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)

Notes: To determine the usefulness of multilocus sequence typing (MLST) in differentiating Candida species during epidemiological studies, the authors investigated the population structure of C. dubliniensis by amplifying the same 10 MLST loci found to be useful in differentiating isolates of C. albicans, a closely related species. PCRs were performed using 1.25 units of GoTaq^® Flexi DNA Polymerase and 1ng of DNA template in a 50µl reaction. (3880)

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: To study how mutations in the quinolone resistance determining region (QRDR) of Staphylococcus epidermidis may have a role in fluoroquinolone resistance, 138 samples of S. epidermidis were swabbed from the conjunctival sacs of 129 patients. These samples were cultured overnight in tryptic soy broth, and genomic DNA isolated using the Wizard^® SV 96 Genomic DNA Purification System. One microliter of the isolated DNA was used in PCR for the QRDR genes (gyrA, gyrB, parC and parE). (3939)

Notes: This article showed that expression of several immune response genes could be induced in lung adenocarcinoma H460 cells by treatment with farnesol and that this induction proceeds through an NF-κB pathway. To determine which MAPKs were involved in the activation of these genes, the authors used the MEK inhibitor U0126 and showed that it inhibited the expression of several of the immune response genes and specifically inhibited the phosphorylation of p65 on Ser²⁷⁶. (3904)

Notes: The authors were interested in testing platinum (Pt) anticancer drugs that do not bind to DNA to see how they worked in cisplatin- and carboplatin-resistant cells. The human ovarian cancer cells, A2780 and A2780/C30, were seeded in T75 cm² flasks with 1.0 × 10⁷ cells. After 24 hours, the cells were treated with 0, 10, 20, 30 and 50µM Pt compounds for 24 hours. Medium was removed, the cells washed, trypsinized and centrifuged. Genomic DNA was isolated using the Wizard^® SV Genomic DNA Purification System and quantitated using absorbance at 260nm. This DNA was using in DNA-Pt binding assessment. (4019)

Notes: Nuclear transport of hypoxia-inducible factors (HIF) allows these factors to activate transcription of genes including epo,vegf an glut1 to maintain oxygen homeostais in cells. In this study the authors synthesized HIF-1α,β and HIF-2α in vitro, and used the expressed proteins in binding studies to determine the nature of HIF binding to nuclear pore complex proteins. The HIF proteins were transcribed and translated in vitro in the presence of ³⁵S-methionine using the TNT^® Coupled Reticulocyte Lysate System according to the protocol. After incubation, 10µl of the reaction batch was allowed to bind to the immobilized fusion-proteins importin alpha3, alpha5, and alpha7. The direct interaction of HIF-1α with alpha importins was dependent on functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS was not effective. In a typical experiment 100µl GST beads were pre-equilibrated in IP buffer (20mM Hepes pH 7.5, 100mM KOAC, 0.5mM EGTA, 5mM MgOAc, 250mM sucrose, 4°C), mixed with 15µg GST-fusion proteins and His-tagged importin beta and incubated at 4° C for 1 h. (3947)

Notes: To examine radiation-bystander responses in neonatal mouse cerebellum, heterozygous radiosensitive Patched-1 (Ptch1) mice were exposed to either whole body (WB) x-rays or shielded head/rest of body (SH) irradiation. Genomic DNA was isolated from tumors and normal tissue using the Wizard^® SV Genomic DNA Purification System. Loss of heterozygosity was tested using PCR and sequencing of exon 23 of the Ptch1 gene. (3940)

Notes: To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus. (3982)

Notes: To examine the role of NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) during greening, the PORA gene was mutated by changing each of the four conserved Cys to Ala using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting genes were subcloned into an expression vector and transformed into XL1-Blue cells. The proteins were expressed either in E. coli cells after IPTG induction or in a wheat germ extract system and tested for interaction with Pchlide molecules and NADPH. (3994)

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: The authors generated population data for unrelated Caucasian-Mestizos from Columbia using the PowerPlex^® 2.1 System and the GammaSTR^® Multiplex. DNA was isolated from whole blood using the Wizard^® Genomic DNA Purification Kit or ReadyAmp™ Genomic DNA Purification System, then amplified per the manufacturer's recommendations. Amplified fragments were detected using a Hitachi FMBIO^® II fluorescence imaging system. (3856)

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: The authors generated population data for 100 unrelated individuals from three main ethnic groups in Bosnia and Herzegovina. Autosomal STR analysis was performed for 100 male and female individuals, and Y-STR analysis was performed with 100 male individuals. DNA was collected as buccal swabs or blood samples, isolated and amplified, and amplification products were detected using an ABI PRISM^® 377 DNA Sequencer. (3877)

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)