Notes: The authors of this paper describe an assay that uses protease biomarkers to assess cell viability and cell death simultaneously in a population of cells. The assay detects an ubiquitous protease activity that is associated with live cells and a second protease activity that is associated with cells that have lost membrane integrity. The readouts are either fluorescent or fluorescent and luminescent. The assay can be performed in multiplex with other assays, such as caspase assays, to gain additional information on the cell population, and it is amenable to high-throughput screening. (3927)

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca²⁺ channel (Ca_V1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM^®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard^® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo^® Luminescent Cell Viability Assay. (3761)

Notes: To study the role of connexin42 (cx43) in testis development, the authors generated a conditional cx43 knockout mouse, which lacked the cx43 gene in Sertoli cells. To confirm that the cx43 gene was deleted in these mice, PCR was performed using primers specific to cx43, 1X Colorless GoTaq^® Flexi Reaction Buffer, 2mM MgCl₂, dNTPs and 0.15µl of GoTaq^® DNA Polymerase. Tissue-specific deletion of cx43 was confirmed by amplifying RNA isolated from mouse testis, heart and tail was also confirmed using the same PCR components. (3713)

Notes: STAT1 is a transcription factor that is involved in a variety of cellular processes and behaves like a tumor suppressor in many ways. It is associated with apoptosis, inhibition of cyclin-dependent kinases, and it may mediate the antitumor effects of IFN-γ. The authors of this study designed a bioluminescent reporter assay to identify small molecules that enhance STAT1-dependent gene expression. The bioluminescent assay was performed in 384-well plates using both stably and transiently transfected cell lines. The screening assay was robust, producing a Z´factor value of 0.92, and it identified three compounds that specifically enhanced STAT1-dependent transcriptional activity. Firefly luciferase activity in stable cell lines was assessed using the Bright-Glo^® Luciferase Assay System; luciferase activity in the transiently transfected cells was monitored using the Dual-Luciferase^® Assay System. Viability of cells in culture was monitored using the CellTiter-Glo^® Assay. (3732)

Notes: The authors examined the effect of ethanol ingestion on the accumulation of proteins containing atypical isoaspartate residues and determined whether betaine prevented the accumulation of proteins with isoaspartate residues. The authors used the ISOQUANT^® Isoaspartate Detection Kit and [³H] methanol to measure the levels of isoaspartate residues in 20–50µg of liver proteins extracted from rats fed control, ethanol- or betaine-supplemented diets. (3972)

Notes: The authors of this study investigated the effects of betulinic acid (BA) and its chemical derivatives on the proteasome in vitro and in cells. They used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to assess the effects of BA derivatives on proteasome activity in MT4 cells. (3871)

Notes: The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase^® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times. (3691)

Notes: This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo^® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis. (3613)

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

Notes: The authors determined the allele frequencies for 21 autosomal STR loci in 936 individuals from the Royal Kingdom of Bhutan using the AmpFlSTR^® Identifiler^® kit, PowerPlex^® 16 System and the F13A01, FESFPS, F13B, LPL (FFFL) Multiplex. DNA was extracted from whole blood samples using the Autopure LS^® kit and amplified as directed by the manufacturers. Amplified products were detected using an ABI PRISM^® 3100 Genetic Analyzer. (3822)

Notes: Allele frequency data for 21 autosomal loci was studied in 953 unrelated individuals belonging to 12 major Nepalese groups. DNA was extracted from whole blood using the Autopure LS^® kit, then amplified using the PowerPlex^® 16 System, AmpFlSTR^® Identifiler^® kit or F13A01, FESFPS, F13B, LPL Multiplex (FFFL) Multiplex. Amplification products were analyzed using the ABI PRISM^® 3100 Genetic Analyzer. Several new alleles not reported in the NIST short tandem repeat database were detected in this population. (3811)

Notes: The authors identified human glucokinase (hGK) as a substrate for the ubiquitin-conjugating enzyme system of rabbit reticulocyte lysate (RRL). Wildtype hGK, His₆-hGK and His₆-hGK mutants were expressed in the TNT^® T7 Quick Coupled Transcription/Translation System in the presence of [³⁵S]methionine and 10µM ubiquitin (in additional to the endogenous ubiquitin in RRL). Ubiquitinated and polyubiquitinated proteins were detected as size-shifted bands by SDS-PAGE and by two-dimensional polacylamide electrophoresis followed by immunoblotting with an anti-ubiquitin antibody. For some experiments, His₆-hGK and His₆-hGK mutants were isolated using the MagZ™ Protein Purification System prior to ubiquitination. (3718)

Notes: The authors generated population data from 200 unrelated individuals in the Sichuan area of China using the PowerPlex^® 16 System. DNA was isolated from blood by phenol:chloroform extraction and quantitated by UV spectrometry. One nanogram of DNA was amplified, and the amplification products were analyzed on an ABI PRISM^® 310 Genetic Analyzer. (3810)

Notes: To determine which proteins are present in DNA replication complexes, the authors used biotin-tagged replication proteins as nanoscale biopointers. T4 DNA replication complexes were recreated in vitro, and biotin-tagged replication proteins were substituted for wildtype replication proteins. Streptavidin was allowed to bind to the biotin-tagged proteins, and the replication complexes were visualized by electron microscopy. The biotin-tagged helicase and DNA polymerase were purified from E. coli using SoftLink™ Soft Release Avidin Resin. (3806)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: When infected with Borrelia burgdorferi, humans respond by producing borreliacidal antibodies against outer surface protein C (OspC). The authors examined the immune response in infected canines to determine if the response was similar. Infected dogs produced borreliacidal antibodies, only some of which were against OspC. To examine the borreliacidal activity of these other antibodies, sera from infected canines were depleted of OspC antibodies, and the borreliacidal activities of the depleted sera were measured. Sera were depleted by passage over an OspC column, which was created by immobilizing 0.5mg of biotinylated OspC protein with 1ml of TetraLink™ Tetrameric Avidin Resin. (3780)

Notes: To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT^® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [³⁵S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel. (3675)

Notes: The authors wanted to examine how the Camelpox virus (CMLV) gene 176R (v-slfn) might affect orthopoxvirus virulence since it shares sequence similarity to murine schlafen (m-slfn) proteins that inhibit T cell and fibroblast growth. HeLa cells were transiently transfected with either pCI Mammalian Expression Vector alone or pCI Mammalian Expression Vector expressing v-slfn with or without a HA tag. Cellular localization of the protein was visualized using either α-v-slfn antiserum or α-HA mAb. (3757)

Notes: The authors tested their hypothesis that Na⁺-H⁺ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

Notes: The role of V₀ subunit isoform and its cellular environment in the disassociation of vacuolar (H⁺)-ATPases (V-ATPases) in yeast was explored in this paper. An EcoRI-BamHI fragment of HA-Stv1p, a hemagglutanin-tagged Golgi-targeting subunit of V-ATPase, had two different mutations introduced (R795K and R795Q) using the Altered Sites^® II in vitro Mutagenesis System. The mutant vectors were then transformed into yeast strain MM112 and used in studies with vacuolar protein sorting (vps) mutants. (3693)

Notes: During casework analysis using the PowerPlex^® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard^® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

Notes: The authors knocked down expression of the hydin gene, which encodes a flagellar protein, to determine the function and location of hydin in Chlamydomonas reinhardtii. Amplification steps in the creation of the RNAi expression vector to target hydin expression were performed using GoTaq^® Flexi DNA Polymerase. (3723)

Notes: Taxol-induced peripheral neuropathy is a common side-effect of treatment that has been associated with disturbed intracellular calcium homeostasis in neuronal cells. These authors investigated whether prolonged exposure to Taxol caused alterations in calcium signaling in human neuroblastoma and rat dorsal root ganglia. They found that expression of the inositol 1,4,5-triphosphate receptor modulator NCS-1 was reduced in Taxol-treated neuronal cells. The authors also found that Taxol treatment activated calpain, which degrades NCS-1, and that the calpain inhibitor AK295 prevented Taxol-mediated suppression of calcium release. These findings suggest that calcium-mediated activation of calpain is responsible for the observed degradation of NCS-1. (3681)

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)