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 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)

Notes: The accumulation of the toxic form of the Amyloid-β peptide is known to induce oxidative damage in the brain. Although treatment with antioxidants has not proven effective at controlling AD symptoms, inducing the natural systems in the brain that protect from oxidative damage may provide a possible therapeutic approach. A host of antioxidant and detoxifying enzymes are upregulated by binding of the Nrf2 transcription factor to the ARE (antioxidant response element) regulatory sequence. The authors used a Dual Luciferase^® Reporter Assay to assess modulation of gene activity through ARE by kavalactones. Kavalactones are compounds found in the roots and rhizomes of Kava (Piper methysticum), a plant cultivated an used in some Pacific societies for medicinal and social uses. The ARE1 region from the rat NAD(P)H:quinone oxidoreductase-1 gene was placed upstream of a pGL3 firefly luciferase reporter construct and cotransfected along with a pRL-TK Renilla control construct into PC12 or C6 cells. The data show induction of luciferase activity by kavalactones. Further investigation shows that the kavalactones promote Nrf2 stabilization possibly through the ERK1/2 pathway. (3859)

Notes: The authors showed that LipL32, a major surface-exposed protein of Leptospira sp., bound to the host extracellular matrix (ECM). They expressed and purified recombinant LipL32 for use in a solid-phase binding assay to test its ability to bind to ECM proteins. LipL32 was expressed with an N-terminal biotin-acceptor tag and purified using the SoftLink™ Soft Release Avidin Resin. (3899)

Notes: Increased expression of midkine (MK), a growth factor normally involved in neural development, is associated with several human malignancies. The authors used quantitative RT-PCR to examine mRNA levels for MK and MK receptors in various soft-tissue sarcoma (STS) cell lines. Reverse transcription was performed using 1µg of total RNA, and 2µl of cDNA was used in qPCR using the PCR Master Mix, primers specific to MK and either glyceraldehyde-3-phosphate or β-actin primers for normalization, and EvaGreen^® dye. To examine the protumorigenic effects of MK, the authors incubated HT1080 and SW684 STS cell lines, which express low levels of MK, with human recombinant MK and measured cell proliferation using the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay. In addition, the authors examined cell proliferation in MK-stably transfected HT1080 cells. The plasmid used for stable transfections was created by reverse transcribing the MK-coding region using the ImProm-II™ Reverse Transcription System, then cloning the resulting cDNA into an expression vector. (3895)

Notes: To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control. (3894)

Notes: The authors identify two partial transcripts that encode granulin-like molecules in Aedes albopictus and Manduca sexta. To test the hypothesis that granulin is a highly conserved growth factor that acts on insect cells, the authors expressed recombinant goldfish granulin with an N-terminal His₆ tag, purified the recombinant protein, exposed A. albopictus Aa23 embryonic cells and M. sexta haemocytes to the purified protein, then monitored cell proliferation using a BrdU proliferation assay. Recombinant goldfish granulin was expressed in E. coli and purified using MagneHis™ Ni Particles. (3964)

Notes: In this study, FuGENE® 6 was used to perform transient transfection of K562 cells using 0.2 or 0.3µg of DNA in a 3:1 ratio of reagent to DNA. The transfections were performed in 24-well plates using 1 × 10⁵ cells/well. FuGENE® HD was used to transiently transfect HepG2 cells using 2µg of DNA in a 3:1 ratio of reagent to DNA using 2× 10⁵ cells  seeded onto coverslips 1 day prior to transfection. (4418)

Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (3905)

Notes: A plastic support suitable for use in microfluidic systems for highly sensitive DNA microarray hybridizations was developed and tested. Human DNA from Hsap-11 cells was isolated using the MagneSil^® KF, Genomic System on a KingFisher ML instrument. Ten nanograms of the isolated DNA was used in RT-PCR. (4021)

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT^® Coupled Reticulocyte Lysate System and labeled with ³⁵S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3752)

Notes: These authors prepared a complete collection of Vibrio cholerae ORF clones using an automated amplification and cloning procedure. They then tested this set of clones for protein expression and capture using a nucleic acid programmable protein array method. To do this, they used the TNT^® T7 Coupled Reticulocyte Lysate System to perform in situ transcription/translation of cDNA clones containing a GST fusion tag. Proteins were captured using an anti-GST antibody and subjected to further analysis. (3879)

Notes: The authors of this paper describe the development of a cell-free assay system derived from Agrobacterium tumefaciens NTL4(pCF218) (pCF372), which expresses β-galactosidase under the control of a tra1 promoter. The tra1 promoter is responsive to quorum-sensing signals. The assay was developed to address time-consuming cell conditioning and incubation times of the standard whole-cell assays used to detect quorum-sensing signals in natural systems. Replacing the X-Gal substrate with the luminescent Beta-Glo^® substrate increased the assay sensitivity by tenfold. (3936)

Notes: The authors of this study used the gene module map method to identify genotype-specific therapies for human cancers. They studied breast cancer cell lines that expressed the "mitochondria" and "wound signatures". These cell lines, which are associated with tumors that have poor prognosis, were also shown to have a "proteasome signature" and were sensitive to the proteasome inhibitor bortezomib. The authors used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that genotype-specific killing of "wound signature" cells was not due to differential degrees of proteasome inhibition, since 1 and 1µmol/L of bortezomib completely inhibited the ability of the wound signature and control cells to cleave the luminogenic substrate. More likely, the wound signature genotype conferred increased susceptibility to death from proteasome inhibition. (3870)

Notes: The authors determined the role of various domains of the hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the inhibition of the protease matriptase. HAI-1 mutants lacking one or more domains were expressed as His-tagged fusion proteins in CHO-K1 or COS-1 cells, and proteins were purified using the HisLink™ Protein Purification Resin. Purified proteins were then used in protease assays with recombinant matriptase. (3788)

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq^® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

Notes: The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System. (3977)

Notes: These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor^® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM^®-T Easy Vector before transfer into an expression vector. (3875)

Notes: To investigate the importance of the zinc knuckle motif of early B cell factor (EBF) in DNA binding and transcription activation of target genes, the authors used a baculovirus system to express wildtype and mutated forms of EBF, then purified the proteins for use in DNA-binding assays. The EBF proteins were expressed with a biotinylated tag and purified using SoftLink™ Soft Release Avidin Resin. (3917)

Notes: These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments. (3872)

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 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: 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)