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Product Focus: BDNF Emax® ImmunoAssay System---Anti-ACTIVE® MAPK pAbBrain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action.Studies on apoptosis, especially in the CNS, continue to amass much information on the growth of neural cells both in normal development and in disease states. This recent report focuses on the interaction of brain-derived neurotrophic factor (BDNF) and NMDA in cerebellar granule (CG) neurons, in particular the role of BDNF in mediating the anti-apoptotic effect of NMDA. NMDA alone can rescue CG neurons in vitro induced to undergo apoptosis. (This effect is thought to be mediated by glutamatergic innervation of developing CG neurons.) The first supportive finding was that the protective effect of NMDA was mirrored by NMDA's ability to induce BDNF. Second, additional reagents that inhibited the action of BDNF, such as a Trk receptor antagonist and a BDNF blocking antibody, in turn diminished the protective effects of both BDNF and NMDA. Moreover, inhibitors of the phosphatidylinositol 3´-OH kinase (PI-3K) signal transduction cascade also reduced the protective effects of BDNF and NMDA. Inhibitors of MAP kinase kinase (MEK) and protein kinase A exhibited no effect. BDNF also increased phosphorylation of Akt, a serine kinase involved in mediating cellular survival and apoptosis in a broad range of cells. NMDA induced phosphorylation of Akt. Akt is a target of PI-3K. The BDNF Emax® ImmunoAssay System (Cat.# G7611) was used to quantitate BDNF in CG neurons. Anti-ACTIVE® MAPK pAb, Rabbit, (pTEpY) (Cat.# V8031) was used to detect active, dually phosphorylated MAPK. Conclusion: the NMDA-BDNF interaction could prove key to the normal development of CG neurons in vivo. Bhave, S.V., Ghoda, L. and Hoffman, P.L. (1999) J. Neurosci. 19,
3277. Product Focus: TransFast™ Transfection Reagent---Tfx™ -50 ReagentA simple method to transfer plasmid DNA into neuronal primary cultures: Functional expression of the mGlu5 receptor in cerebellar granule cells.Neurons, and other so-called terminally differentiated cells, can be extremely difficult to transfect. Virally mediated transfection is the most efficient method, but the production of the recombinant virus remains complex and time-consuming. Also, replication of the virus within the cell can prove toxic. Cationic lipid reagents offer easy reagent preparation as well as easy transfection protocols. Therefore, a study was undertaken to investigate transfection of primary mammalian neuronal cultures with seven commercially available cationic lipid reagents. Green fluorescent protein (GFP) was used as the reporter, and successful transfection was judged by the percentage of cells transfected and the toxicity of the transfection. Mouse cerebellar neuron cultures were grown on poly-L-ornithine-coated glass cover slips (~1 x 105 cells total; 95% granule cells and 5% nongranule cells) for initial optimization experiments. Transfections were optimized for the transfection reagent:DNA ratio and increasing amounts of DNA at the optimized ratio. TransFast™ Transfection Reagent(a) (Cat.# E2431) provided the greatest transfection efficiency with an overall transfection efficiency of 5% (2% granule cells/3% nongranule cells) with no toxicity. The Tfx™ -50 Reagent(b) (Cat.# E1811) ranked second and was non-toxic also. Three of the other reagents were nontoxic to the cells but produced low transfection efficiencies (e.g., 0.3-0.06%). Two of the reagents tested were toxic to the cells. All of the transfection reagents were compared on the same day with the same cell cultures and each data point consisted of nine determinations. The TransFast™ Reagent was most effective at a 1:1 ratio (3µl TransFast™ Reagent/µg DNA) prepared in 1ml of serum-free medium. The TransFast™ Reagent method was also tested on cultured mouse collicular and striatal neurons, and the reagent proved nontoxic and produced comparable transfection efficiencies. The time point of transfection following plating the cells was important: Greatest transfection efficiency into granule cells (~2%) was seen when transfection occurred one day (~24 hours) after plating. Granule cell transfection decreased to 0.2% when transfections were delayed to 48 hours. The number of GFP-expressing cells increased for the first three days after transfection and then remained stable for two weeks. The GFP transfection did not affect the overall electrophysiology of the cells as judged by patch-clamp techniques measuring Na+, Ca2+ and K+ currents. Following optimization trials, cerebellar cultures were transfected with both GFP and the metabotropic glutamate receptor type 5 (mGlu5). Over 90% of the GFP-expressing neurons also expressed the mGlu5 as judged by immunostaining for a hemagglutinin tag fused to the mGlu5 protein cDNA. Stimulation of the mGlu5 receptors produced single big K+ channel activity as it was the case of the native mGlu1 receptor. Therefore, the mGlu5 receptor was functional in the transfected neurons and may share a common coupling mechanism to big K+ channels in neurons. Ango, F.1, Albani-Torregrossa, S.2, Joly, C.1, Robbe, D.1, Michel, J.-M.1, Pin, J.-P.1, Bockaert, J.1 and Fagni, L.1 (1999) Neuropharmacology 38, 793. 1 UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France; 2Department of Pharmacology, University of Florence, Vialle Morgagni, 65, 50134 Florence, ItalyProduct Focus: Recombinant Human BDNF---Anti-Human BDNF pAb---CellTiter 96® Non-Radioactive Cell Proliferation Assay---DeadEnd™ Colorimetric Apoptosis Detection SystemFunctionally antagonistic interactions between the TrkA and p75 neurotrophin receptors regulate sympathetic neuron growth and target innervation.The neurotrophic factor hypothesis states that neuronal growth and survival are regulated by neurotrophic factors and implies that such positive signals are sufficient to determine both the life and death of a developing neuron. Recent studies demonstrated that sympathetic neuron survival is not only determined by TrkA receptor stimulation but is also regulated by negatively acting neurotrophins such as BDNF, which signals through the p75NTR to mediate neuronal apoptosis. In cultured sympathetic neurons that themselves synthesize BDNF, exogenous BDNF inhibits, and function-blocking BDNF antibodies enhance, neuron process outgrowth. Thus, naturally occurring sympathetic neuron death is regulated by positively and negatively acting neurotrophins that signal through TrkA versus p75NTR. The authors of this paper used Recombinant Human BDNF (Cat.# G1491), Anti-Human BDNF pAb (Cat.# G1641), Chicken IgY, Control Immunoglobulin (Cat.# G1161), CellTiter 96® Non-Radioactive Cell Proliferation Assay (Cat.# G4000) and the DeadEnd™ Colorimetric Apoptosis Detection System (Cat.# G7130). Kohn, J.1, Aloyz, R.S.1, Toma, J.G.1, Haak-Frendscho, M.2 and Miller, F.D.1 (1999) J. Neurosci. 19, 5393. 1 Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 Canada, and 2Department of Immunology, Promega Corporation, Madison, WI 53711 USA.Product Focus: pGEM®-T Vector System---Beta-Actin Primer PairInsertion of a retrotransposon in Mbp disrupts mRNA splicing and myelination in a new mutant rat.The ability of a neuron to conduct an action potential is facilitated by the insulating properties of the myelin sheath that surrounds it. This sheath is an extension of the glial cell plasma membrane that has wrapped itself repeatedly around the axon to form a multilamellar structure. Although myelin is composed primarily of lipids, it also contains a significant amount of key proteins that serve to provide structural integrity to the sheath. The most abundant proteins in CNS myelin are proteolipid protein (PLP) and myelin basic protein (MBP). Myelination of the rodent CNS occurs primarily during postnatal development and requires the coordinated expression of the genes synthesizing the structural proteins and enzymes necessary for myelin sheath formation. The study of spontaneous mutants in myelination has greatly enhanced our understanding of the functions of the various myelin proteins. The authors of this paper describe a unique genetic defect in a new myelin mutant called the Long Evans shaker (les) rat that causes severe dysmyelination of the CNS. The myelin defect results from the disruption of the myelin basic protein (Mbp) gene by the insertion of an endogenous retrotransposon into a noncoding region (intron 3) of the gene. This insertion results in altered splicing of the gene and decreased levels of full-length transcript, as well as the presence of improperly spliced chimeric transcripts. The les rat is an excellent model to study the alternative functions of MBP beyond its role in myelin sheath formation. The authors used the pGEM®-T Vector System(c,d) (Cat.# A3600 and A3610) to confirm the identity of the alternatively spliced isoforms of MBP mRNA following amplification. In addition, the levels of MBP mRNA in mutant brains were estimated by quantitative autoradiography of MBP RT-PCR products that had been normalized to actin mRNA levels using the Beta-Actin Primer Pair (Cat.# G5740). O’Connor, L.T.1, Goetz, B.D.1, Kwiecien, J.M.1, Delaney, K.H.2, Fletch, A.L.2 and Duncan, I.D.1 (1999) J. Neurosci. 19, 3404. 1 Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706 USA, and 2Department of Pathology, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5 Canada.Product Focus: T7 RNA Polymerase---Rabbit Reticulocyte Lysate, Nuclease Treated---Mung Bean NucleaseIn vitro virus: Bonding of mRNA bearing puromycin at the 3´-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro.The connection of a genotype molecule to the phenotype molecule is necessary for evolution to proceed smoothly. At present, the following methods have been proposed to allow for evolutionary molecular engineering: phage display, polysome display, ribosome display, encoded combinatorial library, and cellstat. An "in vitro virus" was constructed in which a genotype molecule (mRNA) binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Puromycin resembles the 3´ end of an aminoacyl-tRNA molecule, and this is readily capable of entering the ribosomal A site to be transferred to the nascent polypeptide chains by peptidyl transferase. The scheme for such a process includes: in vitro virus genome synthesis, cell-free translation, puromycin binding to polypeptides, ribosome release, selection process. The genotype molecule was comprised of mRNA encoding a protein (N-terminal half of human tau protein), DNA spacer (105mer), P-acceptor (21mer DNA molecule with 4-mer RNA at the 3´-terminal end), and rCpPur (ribocytidyl-(3´-5´)-puromycin). The mRNA template was synthesized by in vitro transcription using T7 RNA Polymerase (Cat.# P2075). Protein having rCpPur at the C-terminus was efficiently synthesized when a genome without a stop codon and with a DNA spacer was used as an mRNA template in a cell-free translation system consisting of Rabbit Reticulocyte Lysate, Nuclease Treated(e,f) (Cat.# L4960). Covalent binding of a genome having 32P-labeled rCpPur at the 3´-terminal end to the C-terminal of the encoded protein was confirmed by treatment with Mung Bean Nuclease (Cat.# M4311) following translation. The efficiency of intermolecular binding of the genome to the encoded protein was approximately 10%. Thus the in vitro virus is an in vitro alternative to phage display for linking genotype and phenotype in molecular evolution studies. Nemoto, N.1, Miyamoto-Sato, E.1,2, Husimi, Y.3 and Yanagawa, H.1 (1997) FEBS Lett. 414, 405. 1 Mitsubishi Kasel Institute of Life Sciences, Mochida, Tokyo 194 Japan, 2Yokohama National University, Hodogaya-ku, Yokohama 240 Japan, and 3Saitama University, Urawa, Satama 336 Japan.Product Focus: Rabbit Reticulocyte Lysate, Nuclease Treated---TNT® Coupled Reticulocyte Lysate SystemMechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia.The molecular processes involved in long-term facilitation (LTF) in Aplysia are thought to include a cascade leading to the synthesis of proteins needed for an enduring increase in synaptic effectiveness. This cascade begins when the catalytic subunit of cAMP-dependent protein kinase (PKA), together with MAP kinase, phosphorylates and activates cAMP response element-binding protein (CREB) activators and repressors. The authors of this paper were interested in investigating the downstream events produced by CREB. Persistently active PKA is crucial for establishing LTF in Aplysia, and autonomous PKA is generated through the ubiquitin-proteosome-mediated proteolysis of the PKA regulatory subunits. In this multienzyme pathway, protein substrates are targeted for degradation by ligation to ubiquitin and subsequent proteolysis by the 26S proteosome. Using various proteosome inhibitors, the authors were able to demonstrate that the degradation of the regulatory subunits occurs early in the LTF process and appears to be a key function of proteosome in LTF. The regulatory subunits are degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of expression of a ubiquitin carboxy-terminal hydrolase. The authors used Promega’s Rabbit Reticulocyte Lysate, Nuclease Treated(e,f,) (Cat.# L4960), as a source of the ubiquitin-mediated protein degradation machinery, as well as the TNT® Coupled Reticulocyte Lysate System(e,f,g) (Cat.# L4600) for the in vitro expression of 35S-Methionine-labeled recombinant PKA regulatory proteins, which were used in subsequent in vitro proteosome degradation experiments. Chain, D.G.1, Casadio, A.1, Schacher, S.1, Hedge, A.N.1, Valbrun, M.1, Yamaoto, N.1, Goldberg, A.L.2, Bartsch, D.1, Kandel, E.R.1 and Schwartz, J.H.1,2 (1999) Neuron 22, 147. 1 Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, NY 10032 USA, and 2Department of Cell Biology, Harvard Medical School, Boston, MA 02115 USA.Product Focus: Apoptosis Detection System, FluoresceinFas ligand: A sensor for DNA damage critical in skin cancer etiology.Cells that suffer DNA damage induced by UV light can either repair the DNA or undergo p53-dependent apoptosis to eliminate precancerous cells in the skin. The p53 protein is essential for the transcriptional up-regulation of Fas ligand (FasL) in DNA-damaged keratinocytes. Fas ligand is a pro-apoptotic protein. In this paper the authors report that sunburn cell (apoptotic keratinocyte) formation is dependent on Fas ligand (FasL). Exposure of mice to UV-B light (5kj/m2) caused 70% of mutant FasL-deficient mice and 5% of wildtype FasL mice to accumulate p53 mutations in the epidermis. Thus, Fas ligand-mediated apoptosis is important for maintaining skin homeostasis, and dysregulation of the Fas-FasL interaction may be central to the development of skin cancer. The authors analyzed mutations (C to T transitions at codon 270 and 275) in the p53 gene by PCR. In addition, skin sections from UV-B irradiated and nonirradiated mice were harvested at 0 and 24 hours and examined for apoptosis using the Promega Apoptosis Detection System, Fluorescein (Cat.# G3250), which is based on the TUNEL technique to identify apoptotic cells. Hill, L.L., Ouhtit, A., Loughlin, S.M., Kripke, M.L., Ananthaswamy, H.N. and Owen-Schaub, L.B. (1999) Science 285, 898. Department of Immunology, University of Texas, M.D. Anderson Cancer Research, Houston, TX 77030 USA. Product Focus: GeneEditor™ in vitro Site-Directed Mutagenesis System Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.The enzyme acyl-coenzymeA:cholesterol acyltransferase (ACAT) catalyses the formation of cholesteryl esters from cholesterol and long-chain fatty acids. Cholesteryl esters serve as the intracellular storage form of cholesterol. Thus, ACAT is an important regulator of cholesterol levels within cells and ACAT has been the target of the pharmaceutical industry due to its potential role in atherosclerosis. Researchers in this paper tagged the ACAT-1 protein in order to study the topology of the tagged proteins expressed in Chinese hamster ovary cells. Using the GeneEditor™ in vitro Site-Directed Mutagenesis System(h) (Cat.# Q9280), a 67bp primer was used to insert a hemagglutinin tag into the ACAT-1 gene at several sites. The plasmid constructs were transfected into mutant CHO cells lacking endogenous ACAT and the expressed protein was examined by immunocytofluorescence. The ACAT-1 protein localizes mainly to the endoplasmic reticulum, as predicted, as well as to the juxtanuclear region (minor component). The results suggest a seven-transmembrane model for ACAT-1 with one large cytoplasmic domain (N-terminus), one large lumenal domain, three small cytoplasmic loops and three small lumenal domains. Lin, S.1, Cheng, D.1, Liu, M.-S.2, Chen, J.1 and Chang, T.-Y.1 (1999) J. Biol. Chem. 274, 23276. Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755 and 2Beckman Coulter Inc., Fullerton, CA 92634 USA (a)The cationic lipid component of the TransFast™ Transfection Reagent is covered by U.S. Pat. Nos. 5,824,812, 5,869,715 and pending foreign patents. (b)The cationic lipid component of the Tfx™ Reagents is covered by U.S. Pat. Nos. 5,527,928, 5,744,625 and pending foreign patents. (c)U.S. Pat. No. 4,766,072. (d)Licensed under one or both of U.S. Pat. No. 5,487,993 and European Pat. No. 0 550 693. (e)U.S. Pat. No. 5,283,179 and other patents. Certain applications of this product may require licenses from others. (f)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. (g)U.S. Pat. Nos. 5,492,817 and 5,665,563 and other patents. (h)U.S. Pat. No. 5,780,270. Anti-ACTIVE, CellTiter 96, Emax, pGEM and TNT are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. DeadEnd, GeneEditor, Tfx and TransFast are trademarks of Promega Corporation. |
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