Notes: These authors showed that the stomatal initiating factor SPEECHLESSS (SPCH) is a substrate of the kinases MPK3 and MPK6 in vitro, that specific phosphorylation sites on SPCH regulate its activity in vivo, and that components of the stomatal development signaling network modulate SPCH. As part of the study, coomassie-stained gel bands containing the SPCH protein were excised and digested using trypsin and ProteaseMax Surfactant. Mass spectrometry was used to assess phosphorylation at several functionally critical sites. (4087)

Notes: The authors wanted to understand the role of ASAP3, an Arf GTPase-activating protein in cancer cells. FLAG- or hemagglutinin-tagged ASAP3 and GAP-deficient mutants of ASAP3 were cloned in the pCI Mammalian Expression Vector. The constructs were transfected into cells and used in immunofluorescence and Western blotting experiments. (3987)

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP₃R) isoforms InsP₃R1, InsP₃R2 and InsP₃R3. To compare the ATP-binding properties of InsP₃R2 and InsP₃R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi^® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

Notes: The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System. (3803)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM^®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample. (3885)

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq^® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

Notes: The Plautia stali virus contains two open reading frames and includes a 5´ internal ribosome entry site (IRES) and an intergenic IRES region. These authors showed that the 5´ IRES was functional and initiated translation in insect cell lysate, but not in rabbit reticulocyte lysate or wheat germ extract. The efficiency of translation mediated by the 5´ IRES region was tested with and without cap analogue using various firefly and Renilla luciferase reporter constructs. They also used deletion mutants to identify the specific regions required for translation initiation. (3942)

Notes: These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200-400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard^® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis. (3740)

Notes: The authors of this study investigated the role of Lyn, a Src-family tyrosine kinase, in regulating the formation of the phagosome in alveolar macrophages in response to Psuedomonas aeruginosa (PA) infection. The Kinase-Glo^® Assay was used to assess Lyn activity, using acid-denatured enolase as the substrate. The authors found that Lyn kinase activity was increased following infection with PA. (3929)

Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT^® Coupled Transcription Translation System and [³⁵S] methionine. (3889)

Notes: These authors studied the effect of specific amino acid substitutions in the N^pro gene of swine fever virus. The N^pro gene encodes a non-strucutral protein that prevents interferon production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3). N^pro also has an autoprotease function. Deletion of the entire N^pro region attenuates virulence. In this study, the authors showed that degradation of IRF3 and autoprotease activity are independent, structurally overlapping functions. In particular, they investigated the effect of specific amino acid substitutions that eliminated IRF3 interaction and degradation, but did not affect autoprotease activity. They showed that removal of IRF3 degradation activity of N^pro had only minimal effect on virulence in swine. The pGEM-T Vector was used to clone the amplified N^pro gene, and the CheckMate™ Flexi Vector Mammalian Two-Hybrid System was used for protein interaction studies. (3944)

Notes: The authors characterized masculinizing mutations of the female-promoting tra genes in Caenorhabditis briggsae (Cb-tra). Using RT-PCR, the authors monitored the levels of full-length Cb-tra mRNA and a novel splice variant; actin mRNA was amplified as a control. RT-PCR was carried out using the AccessQuick™ RT-PCR System and RNA from 5–10 worms per 50µl reaction. (3892)

Notes: HEK-293 were transiently transfected with adenovirus 5 DNA using FuGENE® 6 Reagent. Cells were seeded at 3 × 10⁶ cells/75 cm² and grown to 60% cell confluency prior to transfection. Cell transfections were 60–70% efficient. (4266)

Notes: The authors of this paper compared time-resolved, fluorescence energy transfer and ATP-based luminescent assays in ultrahigh-throughput screens (1536-well) for Rho-associated kinase II inhibitors. They found that both technologies are suitable for such a screen and perform similarly; however, they note that the ATP-based luminescent kinase assay provides an economical advantage. (3932)

Notes: The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel. (3836)

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

Notes: To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3983)

Notes: The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq^® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry. (3883)

Notes: Somatic cell nuclear transfer technique was used to generate human blastocyst-stage embryos using nuclei from adult male fibroblasts cell lines and enucleated oocytes. Genomic DNA was analyzed using the PowerPlex® 16 system to confirm the genetic identity of the blastocyst cells. (3952)

Notes: The authors describe the developmental validation of the Plexor^® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)

Notes: Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study investigated this link. As part of the study, MCF-7 cells were transfected with various pGL3 firefly luciferase reporter constructs and the control pRL-TK Vector expressing Renilla luciferase. Transfection conditions were as follows: MCF-7 cells were cultured in 24-well plates and transfection was performed using FuGENE® 6 transfection reagentwith a FuGENE:DNA ration of 3:1. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (4362)

Notes: The authors of this study created a cDNA library representative of the extracellular proteome (secreted proteins and the extracellular domains of transmembrane proteins). Each cDNA was individually transfected into 293T cells. Medium from the cDNA of each transfection was used in a suite of cell-based assays. The CellTiter-Glo^® Assay was used to screen for secreted factors from the cell lines expressing the cDNA that affected viability of twelve cell lines: human primary B cells, human primary T cells, human primary NK cells, human primary monocytes, A549 cells, Colo205 cells, U-118 cells, MDA-MB231 cells, PC3 cells, PANC1 cells, human primary skeletal muscle progenitor cells, and rat primary oligodendrocyte precursor cells. (3935)

Notes: The authors performed protein pull-down assays to characterize the interaction of ORAI1 and STIM1, two protein components of the calcium-release calcium current. His₆-STIM1 C terminus and ORAI1 were synthesized using the TNT® Coupled Reticulocyte Lysate System in the presence of ³⁵S, and His₆-STIM1 C terminus was immobilized using MagZ™ Binding Particles. An aliquot of the TNT® reaction expressing ORAI1 was added to the particles, and proteins were washed, eluted using increasing concentrations of imidazole (10–40mM) and analyzed by SDS-PAGE. In a second set of pull-down assays, His₆-STIM1 C terminus was used to pull down ORA1 N- and C-terminal fragments expressed as GST fusion proteins. The His₆-STIM1 C terminus protein was purified from transiently transfected HEK293 cells using the MagneHis™ Protein Purification System. (3781)

Notes: One potential source of antibiotic-resistant bacteria is food-producing animals. The authors examined the ability of protegrin-1 (PG-1), an antimicrobial peptide, to protect wildtype and transgenic mice expressing PG-1 against bacterial infection. As part of the cloning strategy to produce the PG-1 expression construct, the authors amplified and cloned full-length PG-1 into the pGEM^®-T Easy Vector. To test the bactericidal activity of PG-1 expressed in transgenic mice, radial diffusion assays were performed, in which test samples were added to a well containing E. coli and the clear antibacterial zone was measured. Two of the test samples were neutrophil secretions from the PG-1 transgenic mice and purified polyhistidine-tagged PG-1 protein, purified using the MagneHis™ Protein Purification System. (3896)