Product Focus: Anti-ACTIVE^® JNK pAb, Rabbit, (pTPpY)

Regulation of JNK by Src during Drosophila development.

In Drosophila the Hemipterous (Hep) protein, which is homologous to the mammalian MAPK kinase 7, and Basket (Bsk), which is the Drosophila Jun amino-terminal kinase (DJNK), are required for dorsal closure of the embryonic epidermis and thoracic closure of the pupal epidermis. The authors screened a collection of semilethal P-element-inserted mutants with a dorsal midline cleft phenotype. A Src42A mutant (Jp45) with a P-element insertion in the 5´ UTR of the Src42A gene displayed this phenotype. The phenotype of the Src42A^Jp45 mutant was dominantly enhanced by mutations in hep or bsk. In addition, a gene dosage reduction of puckered, which encodes a phosphatase known to inactivate Bsk, suppressed the phenotypic severity of the Src42A^Jp45 mutation. Taken together, these results indicate that Src42A may function in the Bsk pathway during morphogenesis. To investigate whether Src42A can activate Bsk, the authors forced expression of Src42A ubiquitously in larvae via a heat-shock Src42A construct (heat-shock-inducible GAL4 promoter driving expression of Src42A). In immunoblots of larvae carrying the heat-shock construct, the total amount of Bsk protein remained unchanged before and after heat shock. However, the amount of phosphorylated BSK increased with heat-shock, as assayed using Promega's Anti-ACTIVE^® JNK pAb, Rabbit, (pTPpY), (Cat.# V7931, V7932).

Tateno, M., Nishida, Y. and Adachi-Yamada, T. (2000) Science 287, 324.

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 Japan

Product Focus: Wizard^® PureFection Plasmid DNA Purification System---Passive Lysis Buffer

Rac1 regulates Interleukin 1-induced nuclear factor kappaB activation in an inhibitory protein kappaBalpha-independent manner by enhancing the ability of the p65 subunit to transactivate gene expression.

The authors of this study provide evidence that Rac1 does not participate in the IL1-induced signaling pathway leading to IkappaBalpha phosphorylation and degradation. Rather, Rac1 enhances the ability of the NFkappaB p65 subunit to transactivate gene expression. The authors used the Wizard^® PureFection Plasmid DNA Purification System (Cat.# A2150) to purify endotoxin-free plasmid DNA for use in transfection experiments. Using DEAE-dextran, EL4.NOB-1 cells were transfected with purified plasmid carrying an NFkappaB-dependent reporter kappaB-luciferase or cotransfected with kappaB-luciferase and a constitutively active RacV12; IkappaBctag and RacV12 or RacV17 (a dominant-negative mutant); and a Gal-luciferase reporter plasmid, Gal4-p65 and RacV12. After a period of recovery, cells were stimulated with IL1 (10ng/ml for 3 hours) and then lysed with Promega's Passive Lysis Buffer (Cat.# E1941). Luciferase activity was determined using standard procedures. The transfected RacV12 was unable to drive kappaB-dependent reporter gene activity, although it potentiated the IL1 response. In contrast, RacV12 alone drove the response in the AP1 reporter system, possibly by activation of JNK or p42/p44 MAPK pathways.

Jefferies, C.A. and O'Neill, L.A.J. (2000) J. Biol. Chem. 275, 3114.

Department of Biochemistry and Biotechnology Institute, Trinity College, Dublin 2 Ireland

Product Focus: MEK Inhibitor U0126

The MEK pathway is required for stimulation of p21^WAF1/CIP1 by transforming growth factor-beta. 

Transforming growth factor-beta (TGF-beta) is a growth factor that induces growth arrest in late G1 of the cell cycle. This induction of cell cycle arrest may be mediated by increased expression of the cyclin-dependent kinase inhibitors p21 and p15. The authors of this report investigated the signaling pathways involved in p21 induction in HaCaT cells. TGF-beta can regulate the mitogen-activated protein kinase (MAPK) pathway, leading to increased activity of the transcription factor Elk. Constitutively active components of the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway decrease p21 expression in HaCaT cells. The authors used the MEK Inhibitor U0126 (Cat.# V1121) to demonstrate the importance of the MAPK signaling pathway in p21 induction in this cell type. HaCaT cells were either pretreated with the MEK Inhibitor U0126 (10-70µM) for 30-60 minutes prior to treatment with TGF-beta or added at the time of TGF-beta addition for a total incubation time of 18 hours.

Hu, P.P., Shen, X., Lui, Y., Counter, C. and Wang, X. (1999) J. Biol. Chem. 274, 35381.

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC  27710 USA

Product Focus: MEK Inhibitor U0126

MEK inhibitors block BDNF-dependent and -independent expression of GABA_A receptor subunit mRNAs in cultured mouse cerebellar granule neurons.

Brain-derived neurotrophic factor (BDNF) can regulate the maturation of developing cerebellar granule neurons, which generally occurs during postnatal development in rodents. Within 1-2 days of culture, BDNF induces the expression of granule neuron terminal differentiation markers, such as GABA_A receptor alpha6 subunit (GABA_Aalpha-6) mRNA. In the absence of BDNF, GABA_Aalpha-6 mRNA expression does not occur until 3-4 days of culture. The BDNF-dependent expression of GABA_Aalpha-6 mRNA could be effectively blocked by treatment with mitogen activated protein kinase kinase (MEK) inhibitors. These results suggest that both BNDF-dependent and -independent expressions of GABA_A receptor subunits require the activation of MEK and the MAPK signaling pathway. The authors also used the MEK Inhibitor U0126 (Cat.# V1121) in their studies. Cerebellar granule cells were treated with 5-50µM U0126 for 24-48 hours in culture.

Bulleit, R.F.^1,2 and Hsieh, T.¹ (2000) Dev. Brain Res. 119, 1.

¹Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD 21201 USA; ²Promega Corporation, Madison, WI 53711 USA

Product Focus: Rabbit Reticulocyte Lysate, Nuclease Treated---Canine Pancreatic Microsomal Membranes

Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease.

Malaria is caused by a parasitic protozoa of the genus Plasmodium. The merozoite surface protein-1 is an abundant, stably expressed surface protein that is subjected to proteolytic processing and shedding during the invasion of erythrocytes. Pl. falciparum subtilisin-like protease-1 (PfSUB-1) is a protein belonging to the subtilisin-like superfamily of serine proteases and is a possible candidate for the protease responsible for surface protein-1 processing. Proteases that mediate important roles in the life cycle of such pathogenic organisms represent potential targets for protease inhibitor-based drugs. The authors investigated the mode of activation of PfSUB-1 to evaluate its potential as a target for therapeutic drugs. In vitro- translated PfSUB-1 showed no capacity to undergo autocatalytic processing. However, parasite extracts contain a protease that cleaves the in vitro-translated pro-protein. The authors used Rabbit Reticulocyte Lysate, Nuclease Treated^(a,b) (Cat.# L4960), to synthesize the PfSUB-1 pro-protein in vitro. In some experiments, in vitro translation was performed in the presence of Canine Pancreatic Microsomal Membranes^(c) (Cat.# Y4041), and glycosylation of PfSUB-1 in these reactions was inhibited by the addition of the glycosaccharide acceptor peptide Bz-NGT-NH2.

Sajid, M.¹, Withers-Martinez, C.² and Blackman, M.J.² (2000) J. Biol. Chem. 275, 631.

¹Tropical Disease Research Unit, University of California, San Fransisco, San Fransisco, CA  94121 USA; ²Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

Product Focus: Access RT-PCR System

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses.

Feline coronavirus (FCoV) is known to be highly prevalent in the cat population and can result in feline infectious peritonitis (FIP), a potentially fatal infectious disease in cats. The authors developed a one-tube reverse transcription-polymerase chain reaction (RT-PCR) assay for the quantitative detection of feline coronavirus. A fluorogenic probe was designed to bind between the two PCR primers to target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye. During the polymerase extension steps, the Tfl exonuclease activity cleaved the hybridized probe resulting in fluorescence emission of the reporter dye. The resulting fluorescence can then be monitored in real time after each cycle. The authors used the Access RT-PCR System^(d) (Cat.# A1260, A1250, A1280) for the generation of the feline coronavirus RT-PCR product. This citation also demonstrates the compatibility of the Access RT-PCR System with the TaqMan^® probe technology (The Perkin-Elmer Corporation).

Gut, M.¹, Leutenegger, C.M.¹, Huder, J.B.², Pedersen, N.C.³ and Lutz, H.J.¹ (1999) Virol. Meth. 77, 37.

¹Clinical Laboratory, Department of Veterinary Medicine, University of Zurich, Winterthurerstrasse 260, Ch-8057 Zurich, Switzerland; ²Swiss National Center for Retroviruses, University of Zurich, Zurich, Switzerland; ³Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis   95616 USA

^(a)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.

^(b)U.S. Pat. No. 5,283,179, Australian Pat. No. 649289 and other patents. Certain applications of this productt may require licenses from others.

^(c)U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, which claim vectors encoding a portion of human placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation.

^(d)The PCR process is covered by patents issued and applicable in certain countries. Pormega does not enocurage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

Anti-ACTIVE and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.

TaqMan is a registered trademark of Roche Molecular Systems, Inc.

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