Notes: The authors analyzed the transcriptional activity of wild-type Chlamydomonas reinhardtii cultures sampled at different time points during the aerobic and anaerobic phase of the photobiological hydrogen production process under sulfur-depleted conditions. C. reinhardtii were grown in photobioreactors, carefully extracted, centrifuged and flash-frozen in liquid nitrogen. RNA was purified using the SV Total RNA Isolation System following the plant centrifugation protocol without sample grinding. The eluted RNA was quantitated and integrity checked by gel electrophoresis and qRT-PCR. Total RNA was used to synthesize labeled cDNA using the ChipShot™ Indirect Labeling and Clean-Up System. The labeled cDNA was used for probing microarrays. (4022)

Notes: Since tumor necrosis factor alpha (TNF-α) is known to have a protective effect on herpes simplex virus type 1 (HSV-1) infection in mouse eyes and brains, a recombinant virus constitutively expressing TNF-α was generated to see if it affected virus location and spread in the eyes and brain. Mouse TNF-α DNA was subcloned from one vector using EcoRI and placed into the pCI Mammalian Expression Vector downstream of the CMV immediate/early enhancer/promoter region. Then the CMV enhancer/promoter::TNF-α region was digested with BamHI and BglII to be placed in a third vector between the UL49 and UL50 genes of HSV-1. This construct and two controls were used to create recombinant HSV-1 particles that were then injected into the eyes of BALB/c mice. (3984)

Notes: The authors infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples. (3888)

Notes: The authors identified a Y-chromosome-specific polymorphism, the M293 mutation, that defines the Y haplotype Eb1f-M293. They examined the geographic distribution of this and other Y haplotypes to determine the date of origin of the E3b1f-M293 haplotype and track the migration of the pastoralist lifestyle in Africa. Y-STR analysis was performed using the PowerPlex® Y System and two additional sets of primers. (4039)

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

Notes: PDILT is a protein disulfide isomerase (PDI) homolog under developmental control and is induced during puberty. To determine whether PDILT expression coincides with the first wave of spermatogenesis, the authors examined expression of PDILT in 2-, 15-, 30-, 45- and 58-day old rats using RT-PCR. Total RNA was isolated from rat testis, and 50ng was amplified using the AccessQuick™ RT-PCR System and primer pairs specific for PDILT, PDI and calmegin, a testis-specific chaperone that is expressed upon the appearance of spermatocytes. Primer pairs were designed to span introns to avoid amplification of genomic DNA. PCR products were analyzed on a 1% agarose gel. Results showed that PDILT mRNA was first detected during the onset of spermatogenesis. (3765)

Notes: The authors studied the role of the Erk1/2 signaling pathway during neural specification in mouse embryonic stem (ES) cells. Undifferentiated ES cells express high levels of the pluripotent marker Nanog but do not express fibroblast growth factor (Fgf5), an early marker of differentiation of ES cells, or Sox1, an early neural transcription factor gene. Using quantitative PCR, levels of Nanog, Fgf5 and Sox1 mRNA were quantitated during ES differentiation in the presence and absence of a MEK inhibitor. Prior to quantitative PCR, 1µg of total RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase. By measuring these mRNA levels, the authors determined that inhibition of the Erk1/2 pathway blocked ES differentiation. (3726)

Notes: These authors screened a library of 15,000 expression cDNAs in neuroblastoma IMR-32 cells searching for genes that activate the antioxidant response element, ARE. ARE is a cis-acting enhancer element found in the 5´ flanking region of many genes that are involved in protection from oxidative stress. The library was screened using a luciferase reporter construct under the control of an ARE-containing promoter. Luminescence, indicating the presence of cDNA-activating ARE, was measured using the Bright-Glo™ Luciferase Assay System. cDNA clones showing reproducible activation were selected for further analysis. The authors tested the effect of over expression of these ARE activators on the ability to resist oxidative stress. IMR-32 cells expressing the various cDNAs were exposed to hydrogen peroxide or rotenone, and the effect on cell viability was measured using the CellTiter-Glo^® Assay. Cells overexpressing the ARE-activators were more resistant to oxidative stress than controls. (3629)

Notes: These authors used the Kinase-Glo^® Luminescent Kinase Assay to perform a high-throughput screening assay for inhibitors of glycogen synthase kinase-3β in 96-well plates. They used a 1µM ATP concentration and screened 55,000 compounds at 10µM. The assay sensitivity and IC₅₀ values of reference compounds were comparable to radioactive methods; the final optimized assay had an average Z´-factor of 0.72. (3591)

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)