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In this paper, lentiviral delivery of glial cell line-derived neurotrophic factor ("lenti-GDNF") was administered to nonhuman primate model of Parkinson's disease (MPTP-treated) in efforts to detect any trophic effects. Extensive GDNF expression was seen in all animals, control as well as test subjects. The GDNF Emax® ImmunoAssay System (Cat.# G7620) was used to measure GDNF levels in tissue extract (brain punch) supernatants. Some lysates were diluted to ensure samples were in the linear range of the assay (using the standard curve), and concentrations of GDNF were calculated against a standard curve constructed using six data points and then adjusted to GDNF (pg) / total protein (mg).
Kordower, J.H.1*, Emborg, M.E.1, Bloch, J.2, Ma, S.Y.1, Chu, Y.1, Leventhal, L.1, McBride, J.1, Chen, E.-Y.1, Palfi, S.1, Roitberg, B.Z.1, Brown, W.D.4, Holden, J.E.3,4, Pyzalski, R.4, Taylor, M.D.3, Carvey, P.5, Ling, Z.5, Trono, D.6, Hantraye, P.7, Déglon, N.2 and Aebischer, P.2,8 (2000) Science 290, 767–773.
1Department of Neurological Sciences and 5Department of Pharmacology, Rush Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA; 2Division of Surgical Research and Gene Therapy Center, Lausanne University Medical School, Lausanne, Switzerland; 3Department of Medical Physics and 4Department of Radiology, University of Wisconsin, Madison, WI 53706, USA; 6Department of Genetics and Microbiology, Faculty of Medicine, University of Geneva, Geneva, Switzerland; 7Commissariat a l'Energie Atomique (CEA), CNRS, Unite de Recherche Associe (URA), 2210 Service Hospitalier Frederic Joliot, CEA, Direction des Sciences du Vivant (DSV), Departement de Recherche Medicale (DRM), Orsay cedex, France; and 8Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland.
*To whom correspondence should be addressed. E-mail: jkordowe@rush.edu
Here the researchers were interested in physiological actions following substitution of the NT-4 (neurotrophin-4) gene for the BDNF (brain-derived neurotrophic factor) gene, two neurotrophic factors that engage the TrkB and p75NTR receptors. The coding sequence of BDNF was replaced with that for NT-4 in mice (Bdnfnt4-ki). These animals were viable, and NT-4 supported a greater number of sensory neurons (compared to BDNF) and promoted functional synapse formation in hippocampal neurons in vitro. Mice homozygous for the genetic substitution showed reduced body weight, infertility and skin lesions indicating differential response by the TrkB receptor. ELISAs for BDNF and NT-4 were performed using the BDNF (Cat.# G7611) and NT-4 (Cat.# G7651) Emax® ImmunoAssay Systems. In vitro gene reporter assay studies were also performed using the Dual-Luciferase® Reporter Assay System(*) (Cat.# E1910) on cultures of hippocampus and cortical cells treated with NT-4 or BDNF.
Fan, G.1, Egles, C.2,3, Sun, Y.4, Minichiello, L.5, Renger, J.J.2,3, Klein, R.5, Liu, G.2,3 and Jaenisch, R.1,2* (2000) Nat. Neuroscience 3, 350–357.
1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; 2Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA; 3Center for Learning and Memory and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 4Division of Neuroscience, Children's Hospital and Department of Neurobiology, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA; and 5European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
*To whom correspondence should be addressed. E-mail: jaenisch@wi.mit.edu
Many apoptotic molecules relocate subcellularly in cells undergoing apoptosis. The pro-apoptotic protein BID underwent posttranslational (rather than classic cotranslational) N-myristoylation when cleavage by caspase-8 caused exposure of a glycine residue. N-myristoylation enabled the targeting of a complex of p7 and myristoylated p15 fragments of BID to artificial membranes bearing the lipid composition of mitochondria as well as to intact mitochondria. This postproteolytic N-myristoylation serves as an activating switch, enhancing BID-induced release of cytochrome c and cell death. The TNT® T7 Coupled Reticulocyte Lysate System(*) (Cat.# L4610) was used for transcription and translation.
Jiping, Z., Weiler, S., Oh, K.J., Wei, M.C. and Korsmeyer, S.J.* (2000) Science 290, 1761–1765.
Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Departments of Pathology and Medicine, Harvard Medical School, Boston, MA 02115, USA.
*To whom correspondence should be addressed. E-mail: stanley_korsmeyer@dfci.harvard.edu
In this report, binding partners of b-arrestin 2 were identified by yeast two-hybrid screening as well as by coimmunoprecipitation in both mouse brain extracts and cotransfected COS-7 cells. JNK3 (c-Jun amino-terminal kinase 3) as well as the upstream JNK activators, ASK1 (apotosis signal-regulating kinase 1) and MAPK (mitogen-activated protein kinase), were found associated with a b-arrestin 2 complex. Stimulated angiotensin II type 1A receptor activated JNK3, leading to the colocalization of b-arrestin 2 complex and active JNK3 in intracellular vesicles. Anti-ACTIVE® JNK pAb (Cat.# V7931) was used to to detect phosphorylated (active) JNK in HEK-293 cells transfected with angiotensin II type 1A receptor, b-arrestin 2 and JNK3.
McDonald, P.H.1, Chow, C-W.2, Miller, W.E.1, Laporte, S.A.3, Field, M.E.1, Lin, F-T.1, Davis, R.J.2 and Lefkowitz, R.J.1* (2000) Science 290, 1574–1577.
1Howard Hughes Medical Institute and Departments of Medicine, Cardiology, and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710, USA; 2Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA; 3Department of Cell Biology, Duke University Medical Center, Box 3827, Durham, NC 27710, USA.
*To whom correspondence should be addressed.