ABSTRACT
The substantia nigra in Parkinson's disease (PD) is depleted of dopaminergic neurons and contains fibrillar Lewy bodies comprising primarily alpha-synuclein. We screened a library to identify drug-like molecules to probe the relation between neurodegeneration and alpha-synuclein fibrilization. All but one of 15 fibril inhibitors were catecholamines related to dopamine. The inhibitory activity of dopamine depended on its oxidative ligation to alpha-synuclein and was selective for the protofibril-to-fibril conversion, causing accumulation of the alpha-synuclein protofibril. Adduct formation provides an explanation for the dopaminergic selectivity of alpha-synuclein-associated neurotoxicity in PD and has implications for current and future PD therapeutic and diagnostic strategies.
Subject(s)
Dopamine/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Animals , Antioxidants/pharmacology , Biopolymers/chemistry , Biopolymers/metabolism , Catecholamines/pharmacology , Cytoplasm/metabolism , Dopamine/chemistry , Dopamine/pharmacology , Humans , Levodopa/pharmacology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Oxidation-Reduction , Oxidative Stress , Parkinson Disease/etiology , Parkinson Disease/metabolism , Parkinson Disease/therapy , Quinones/metabolism , Spectrometry, Fluorescence , Synaptic Vesicles/metabolism , Synucleins , alpha-SynucleinABSTRACT
We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus alpha-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Trehalose/pharmacology , 3T3 Cells , Animals , Bacterial Toxins/genetics , Biological Transport , Cell Membrane , Cell Survival , Genetic Engineering , Hemolysin Proteins/genetics , Humans , Keratinocytes , Mice , Staphylococcus aureus/geneticsABSTRACT
The disaccharide trehalose is found to be effective for stabilization of active recombinant retroviruses in an amorphous dry state achieved through ambient-temperature vacuum dehydration of retroviral supernatants. Studies revealed that trehalose is a significantly better desiccation protectant than sucrose, glucose, and dextran: dextran has essentially no protective effect on retroviral survival after drying and rehydration. X-ray diffractometry of the retroviral supernatant dried with trehalose demonstrated its amorphous nature. The ability to dehydrate retroviral stocks at ambient temperatures into a stable glassy state will have a profound effect for researchers and commercial biotechnology companies which supply retroviral vectors for human gene therapy and basic research.
Subject(s)
DNA, Recombinant/chemistry , Retroviridae/chemistry , Retroviridae/drug effects , Trehalose/pharmacology , 3T3 Cells , Animals , Cryopreservation/methods , Dextrans/pharmacology , Dose-Response Relationship, Drug , Freeze Drying/methods , Glucose/pharmacology , Lac Operon/genetics , Mice , Sucrose/pharmacology , X-Ray DiffractionABSTRACT
Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.
