Notes: The authors developed a single nucleotide polymorphism (SNP)-based assay to distinguish four Sclerotinia species. The assay consisted of amplification of a 300bp intergenic spacer and portions of the calmodulin and ras genes, followed by Southern blot using species-specific, radiolabeled probes. Amplifications were performed using the GoTaq® Colorless Master Mix, 0.2µM of each primer and 10–20ng of template DNA. (4098)

Notes: The authors characterized differential expression of several carcinogen metabolism genes in human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissues in smokers, former smokers and people who have never smoked. They combined laser capture microdissection (LCM) and quantitative RT-PCR. RNA was isolated from paired microdissected malignant and nonmalignant lung tissue, 100ng of total RNA was reverse transcribed in a 20µl reaction, then 1µl of cDNA was amplified by real-time PCR using an ABI PRISM® 7500HT sequence detection system, GoTaq® Flexi DNA Polymerase and gene-specific primers. The expression level for each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that expression of cytochrome P450 1B1 and glutathione-S-transferase P1 in AC, but not BEC, tissue was strongly associated with exposure to tobacco. (4095)

Notes: In this study, the effect of solar disinfection on Shigella flexneri and Salmonella typhimurium in drinking water samples was evaluated. A variety of viability indicators were used to investigate the effectiveness of the disinfection method, including measurement of cellular ATP levels. The BacTiter-Glo Assay was used for ATP detection. (4104)

Notes: This study examined the effect of lactic acid bacteria on gene expression of Staphylococcus aureus in mixed cultures. Total RNA was isolated from a mixed culture of S. aureus and Lactococcus lactis, labeled with 5µg of RNA with Cy^®3- or Cy^®5-dCTP using the ChipShot™ Labeling and Cleanup System, the cDNA was dried and stored at –20°C. Genomic DNA was isolated from S. aureus and L. lactis cultures, digested with HinPII, purified, and 400ng of the digested gDNA labeled with Cy^®3- or Cy^®5-dCTP and the Prime-a-Gene^® Labeling System. The level of gene expression was assessed using a S. aureus microarray. (4068)

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gα_q or Gα_i/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the G_i-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC₅₀ values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC₅₀ for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gα_i subunit binding responsible for enhanced GTPase activity.
(4148)

Notes: To better understand which lysine residues in monocarboxylate transporter 1 (MCT1) were involved with binding di-isothiocyanostilbene disulfonate (DIDS), a transport inhibitor, and crosslinking to embigin, rat MCT1 and embigin were subcloned into the pCI-neo Mammalian Expression Vector via the EcoRI site. The vector was then subjected to site-directed mutatgenesis of each of six lysines either singly or in combination, and the effect on the binding activity or inhibition was tested. (4069)

Notes: The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER^® Vector and amino acid substitutions were introduced using the Altered Sites^® II in vitro Mutagenesis System. The pGEM^®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase^® Reporter Assay System. (4023)

Notes: The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM^®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay. (4033)

Notes: MCF7 cells were grown in a 35mm dish to 80% confluence. Four micrograms pCMV6-XL5/RNF146 or pCMV6-XL5 (Origen Technologies Inc.) was mixed with 8 μL FuGene HD transfection reagent and added to the cultured cells. After 48 or 72 h transfection, the cells were washed with 1X PBS and lysed. The lysates were centrifuged at 13,200 rpm and the supernatants used in Western blot analysis. (4424)

Notes: These authors examined conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase as well as the conformation of the DNA-gate itself. Negatively supercoiled pUC18 plasmid was purified using the PureYield™ Plasmid Midiprep System and used in single-molecule FRET experiments. (4061)

Notes: These authors used the Maxwell^® 16 System to isolate genomic DNA from whole blood and normal colonic tissue samples. The DNA was used in genotype analysis, testing for wildtype and mutant KRAS genes, and for various EGFR-related polymorphisms. The results were used in a research study testing the relationship between various genotypes and response to different treatment regimens. (3961)

Notes: The human sepiapterin reductase (SPR) gene has been mapped at the PARK3 locus, which is related to the onset of Parkinson disease. The silkworm Bombyx mori body color mutant lemon (lem) has been associated with a lack of SPR activity; lem lethal is a homozygous lethal allele of lem. Genetic linkage analysis was performed with normal silkworm strain p50T, lem strain l70, and lem^l strain a65 to more closely examine the relationship with SPR. DNA from the F1 and F2 crosses were isolated using the Wizard^® SV 96 Genomic DNA Purification System and the genome sequenced. (4017)

Notes: The authors examined how cytotoxicity differed between cancer cell lines treated with drugs that induced either apoptosis or paraptosis, non-apoptotic cell death. HT1080 fibrosarcoma epithelial cells were seeded in 10cm dishes and grown until 40% confluent before transfection with 15µl of FuGENE® 6 Transfection reagent and 4.5µg of wild type or mutant vector + 0.5µg of GFP vector for a 3:1 reagent-to-DNA ratio. After an eight-hour incubation, the cells were trypsinized, seeded into two 24-well plates at 100,000 cells per well), and treated with drugs 24 hours later. Sixteen hours after drug treatment, one set of plates were assessed for viability, the other, cell death. (4381)

Notes: The authors attempted to isolate DNA from samples tested with Hemastix® reagent strips, which are commonly used to test for the presence of blood, using the DNA IQ™ System. Yields from samples that had been tested with Hemastix® strips were dramatically lower than those from untested samples. The experiments suggested that 3,3´,5,5´tetramethylbenzidine (TMB) irreversibly binds to the magnetic DNA IQ™ Resin to prevent DNA recovery. To circumvent this, the authors implemented an effective indirect screening method, where the unknown sample was rubbed with dry filter paper, which was then tested with a prewetted Hemastix® strip. (4035)

Notes: To identify molecules that affect metastasis signaling pathways downstream of HER2-Y1248 phosphorylation, suppression subtractive hybridization assays (SSH) were
performed using MDA-MB-468 cells overexpressing HER2 and control MDA-MB-468 cells expressing HER2 without the Y1248 phosphorylation site. Reactions were cloned
using a T-vector system, transformed and plated. Positive clones from each assay were selected and grown overnight in 2ml deep-well plates. The Wizard^® Magnesil^® Plasmid Purification System was used to isolate plasmids for BigDye™ sequencing. (4134)

Notes: These authors used the Maxwell^® 16 System to isolate DNA from frozen whole blood samples infected with Plasmodium falciparum. The isolated DNA was used in a qPCR-based SNP genotyping assay that sought to uniquely identify the parasites based on their SNP marker profile. (3962)

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag^® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag^® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag^® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase^® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag^®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase^® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

Notes: The authors of this study investigated the mechanism of action of syringolin A (SylA), which is secreted by virulent strains of the plant pathogen Pseudomonas syringae. They show that SylA inhibits all three activities of the proteasome in vitro. They also used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that SylA inhibits the chymotrypsin-like activity of the proteasome in SK-N-HS neuroblastoma cells. (3846)

Notes: To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues. (3989)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System. (3993)

Notes: The authors examined protein interactions between splice variants of the SH3A domain of intersection 1 (ITSN1) and the main ITSN1 protein partners using protein pull-down assays. In one set of pull-down assays, SH3A splice variants were expressed as polyhistidine-tagged proteins, and the proline-rich domain of dynamin 1, a known ITSN1 protein partner, was expressed as a glutathione-S-transferase (GST) fusion protein. In a second set of experiments, the SH3A domain variants were expressed as GST-fusion proteins, immobilized, then used to capture endogenous dynamin 1, SOS1, c-Cbl and Cbl-b from cell lysates. Recombinant GST-fusion proteins were purified using glutathione-Sepharose^® 4B or the HisLink™ Protein Purification Resin. Based on the data, the authors concluded that alternative splicing of ITSN1 can change the binding properties and its protein partners. (3950)

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard^® SV Gel and PCR Clean Up System and then subcloned into the pGEM^®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

Notes: The authors developed a new human prostate cancer cell line, CWR22Pc, that is both androgen-dependent and able to produce tumors in dihydrotestosterone-supplemented nude mice. To confirm that CWR22Pc cells are derived from primary CWR22 human prostate xenograft tumors, the authors performed genotyping at 8 STR loci and amelogenin using the PowerPlex^® 1.2 System. DNA purification from the cell line and original tumor samples was performed using the Wizard^® Genomic DNA Purification Kit. (4041)

Notes: The authors wanted to examine the role of plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1) in apoptosis. API cells, a NHE1-deficient Chinese hamster ovary cell line, was cotransfected with wild-type NHE1 or mutant NHE1 constructs and destabilized yellow fluorescent protein (YFP). Cells were plated at a density of 1 × 10⁴ cells/well in a 96-well plate with or without FBS. To induce apoptosis in the cells, serum was withdrawn for 24 hours. The ratio of dead-to-live cells was measured using the MultiTox-Fluor Multiplex Cytotoxicity Assay. Cell death was also determined by examining the loss of YFP fluorescence under a microscope. (3937)

Notes: The authors of this study investigated the role of the genes CLV1 and CLV3 stem cell renewal and differentiation in self-renewing shoot apical meristem (SAM) in Arabidopsis. CLV1 encodes a receptor kinase that is a negative regulator of cell growth, and therefore cannot be overexpressed in plant cells. CLV3, which encodes a protein that is processed to a 12-amino acid peptide, is believed to be a ligand for CLV1. In this study, the authors made a construct in which the CLV1 kinase domain was replaced with the HaloTag™ protein and expressed the fusion protein in tobacco BY-2 cells. The fusion protein was detected using the HaloTag™ TMR ligand. They next incubated membrane extracts from wildtype and transgenic BY-2 cells (expressing the HaloTag™-CLV1 fusion) with tritium-labeled CLV3. The transgenic cells showed significantly higher CLV3 binding than wildtype, and the authors show that CLV3 specifically labels a 130-kd band that corresponds to the HaloTag™-CLV1 fusion protein, indicating that CLV3 binds the ectodomain of CLV1. (3763)