Notes: These authors describe a method for cell-surface labeling with two genetically expressed tags, a biotin acceptor peptide (BAP) and HaloTag (HT). BAP and HT were fused with the N-terminus of the mouse Ig κ-chain leader sequence to direct them to the secretory pathway, and the c-terminus of the platelet-derived growth factor receptor transmembrane domain to anchor them to the plasma membrane. Fluorescent proteins were fused with BAP and HT to create the two distinct fluorescent transmembrance proteins BAP-YFP-TM and HT-CFP-TM. The constructs were introduced into HeLa cells and imaged simultaneously. The authors then demonstrated in vivo imaging by injecting the doubly-tagged HeLa cells into nude mice and imaging with near infrared fluorescent probes, and with magnetic resonance probes. (3998)

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard^® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (T_c), which is lower than the melting temperature (T_m) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq^® Flexi DNA Polymerase and mutation-specific TaqMan^® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan^_® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

Notes: The authors created a full-length infectious cDNA clone of a genotype 3 hepatitis E virus (strain JE03-1760F) for use in cell culture. The full-length ORF2 sequence of the JE03-1760F genome was amplified and cloned into the pCI Mammalian Expression Vector. The construct was transfected into PLC/PRF/5 cells for 3 days then analyzed by Western blotting. (4029)

Notes: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase^® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 10³ in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter^® 96 AQ_ueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

Notes: The authors tested if CYP1B1 removed cellular oxygenation products that induce oxidative stress and promote the release of antiangiogenic factors. The P450-Glo™ CYP1B1 Assay was used to determine CYP1B1 activity. The presence of glutathione was assessed using either 10⁴ retinal endothelial cells or 50µl of mouse retinal extracts dispensed into each well of a 96-well plate with the GSH-Glo™ Glutathione Assay. (4010)

Notes: The protein phosphatase Cdc14 is sequestered in the nucleolus during interphase and, after successful interphase, is dispersed from the nucleolus throughout the cell by an unknown mechanism to drive a cell's exit from mitosis. The authors determined that Cdc14 contains a nuclear localization signal (NLS), but phosphorylation of serine and threonine residues adjacent to the NLS interferes with localization. These phosphorylation sites were mapped using trypsin and AspN digestion, followed by mass spectrometry. The authors showed that phosphorylation of Cdc14 is mediated by the protein kinase Dbf2-Mob1 by co-incubating purified Dbf2-Mob1 and Cdc14 in the presence of γ-[³²P]ATP. Dbf2-Mob1 was expressed with a His6 tag and purified using MagneHis™ Ni-Particles. (4120)

Notes: This paper explored the effect of methyl-CpGmodified single-stranded oligonucleotides (ssODN) on gene repair. The Wizard^® Genomic DNA Purification Kit was used to isolate gDNA from methylCpG-ssODN-treated myoblasts derived from limb muscle of neonatal C57 mice that stably expressed the mismatch repair site. One microgram of purified gDNA was digested overnight with 5U of restriction enzyme, purified, resuspended in 20µl of water and 5µl used in real-time PCR to determine if the target had been repaired. (4062)

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)

Notes: The authors were interested in examined the relationship between pregnane X receptor (PXR) and protein arginine methyltransferase 1 (PRMT1). Cells were transiently transfected for 60 hours including chemical treatment, and the Luciferase Assay System was used to analyze reporter activity. For the Checkmate™ Mammalian Two-Hybrid Assay, CV-1 cells were transfected with the pBIND Vector containing PXR, pACT Vector containing PRMT1 and pG5luc Vector. After 12 hours, the cells were treated with rifampicin and 48 hours later, luciferase activity was measured. Full-length PRMT1 protein was synthesized using the TNT^® SP6 Coupled Reticulocyte Lysate System and used in a GST pulldown assay. (4027)

Notes: The authors examined the role of two promoters in the regulation of fabA, an enzyme involved in unsaturated fatty acid synthesis. fabA transcript levels were quantified using real-time quantitative RT-PCR using ImProm-II™ Reverse Transcriptase, followed by a SYBR^® Green method. (4053)

Notes: In this study, the authors wanted to examine the ability to trace the origin of tomato goods from fresh to processed. They tested several DNA extraction procedures for fresh tomato, tomato sauce, tomato puree, tomato pulp, whole peeled S. Marzano PDO (Protected Designation of Origin) tomato, whole peeled tomato, tomato concentrate and ‘‘Arrabbiata sauce”. Homogenized material (200mg) was extracted in three replicates using seven different methods including the Wizard^® DNA Clean-Up System. The DNA extracted was then analyzed by agarose gel electrophoresis, quantified and tested in PCR using SSR loci. The authors concluded that the Wizard^® DNA Clean-Up System was the most effective of the DNA extraction methods tested and yielded the greatest number of successful amplification reactions with lowest investment of personnel time and money. (4003)

Notes: This study examined the effects of folic acid supplementation on the offspring of pregnant rats fed a protein-restricted diet. Genomic DNA was extracted from rat adipose tissue using the Wizard^® SV Genomic DNA Purification System. The purified DNA was then incubated with methylation-sensitive restriction enzymes and then used in real-time PCR. (4066)

Notes: The lymphocytic choriomeningitis virus (LCMV) was used as a model to create a trisegmented recombinant arenavirus in which viral genes were replaced by a gene of interest. One such engineered virus, r3LCMV CAT/FLuc, was used in a pilot screen to identify anti-arenaviral compounds. Firefly luciferase (FLuc) activity was measured using the ONE-Glo™ Luciferase Assay System. (3957)

Notes: The authors were interested on confirming the identity of the exhumed skeletal remains thought to be Nicolaus Copernicus. DNA isolation from teeth was performed by pulverizing the tooth in liquid nitrogen, soaking the powdered sample in 0.5M EDTA, 5% SDS and 3 mg proteinase K at 37 °C and extracting genomic DNA using the Wizard^® Genomic DNA Purification Kit. Bone and other tooth samples were extracted using another method and the Wizard^® Genomic DNA Purification Kit used for a salting-out method. Purified DNA was subjected to mitochondrial, autosomal and Y chromosome STR amplification and analysis. (4063)

Notes: In 1991, the remains of murdered Emperor Nicholas II, Empress Alexandra and three of their five children were discovered. Until recently, the remains of the two other children were never found. In July of 2007 human bone fragments were discovered at a second grave site in the Ural region of Russia. The authors performed DNA typing to determine if these remains were those of the two missing children. Bone fragments and teeth were subjected to mitochondrial and nuclear DNA typing. DNA was quantitated using the Plexor® HY System. Nuclear DNA analysis was performed, in part, using the PowerPlex® S5 System. A comparison of mitochondrial DNA sequences from remains in the first and second graves and from maternal reference samples confirmed that the remains constituted a family with a "Queen Victoria" mitotype (Empress Alexandra was the granddaughter of Queen Victoria). Y-STR analysis of both sets of remains was performed, and the results confirmed that the Y-STR haplotypes of the two sets of male remains matched, and this haplotype matched that of several descendants from an unbroken paternal lineage of Nicholas I, father of Nicholas II. The mitochondrial and Y-STR haplotypes and autosomal STR profile also matched those obtained from a bloodstained shirt that Nicholas II was wearing during an assassination attempt. (3967)

Notes: The authors note that while many protein fusion tags are available to assist in purifying functional, recombinant proteins in soluble form and adequate amounts, none of the available tags are ideal when applied to the diversity of proteins studied. Available tags are often best-suited to specific aspects of the overall process, be it expression, solubilization, protein capture, etc. HaloTag was designed to address multiple features. The authors demonstrated that HaloTag provided enhanced expression and solubility in E. coli, with efficient protein purification and labeling for screening and quantitation. Enhanced solubility coupled with covalent capture gives HaloTag advantages over conventional affinity fusion tags, demonstrated by the successful purification of a wide variety of proteins, including low expressers, with higher yield and purity compared to the other tested tags. Covalent capture coupled with efficient tag removal to release the target protein, provides higher purity by eliminating tag contaminants. Using full-length cDNAs encoding 23 human proteins of varying size and function, the authors compared the efficacy of HaloTag as an expression and purification tag to the frequently used solubility enhancers MBP and GST. As reported previously, the three larger tags (HaloTag, MBP and GST) provided higher total and soluble expression compared to the smaller His6tag. In comparing soluble protein expression levels for the three larger tags, it was found that HaloTag solubilized 74% of the 23 human proteins examined, compared to 52% for MBP and 39% for GST. G2681 was one of the T7, bacterial promoter-based Flexi vectors used.
(4005)

Notes: These authors used proteolysis and mass spectrometry to identify the specific protein domains involved in copper-dependent phosphorylation. They expressed wildtype and mutant ATP7B in COS1 cells. The band of interest was excised from a gel, cut into small pieces and subjected to tryptic digestion in the presence of ProteaseMax Surfactant. (4086)

Notes: The authors use the Flexi^® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag^® Technology. They also describe a method of transferring ORFs between Flexi^® Vectors in a 96-well plate format. They also used Wizard^® SV 96 Plasmid DNA Purification, Wizard^® SV PCR Clean-Up, and Wizard^® SV Gel and PCR Clean-Up Systems. (4056)

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq^® Hot Start DNA Polymerase and Green GoTaq^® Flexi Reaction Buffer. (4037)

Notes: These authors used short heteronuclear RNAs (shRNA) in multiple cancer cell lines to silence hypoxia-inducing factor-2α (HIF-2α), a gene that many cancers exploit to increase angiogenesis and activate fundamental receptor tyrosine kinase signaling pathways to gain growth signal autonomy. Western blotting and RT-PCR were used to monitor the level of HIF-2α silencing. RT-PCR was performed using the AccessQuick™ RT-PCR System. The authors also examined the level of tyrosine kinase activation in shRNA-treated cells by Western blot analysis. To examine levels of ERK1/2 phopshorylation, the authors used the Anti-ERK 1/2 pAb to compare levels of activated and unactivated ERK1 and 2. (4050)

Notes: The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing. (4030)

Notes: The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjunct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega. (4032)

Notes: In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured. (4004)