Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard^® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq^® Hot Start Polymerase. (4164)

Notes: The authors of this paper demonstrate the utility of the HaloTag^® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag^® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

Notes: The authors examined the heritability of certain eye characteristics that may be associated with glaucoma using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4044)

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

Notes: These authors characterized the venom composition of the water snake Cerberus rynchops. As part of the study, whole venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis. For in-solution tryptic digestion, lyophilized crude venom (500 μg) was dissolved in ammonium bicarbonate and precipitated with ice-cold acetone for 3 h at -15 °C. The pellet was resuspended in 1 ml 50 mM ammonium bicarbonate containing 20 μl of 1% ProteaseMAX Surfactant. After reduction with 10 μl 0.5M DTT (at 56 °C for 20 min) and alkylation with 27 μl of 0.55M iodoacetamide (at room temperature for 15 min in the dark), the venom was digested at 37 °C for 3 h using 18 μg trypsin (1 μg/μl) and 10 μl of 1% ProteaseMAX surfactant to improve tryptic cleavage activity. (4081)

Notes: Dysregulation of the NF-kB pathway has been associated with the formation of a wide variety of tumors and other cancers, as well as diseases, including chronic inflammation and immunodeficiency. Because of the association of constitutive NF-kB regulation and tumors, inhibition of the NF-kB pathway by small molecule antagonists was thought to be a means of reversing or halting the growth and spread of tumors. The authors screened compounds from a database (the NCGC Pharmaceutical Collection or NPC) of small molecule compounds: 52% of the compounds have been approved for human or animal use by the FDA, 22% were drugs approved for use in Europe, and another 25% either drugs approved in other countries or compounds that have been tested in clinical trials. The database served as a source from which to rapidly and efficiently identify already approved drugs that inhibited NF-kB. They used a quantitative high-throughput screening format. To identify small molecules that could inhibit the NF-kB pathway, the compounds were initially screened using a cell-based NF-kB lactamase reporter gene assay, with TNFalpha and MG132 as positive controls. (TNFalpha induced NF-kB coupled beta-lactamase activity, while MG132 blocked TNFalpha induction NF-kB-coupled beta-lactamase activity.) After several rounds of screening, 20 compounds were further studied for their NF-kB inhibition, with NF-kB luc2P HEK293 cells. After a concordance rate of 95% between the luciferase and beta-lactamase tests, compounds were additionally examined for their ability to affect caspase 3/7 activity, for the ability to disrupt the electrochemical gradient across the mitochondrial membrane in relation to cellular apoptosis, as well as tests of the inhibitors on cancer cell viability and affects on LDH release, an indicator of cell necrosis.
(4049)

Notes: pGL4.15[luc2P /Hygro] or pGL4.14 [luc2/Hygro] were used. (4265)

Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

Notes: These authors characterized inflammatory caspase substrates using an enzymatic enrichment method for caspase 1,-4 and -5 cleaved proteins and mass-spectrometry. To confirm that caspase-1 cleaved the putative substrates, some substrates were expressed and fluorescently labeled using the TNT® T7 Transcription/Translation System and the FluorTect™ Green_Lys System. The proteins were then treated with recombinant caspase-1, and the progression of the reaction tracked via SDS-PAGE. The authors also used the CytoTox™ ONE Homogeneous Membrane Integrity Assay to track membrane permeabilization in relation to caspase cleavage and IL-1B release. (4252)

Notes: To help understand the molecular mechanism behind the biogenesis and assembly of photosystem II (PSII), the authors characterized the high chlorophyll fluorescence low psii accumulation19 (lpa19) mutation, which results in defective PSII biogenesis. To examine Lpa19 expression patterns in lpa19 mutant plants, the authors performed Western blot analysis using an anti-Lpa19 antibody raised against purified recombinant His-tagged LPA19 protein. This antigen was purified using the HisLink™ Resin. (4101)

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

Notes: RNA was isolated from plant tissue using the SV Total RNA Isolation System. (4544)

Notes: In this study the luminescence-based BacTiter-Glo System and the GloMax 20/20 luminometer were used to detect ATP concentrations as low as 0.0001 nM in various water samples. The results correlated with those generated using traditional microbial detection methods. (4103)

Notes: To better understand early onset colorectal cancer (CRC), the researchers examined mismatch repair (MMR) deficiency by analyzing microsatellite instability (MSI) of five mononucleotide markers BAT25, BAT26, NR21, NR24, and MONO27 from tumors using the MSI Analysis System, Version 1.2. Instability at ≥3 markers classified the tumors as high MSI, while those unstable at ≤2 markers were designated as microsatellite stable. These results were compared with immunostaining analysis of MMR proteins. (4113)

Notes: 293T cells were seeded in 12-well plates and transfected using 0.4µg of an expression plasmid, 0.6µg of a luciferase reporter plasmid and 60ng of the pRL-SV40 or pRL-TK control vector using FuGENE® 6 transfection reagent. At 24 hours posttransfection, cells were infected with Sendai virus or mouse hepatitis virus, and 8 hours postinfection, luciferase activity was measured using a dual luciferase reporter assay (Promega). (4277)

Notes: The authors investigated the role of the ubiquitin E3 ligase PDLIM2 in the degradation of signal tranducer and activator of transcription 1 (STAT1). They showed that activation of PDLIM2 and subsequent STAT1 ubiquitination require protein kinase C-mediated phoshorylation of PDLIM2 on serine 137. Polyhistidine-tagged PDLIM2 and polyhistidine-tagged mutants PDLIM2-S137A and PDLIM2-S137D were purified using the MagneHis™ Protein Purification System for use in in vitro phosphorylation and ubiquitination assays. One mechanism used to assess levels of STAT1 was a reporter assay using RAW264.7 cells transfected with a pGL3-based construct containing a interferon γ-activated sequence (GAS) upstream of the firefly luciferase reporter gene. Expression of wildtype PDLIM2, but not the mutant forms, resulted in much lower levels of STAT1 protein, and thus lower luciferase activity, when cells were challenged with lipopolysaccharide. The pRL-TK Vector was used to normalize for transfection efficiency. Luciferase assays were performed using the Dual Luciferase^® Reporter Assay System and Reporter Lysis Buffer. (4152)

Notes: The vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl₂ and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

Notes: The authors investigated the role of pioglitazone on transcriptional regulation of the apoA-I gene and looked at the biological properties of pioglitazone-induced apoA-I-containing high-density lipoprotein particles (HDL). To investigate the biological properties of the HDL particles, the authors treated THP-1 cells with conditioned medium from HepG2 cultures treated or untreated with pioglitazone and looked at adhesion to human aortic endothelial cells (HAEC). During the experiment, HAEC viability and proliferation were monitored using the CellTiter-Glo^® Luminescent Cell Viability Assay. Additionally, to determine whether pioglitazone stimulates apoA-I transcription, a luciferase reporter construct was made containing the apoA-I gene promoter. Transfected cells were treated with pioglitazone, and luciferase expression was monitored using the Dual-Luciferase^® Reporter Assay System. (4173)

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m⁷G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m⁷G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

Notes: The microtubule-based cytoskeleton of Toxoplasma gondii is important for cellular invasion. The authors of this paper investigated the tubulin composition and structure
and showed that T. gondii tubulin is extensively altered by post-translational modification. These modifications were analyzed by mass spectrometry. Trypsin digestion of dried gel pieces containing tubulins was performed overnight at 37 °C with the ProteaseMAX Surfactant trypsin enhancer. (4082)

Notes: The authors expressed the seven subunits comprising the middle module of Mediator, the 1.4MDa coactivator complex required for regulated transcription by RNA polymerase II, in E. coli. To detect and characterize the interaction of these subunits within the module, they coexpressed combinations of these subunits and performed pull-down assays. This enabled them to publish a subunit interaction map. Coexpression and pull-down assays were performed using proteins purified using MagneHis™ Ni-Particles. (4122)

Notes: As part of this study, the authors performed in-gel digestion of expressed protein fragments using trypsin and ProteaseMax Surfactant. The protein of interest was first run on an SDS-PAGE gel and the band excised, cut into 1 x 1mm pieces, reduced with 50 mM DTT and alkylated with 100 mM acrylamide. After destaining with 50 mM 1:1 acetonitrile:ammonium bicarbonate solution, the samples were dried and reconstituted in 12 ng/μL trypsin in the presence of ProteaseMax. The mixture was incubated at 50°C for 1 h before being spun down at 12g for 30 s. The isolated peptides were dried, then reconstituted in 10 μL of 2% acetonitrile/water with 0.1% formic acid before LC-MS/MS analysis. (4079)

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)