ABSTRACT
Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.
Subject(s)
MAP Kinase Kinase Kinase 1 , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , Apoptosis , Cell Division , Embryo, Mammalian/pathology , Genes, Essential , Genotype , Heterozygote , Homozygote , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Phenotype , Proto-Oncogene Proteins c-raf/genetics , Signal Transduction , Yolk Sac/blood supply , Yolk Sac/pathologyABSTRACT
The gene encoding E. coli nitroreductase (NTR) was expressed in the luminal cells of the mammary gland of transgenic mice using the ovine beta-lactoglobulin promoter. Treatment of NTR expressing animals with the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) resulted in a rapid and selective killing of this population of cells whereas the closely associated myoepithelial cells were unaffected. NTR-mediated inducible cell ablation offers a number of advantages over the use of HSV1-tk for the selective killing of cells in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Aziridines/pharmacology , Genetic Therapy/methods , Nitroreductases/genetics , Prodrugs/pharmacology , Animals , Apoptosis/drug effects , Cell Culture Techniques , Escherichia coli/enzymology , Escherichia coli/genetics , Female , Gene Expression , Immunoenzyme Techniques , Lactation , Lactoglobulins/genetics , Mammary Glands, Animal/cytology , Mammary Glands, Animal/drug effects , Mice , Mice, Transgenic , Nitroreductases/metabolismSubject(s)
Arthritis, Rheumatoid/immunology , Immunoglobulin G/chemistry , Oligosaccharides/chemistry , Arthritis, Rheumatoid/blood , Chromatography, DEAE-Cellulose , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes , Glycosylation , Humans , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Oligosaccharides/isolation & purification , Reference ValuesABSTRACT
Following previous studies of verapamil reversal of chloroquine resistance in malaria and multi-drug resistance in cancer cells, the effect of verapamil was investigated on nifurtimox-resistant Trypanosoma cruzi in vitro and antimony-resistant Leishmania donovani in vitro and in vivo. Verapamil alone was not active against either parasite, but in combination with nifurtimox it reversed the drug resistance of T. cruzi and in combination with sodium stibogluconate reversed the drug resistance of L. donovani.
Subject(s)
Antimony Sodium Gluconate/pharmacology , Gluconates/pharmacology , Leishmania donovani/drug effects , Nifurtimox/pharmacology , Nitrofurans/pharmacology , Trypanosoma cruzi/drug effects , Verapamil/pharmacology , Animals , Drug Resistance , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Mice , Mice, Inbred BALB CABSTRACT
Sinefungin, a C-nucleoside antibiotic, displays a high antiparasitic action. The effect of free and microencapsulated sinefungin was compared on BALB/c mice infected with Leishmania donovani; the encapsulated form proved more effective by one order of magnitude.
