The aim of the present study was to investigate the effects of glucosamine on lipopolysaccharide (LPS)-induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. Lipopolysaccharide (100 ng/mL) was used for the activation of primary cultured rat microglial or BV2 microglial cells. Changes in intracellular Ca2+ levels and outward K+ currents were measured using fura-2/AM and whole-cell patch-clamp methods, respectively. Lipopolysaccharide-induced expression of tumour necrosis factor (TNF)-alpha mRNA was analysed by reverse transcription-polymerase chain reaction. Lipopolysaccharide transformed cell morphology into an amoeboid shape in vitro and induced microglial activation in vivo, as measured by immunohistochemical staining, but glucosamine inhibited this activation. Glucosamine also inhibited LPS-induced Ca2+ influx, outward K+ currents and TNF-alpha mRNA expression, which are typically representative of microglial activation. 4. The results suggest that the inhibitory mechanisms of glucosamine on LPS-induced microglial activation include inhibition of Ca2+ influx and outward K+ currents, as well as downregulation of the microglial activator gene TNF-alpha.
Familial British dementia (FBD), is an autosomal dominant neurodegenerative disorder,with biochemical and pathological similarities to Alzheimer's disease. One notable similarity is the production of an amyloid peptide (ABri) due to point mutation of a stop codon in the BRI gene. The mutation extends the wild-type protein, and following proteolytic cleavage, results in the 34 residue ABri peptide isolated from plaques in FBD brain sections. Examination of the ABri sequence and the limited literature suggested that a constraining intramolecular disulphide bond might exist in ABri. Therefore we synthesized oxidized (cyclic) and reduced forms (linear) of both the ABri peptide and the analogous wild-type peptide. Fibrils were observed by negative-staining EM of aged solutions of ABri, and Congo Red stained samples of the same solution exhibited birefringence. Furthermore, CD spectra of solutions of the cyclic ABri peptide showed p structure, whereas the reduced form was random coil.Based on literature of amyloid proteins, and knowledge of p-strand register and turn prediction, we propose a model for ABri monomers that is consistent with a two or three stranded p-sheet monomer with a predominantly hydrophobic face for p-sheet aggregation. Further structure/function analysis of ABri peptides will complement the study of causes of dementia in diseases such as Alzheimer's, that are related to changes in protein aggregation and folding.
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