirus type 1 (MAV-1) was detected in DNA extracted from the lungs of mice after PCR amplification of the E1A region of MAV-1. For these assays, 80ng of total DNA was added to a 20µl PCR reaction containing 0.5 units of GoTaq^® DNA Polymerase, 4µl of 5X GoTaq^® Buffer, dNTPs and primers for MAV-1 E1A. The amplified products were separated on a 1.8% agarose gel and stained with ethidium bromide. (3381)

Notes: To monitor the effects of targeted degradation of micro RNA (miRNA) on Drosophila embryos, the authors cloned the full-length 3'UTRs of the proapoptotic factors hid and reaper into the psiCHECK™-2 Vector. The constructs were injected as plasmids (1µg/ml) mixed with 400µM sense and antisense miR-2 2'O-methyl oligoribonucleotides in early embryos. The total volume injected was equal to 5% of egg volume. After 10 hours development, the embryos were washed and lysed under agitation using 60µl lysis buffer and shaken at 750 rpm at room temperature for 30 minutes. The resulting lysate was cleared by centrifugation and three aliquots were tested using the Dual-Luciferase^® Reporter Assay System. The Renilla-to-firefly luciferase ratios from three to five independent replicates were averaged and normalized to the value of the miR-6 sense control, the most severe apoptotic phenotype when targeted for depletion. Statistical significance was assessed using the t test. (3292)

Notes: A transposase protein (Tnp) open reading frame (amino acids 1 to 345) was amplified from the Drosophila mauritiana Mos1 mariner transposable element using GoTaq^® DNA polymerase. The Tnp open reading frame was then cloned with into the pGEM^®-T Easy Vector and sequenced before further subloning. Tnp protein was purified and used in gel-shift assays to demonstrate Tnp binding regions in the Mos1 transposable element. (3344)

Notes: This study investigated the signaling mechanisms involved in 17β-estradiol- (E2) and tamoxifen-induced release of neurotrophic factors from rat cortical astrocyte cultures. The TGFβ1, TGFβ2, BDNF and GDNF E_max^® ImmunoAssay Systems were used to measure the release of these neurotrophic factors from astrocyte cultures treated with E2 or tamoxifen. Both E2 and tamoxifen induced the release of TGFβ1 and TGFβ2, but did not stimulate release of BDNF or GDNF. The PI3K inhibitors LY294002 and wortmanin, and the Akt inhibitor blocked TGFβ release, suggesting the involvement of the PI3K/Akt signaling pathway. The MAPK kinase inhibitors PD98059 and U0126 had no effect. E2 was found to induce transient Akt ^Ser473 phosphorylation, and this effect was blocked by LY294002 pretreatment, demonstrating a role for PI3K in the observed E2-mediated Akt phosphorylation. (3479)

Notes: The Wizard^® SV 96 Plasmid Purification System was used to purify plasmid DNA on the Beckman Coulter Biomek^® NX Laboratory Automation Workstation. Plasmid DNA quantity and quality were assessed by spectrophotometry, restriction digestion and PCR. The Wizard^® SV 96 Plasmid DNA Purification System was also used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening. (3300)

Notes: The host gene E3L is required for vaccinia virus infection and has anti-apoptosis activity. The authors examine the ability of E3L to regulate transcription of several genes related to apoptosis, immune response and viral pathogenesis. IL-6, NF-AT, p53, NF-κB, Ap-1 and cAMP response elements were cloned upstream of a TATA box and the firefly luciferase reporter gene. Renilla luciferase (pRL-null Vector) was used to normalize for transfection efficiency. Luciferase activities were measured using the Dual Luciferase^® Reporter Assay System. The authors also show that the Z-DNA binding region of E3L is important for transcriptional regulation. HeLa cells were transfected with an expression vectors expressing full-length E3L, E3L with a deletion of the Z-DNA binding domain or E3L with point mutations in residues important for Z-DNA binding. The AccessQuick™ RT-PCR System was used to quantitate IL-6, NF-AT and p53 mRNAs; β-actin was used as a control for RNA input. (3452)

Notes: In early stage of Lyme disease in humans, the primary immune response is the production of antibodies to outer surface protein C (OspC). In previous work, the authors demonstrate that the antibody response is specific to the 50 amino acids nearest to the OspC C-terminus. In this study, the authors used smaller OspC fragments of 7 and 16 amino acids to more specifically locate the epitope. Proteins consisting of the final 7 C-terminal amino acids (C7) or 16 C-terminal amino acids (C16) were expressed as biotinylated proteins in E. coli. To purify C7 and C16, the E. coli cultures were sonicated and passed over columns of SoftLink™ Soft Release Avidin Resin at a rate of 0.5ml/minute at 4°C. The captured proteins were analyzed by SDS-PAGE and Western blot analysis using anti-OspC antibodies to confirm protein purity. An affinity column for OspC antibodies was generated by passing biotinylated C7 or C16 over a column of TetraLink™ Tetrameric Avidin Resin, which irreversibly binds biotinylated proteins. Human sera were passed over this column, and the flowthrough fractions were tested in an ELISA to determine the extent to which the OspC antibodies were adsorbed to C7 or C16 fragments. (3663)

Notes: An open reading frame encoding a putative DNA polymerase, SpPol4, was amplified from S. pombe genomic DNA by PCR. The resulting PCR product was cloned into the pGEM^®-T Easy Vector, and the sequence was verified. Based on sequence analysis, the authors hypothesize that SpPol4 has deoxyribose phosphate (dRP) lyase activity, suggesting that the enzyme plays a role in base excision repair. The authors perform a dRP lyase activity assay with an oligonucleotide substrate labeled using [³²P]ddATP and terminal deoxynucleotidyl transferase. (3465)

Notes: Production of extracellular proteins in E. Carotovora subspecies that are critical for the development of soft-rotting disease of plants, is controlled by quorum-sensing signals, plant signals and assorted transcriptional factors and post-transcriptional regulators. Of these, post-transcriptional regulation by the RsmA-RsmB RNA pair is critical. RsmA is a small RNA-binding protein that promotes decay of RNA. RsmB specifies an untranslated regulatory RNA that binds RsmA and neutralizes its regulatory effect. Many of the transcription factors and QS signals known to regulate extracellular protein production actually act via these post-transcriptional regulators. ExpR, the putative AHL (N-acetyl homoserine lactone) receptor of E. carotovora subspecies carotovora, activates transcription of rmsA and AHL prevents this activation. The authors generated PCR fragments using primers for rsmA71 and rsmA153 and then used the Wizard(R) SV Gel and PCR Clean-Up System (Cat.# A9281) to purify PCR-generated DNA fragments used in gel mobility shift assays. Band shift assays revealed that ExpR is a DNA-binding protein and that its DNA-binding property is modified by AHL. In addition they showed that RsmA overproduction is responsible for inhibition of extracellular protein. (3572)

Notes: The Kinase-Glo^® Luminescent Kinase Assay was used to screen inhibitors of glycogen synthase kinase-3 beta (GSK-3β). Reactions were performed in the presence of 25µM substrate, 1µM ATP, 50 mM Hepes, (pH 7.5), 1mM EDTA, 1mM EGTA, and 15mM magnesium acetate. Purified glycogen synthase kinase-3β was obtained from Upstate (catalog# 14-306). The substrate used in the assays was a synthesized prephosphorylated polypeptide. Inhibitors in DMSO vehicle were added to the reactions so that the final DMSO concentration did not exceed 1%. Reactions were incubated at 30°C for 30 minutes. Results were read on a FLUOstar POLARstar Optima (BMG Labtech) multimode reader. (3387)

Notes: To examine the influence of cyclin E overexpression on gene expression in breast cancer, the breast cancer cell line MDA-MB-468 was stably transfected with a cyclin E-GFP fusion construct or GFP alone. RNA from two of these clones was isolated and pooled prior to reverse transcription and labeling. The microarrays were filtered and used in GoMiner analysis. Attachment assays were also performed with the stable cell lines. In these assays, the cells were allowed to adhere to 96-well plates for 1 hour, washed with PBS and then incubated with the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay for 1.5 hours. Absorbance was used to measure the number of attached cells. (3388)

Notes: In this study, the role of Cyclophilin-D (CypD) in the mitochondrial permeability transition (mPT) response was investigated using CypD-deficient mice. The CellTiter™ Blue Cell Viability Assay was used to measure the viability of mouse embryonic fibroblasts (MEF) and hepatocytes isolated from normal and CypD-deficient mice after exposure to various apoptotic stimuli and H₂O₂. (3398)

Notes: To examine the role of cyclin-dependent kinase 11 in Fas-induced apoptosis, the authors studied caspase-3 activation in A375 (human melanoma) cells after inducing cell death via Fas. Twenty-four hours after seeding 1 ×10⁴ cells per well in a 96-well plate, 0.5µg/ml anti-Fas antibody was added and the cells incubated 3–72 hours. Activation was determined by adding 100µl Caspase-Glo® 3/7 reagent to each well, incubating for 1 hour and measuring luminescence using a Sirius Luminometer (Berthold Detection System). (3315)

Notes: The presence or absence of intercellular adhesion genes (icaA and icaD) in various strains of Staphylococcus epidermidis isolated in medical facilities was investigated using PCR amplification and GoTaq^® DNA Polymerase. Each reaction contained 150ng of template. GoTaq^® Green Reaction Buffer was used in the PCR. (3359)

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM^®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM^®-7Zf Vector. Transcription/translation experiments were performed using the TNT^® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ Green_Lys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe^® System –T7 and the Ribo m⁷G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

Notes: This study investigated the mechanisms of acute and long-term synaptic modulation by neurotrophin-3 (NT-3). The investigators used the Xenopus neuromuscular junction synapse as a model system in which to characterize the effects of short- and long-term exposure to NT-3. Three features were found to be required for long-term, but not acute, effects: 1) endocytosis of the NT-3-receptor complex, 2) Akt activation, and, 3) new protein synthesis. As part of the investigation biotinylated NT-3 was supplied to cultures on streptavidin-coated beads, which were too large to be internalized by endocytosis. In this experiment long-term effects were eliminated but acute effects were unchanged, supporting the requirement for endocytosis of NT-3 for long-term effects. To confirm that NT-3 was tightly bound to the beads, the NT-3-coated beads were vortexed for 30 seconds, and the supernatant collected by centrifugation (5 minutes) and assayed for NT-3 content using the NT-3 E_max^® ImmunoAssay System. (3501)

Notes: The firefly and Renilla luciferase coding regions were amplified from the pGL3 and pRL-CMV Vectors and cloned into various yeast expression vectors. Strains of Saccharomyces cerevisiae were transformed with these constructs and analyzed with the Dual-Luciferase^® Reporter Assay System. The authors created yeast lysates for the Dual-Luciferase^® Reporter Assay System using 1X Passive Lysis Buffer. Several factors important to assay performance as well as firefly and Renilla luciferase expression were explored, including the stability of both luciferases stored in lysates at room temperature for various periods of time, optimal culture density before lysis of transformants and the firefly luciferase half-life in S. cerevisiae. (3298)

Notes: To rapidly characterize somatic mutations in the EGF receptor in lung cancer tissues, genomic DNA was extracted from lung biopsies using the Wizard^® SV Genomic DNA Purification System. The extracted DNA was used in a LightCycler SNP genotyping assay to determine the genotype of common loci associated with improved patient response to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. (3588)

Notes: Immunohistochemistry, Western blotting, and real-time PCR were used to determine levels of COX-2 in papilloma and normal laryngeal tissue. Explant cultures of human normal laryngeal and papilloma cells were used to define the signaling pathways that regulate COX-2 expression and investigate the potential of targeting COX-2 as a strategy to suppress papilloma growth. To measure the effects of prostagladin E₂ (PGE₂) on cell number, papilloma cells were cultured in keratinocyte growth media containing 250 or 500 nmol/L PGE₂ or an equal volume of DMSO for 24 hours. The relative measure of viable cells was determined by the CellTiter 96™ Non-Radioactive Cell Proliferation Assay.
(3306)

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

Notes: The authors used modified baculoviruses containing mammalian expression cassettes in cell-based reporter assays for nuclear receptor activity. An MMLV-firefly luciferase was used as a reporter construct. Activity of the reporter was assessed using the Steady-Glo^® Reagent. (3945)

Notes: To simulate isolating Salmonella typhimurium genomic DNA from various sample types, 2 to 2 × 10⁶ genome equivalents and 200µl lysis buffer were added to pelleted material from fishmeal, pig feces and chicken neck skin. After vortexing the samples, the genomic DNA was isolated using the MagneSil^® KF, Genomic System on a KingFisher^® workstation. To analyze the purified DNA, 5µl was used in real-time PCR with four replicates. (3425)