ABSTRACT
Vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that can be activated by capsaicin and other vanilloids as well as by protons and heat. In the present study, we have analyzed the oligomeric state of VR1. Co-immunoprecipitation of differently tagged VR1 molecules indicated that VR1 can form oligomers. Using two different heterologous VR1 expression systems as well as endogenous VR1 expressed in dorsal root ganglion cells, we analyzed oligomer formation using perfluoro-octanoic acid polyacrylamide gel electrophoresis. Results were confirmed both with chemical cross-linking agents as well as through endogenous cross-linking mediated by transglutaminase. Our results clearly show that VR1 forms multimers in each of the expression systems with a homotetramer as a predominant form. The oligomeric structure of VR1 may contribute to the complexity of VR1 pharmacology. Finally, differences in glycosylation between the systems were observed, indicating the need for caution in the use of the heterologous expression systems for analysis of VR1 properties.
Subject(s)
Receptors, Drug/chemistry , Animals , Blotting, Western , CHO Cells , COS Cells , Caprylates/chemistry , Cricetinae , Cross-Linking Reagents/pharmacology , Dimerization , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fluorocarbons/chemistry , Green Fluorescent Proteins , Ligands , Luminescent Proteins/metabolism , Plasmids/metabolism , Precipitin Tests , Protein Structure, Quaternary , Protons , Recombinant Fusion Proteins/metabolism , TRPV Cation Channels , Transfection , Transglutaminases/metabolismSubject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Endopeptidases/metabolism , Norbornanes/chemical synthesis , Protease Inhibitors/chemical synthesis , Sulfonamides/chemical synthesis , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/biosynthesis , Aspartic Acid Endopeptidases , Cell Line , Humans , Norbornanes/chemistry , Protease Inhibitors/chemistry , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to down-regulate GDNF-induced biological activity.
Subject(s)
Antibodies, Monoclonal/immunology , Drosophila Proteins , Nerve Growth Factors , Nerve Tissue Proteins/immunology , Animals , Antibody Specificity , Biosensing Techniques , Blotting, Western , Choline O-Acetyltransferase/pharmacokinetics , Dopamine/pharmacokinetics , Enzyme-Linked Immunosorbent Assay/methods , Female , Glial Cell Line-Derived Neurotrophic Factor , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/pharmacology , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ret , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant ProteinsABSTRACT
Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.
Subject(s)
Dopamine/physiology , Mesencephalon/physiology , Nerve Tissue Proteins/pharmacology , Animals , Behavior, Animal/drug effects , Glial Cell Line-Derived Neurotrophic Factor , Immunohistochemistry , Male , Mesencephalon/metabolism , Nerve Growth Factors/pharmacology , Neural Pathways/drug effects , Neural Pathways/physiology , Rats , Rats, Inbred F344ABSTRACT
A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific; GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration of midbrain dopaminergic neurons.
Subject(s)
Dopamine/biosynthesis , Mesencephalon/drug effects , Nerve Growth Factors , Nerve Tissue Proteins/pharmacology , Neuroglia/metabolism , Neurons/drug effects , Amino Acid Sequence , Animals , Astrocytes/cytology , Astrocytes/drug effects , Base Sequence , Cell Differentiation/drug effects , Cell Line , Cell Survival/drug effects , Cells, Cultured , Cloning, Molecular , Glial Cell Line-Derived Neurotrophic Factor , Humans , Mesencephalon/cytology , Mesencephalon/metabolism , Molecular Sequence Data , Molecular Weight , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Neurons/cytology , Neurons/metabolism , Parkinson Disease/drug therapy , RatsABSTRACT
Ciliary neurotrophic factor (CNTF) is one of a small number of proteins with neurotrophic activities distinct from nerve growth factor (NGF). CNTF has now been purified and cloned and the primary structure of CNTF from rabbit sciatic nerve has been determined. Biologically active CNTF has been transiently expressed from a rabbit complementary DNA clone. CNTF is a neural effector without significant sequence homologies to any previously reported protein.
Subject(s)
Nerve Growth Factors/genetics , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Cell Line , Ciliary Neurotrophic Factor , Cloning, Molecular , DNA/genetics , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/isolation & purification , Rabbits , Recombinant Proteins/biosynthesis , Sciatic Nerve/metabolism , TransfectionABSTRACT
The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED50 = 20-25 mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED50 = 10.8 +/- 7.0 nM). In contrast, chemically closely related dihydropyridines, PN200-110 (ED50 = 0.33 +/- 0.15 nM) and nitrendipine (ED50 = 1.3 +/- 0.3 nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED50 = 0.78 +/- 0.38 microM), the benzothiazepine diltiazem (ED50 = 1.7 microM), and the inorganic ion cadmium (ED50 = 5.8 microM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve growth factor, basic fibroblast growth factor, or ciliary neurotrophic factor, also suggesting that voltage-gated calcium channels are not involved in the action of these factors. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.
Subject(s)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Calcium Channels/physiology , Calcium/physiology , Dihydropyridines/pharmacology , Neurons/cytology , Potassium/physiology , Animals , Calcium Channel Blockers , Calcium Channels/drug effects , Cell Count , Cell Survival , Cells, Cultured , Chick Embryo , Membrane Potentials/drug effects , Neurons/drug effects , Neurons/physiologyABSTRACT
Exoenzyme S is an extracellular ADP-ribosyltransferase enzyme produced by Pseudomonas aeruginosa. Mutants of Pseudomonas aeruginosa deficient in this enzyme have been shown to have reduced virulence in infections of burned mice. The contribution of exoenzyme S to the pathogenesis of chronic lung infections with this organism was evaluated by examining the incidence of exoenzyme S production by Pseudomonas aeruginosa strains isolated from cystic fibrosis patients and comparing an exoenzyme S deficient mutant and its exoenzyme S producing parent in a rat chronic lung infection model. Of 51 isolates examined, 43% produced detectable levels of exoenzyme S. While both the exoenzyme S deficient mutant and its parent strain were equally capable of colonizing and persisting in rat lungs, the exoenzyme S producing parent elicited a greater degree of lung damage. These data suggest that exoenzyme S contributes to the pathogenesis of chronic lung infections.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Cystic Fibrosis/microbiology , Nucleotidyltransferases/metabolism , Pneumonia/microbiology , Pseudomonas aeruginosa/pathogenicity , Animals , Chronic Disease , Cystic Fibrosis/enzymology , Humans , Pneumonia/enzymology , Poly(ADP-ribose) Polymerases , Pseudomonas aeruginosa/enzymology , RatsABSTRACT
The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.
