Notes: In this study of the frequency of human papilloma virus (HPV) in patients with oral squamous cell carcinoma (OSCC), the MagneSil^® Genomic, Fixed Tissue System was used to isolate DNA from formalin-fixed paraffin-embedded tissue samples from primary tumors and matched samples. Five microliters of genomic DNA was amplified using PCR Master Mix and primers for both a 110 bp fragment of human ß-globin gene and HPV genotype. After PCR, the product was analyzed on an 8% nondenaturing polyacrylamide gel and stained with AgNO₃. (3938)

Notes: This paper demonstrates use of a calpain assay in a cell-based format. (Calpain-Glo™ Assay). (3941)

Notes: RBCK1 is a RING-IBR (ring in between ring fingers) protein previously shown to have transcriptional activity and to bind protein Kinase Cβ. This paper demonstrates that RBCK1 also possesses ubiquitin ligase E3 activity. Both FLAG-tag and HaloTag® labeled proteins were used to demonstrate this activity. To demonstrate the interaction between RBCK1 and ubiquitinated proteins, HaloTag®-ubiquitin and FLAG-RBCK1 were coexpressed in HEK293 cells in the presence or absence of a proteasome inhibitor. The anti-FLAG immunoprecipitates isolated from these cells were analyzed by SDS-PAGE and Western blotting using anti-HaloTag® and anti-FLAG antibodies and self-ubiquitination of RBCK1 was demonstrated. RBCK2, a splice variant of RBCK1 lacking the RING domain showed no self ubiquitination activity but was demonstrated to interact with RBCK1 in vivo and in vitro, and to inhibit the self-ubiquitination activity of RBCK1 in a FLAG-tag assay. Pulse-chase experiments, using HEK293 cells expressing HaloTag®-RBCK1 with or without RBCK2 and treated with HaloTag®-TMR ligand, were used to show that the half-life of RBCK1 was extended by overexpression of RBCK2. (3874)

Notes: In this review, the author discusses the advantages of the Promega MSI Analysis System, including the use of mononucleotide markers that are monomorphic (i.e., almost all individuals are homozygous for the same common allele) to simplify data analysis. In addition, the inclusion of pentanucleotide repeat markers ensures that the normal and tumor tissue are from the same individual. The author shows representative data generated using MSI Analysis System, Version 1.1. (4118)

Notes: The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures. (3953)

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4^oC but slides stored at -20^oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70^oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

Notes: Trivalent arsenic (As³⁺) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As³⁺-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR. (3766)

Notes: In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting. (3986)

Notes: The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75^NTR) using GoTaq® Green Master Mix. RT-PCR results were confirmed by Western blot analysis. (3884)

Notes: In this study, the Maxwell^® 16 Instrument was used to purify genomic DNA from blood samples. The extracted DNA was amplified by PCR for subsequent genotype analysis. (3902)

Notes: The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors. (3886)

Notes: The authors characterized five open-reading frames flanking the alcohol-inducible alkane monooxygenase (BMO) structural gene of Pseudomonas butanovora. Strains with mutated bmoR, which encodes a putative transcriptional regulator, or bmoG, which encodes a putative chaperonin, were created by gene inactivation. The bmoR gene was amplified and cloned into the pGEM^®-T Vector for disruption with a kanamycin cassette. The two termini of the bmoG gene were amplified separately, ligated to the kanamycin cassette and cloned into the pGEM^®-T Easy Vector. Plasmids encoding the disrupted genes were transformed into Pseudomonas butanovora by electroporation. (3893)

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 asparagine-glycine-arginine (NGR) sequence can be converted to isoaspartate-glycine-arginine (isoDGR) by asparagine deamidation, and isoDRG can interact with αvβ3, an integrin involved in angiogenesis. The authors tested the hypothesis that a cyclic isoDGR peptide (CisoDGRC) can be used to target delivery of drugs or nanoparticles to tumor neovasculature. The authors produced tumor necrosis factor α tagged with NGR (NGR-TNF) and subjected it to conditions favoring asparagine deamidation to produce CisoDGRC-TNF. The isoaspartate content was determined using the IsoQuant® Isoaspartate Detection Kit. (3900)

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: To amplify yeast mutarotase, S. cerevisiae was used, and E. coli strain JM109 (Promega Cat.# L2001) was used for plasmid propagation. Fungal mycelia were harvested by filtration, washed, frozen and ground under liquid nitrogen. Genomic DNA was extracted using the Wizard Genomic DNA Purification System (Promega Cat.# A1120). RNA for hybridization and RT-PCR was extracted from mycelia using the SV Total RNA Isolation System (Promega Cat.# Z3101) and plasmid DNA isolated using the PureYield(TM) Plasmid Midiprep System (Cat.# A2492). (3919)

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: The C-terminus of free methionine-R-sulfoxide reductase (fRMsr) gene, a yeast gene that can generate methionine from methionine-S-sulfoxide and methionine-R-sulfoxide, was tagged with six histidines before being cloned into the pCI-neo Mammalian Expression Vector. This construct was transfected into SK-Hep1 hepatocytes with or without the Clonegene pEGFP-N1 Vector. Using 800 μg/ml G418 sulfate, a stable cell line was selected. This cell line was then tested for response to oxidative stress. (3985)

Notes: To examine the methylation state of DNA of cultured cells, genomic DNA was purified using the Wizard® Genomic DNA Purification Kit. The extracted DNA was restriction enzyme digested and then treated with bisulfite. (3978)

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: To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR. (3992)

Notes: In this study, in vivo selection of a phage display random peptide library was performed in mice, isolating specific peptides that homed to mouse thymus. A thymus-homing and kidney-recovered peptide were injected into BALB/c mice, and peripheral blood removed at intervals of 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours. DNA was extracted using the ReadyAmp™ Genomic DNA Purification System. Thymus activity was assessed by using real-time PCR. (4020)