Effect of aminoglycoside antibiotics on erythrocyte membrane potassium ion transport

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.

Ankyrin is the major oxidised protein in erythrocyte membranes from end-stage renal disease patients on chronic haemodialysis and oxidation is decreased by dialysis and vitamin C supplementation

Chronically haemodialysed end-stage renal disease patients are at high risk of morbidity arising from complications of dialysis, the underlying pathology that has led to renal disease and the complex pathology of chronic kidney disease. Anaemia is commonplace and its origins are multifactorial, involving reduced renal erythropoietin production, accumulation of uremic toxins and an increase in erythrocyte fragility. Oxidative damage is a common risk factor in renal disease and its co-morbidities and is known to cause erythrocyte fragility. Therefore, we have investigated the hypothesis that specific erythrocyte membrane proteins are more oxidised in end-stage renal disease patients and that vitamin C supplementation can ameliorate membrane protein oxidation. Eleven patients and 15 control subjects were recruited to the study. Patients were supplemented with 2 × 500 mg vitamin C per day for 4 weeks. Erythrocyte membrane proteins were prepared pre- and post-vitamin C supplementation for determination of protein oxidation. Total protein carbonyls were reduced by vitamin C supplementation but not by dialysis when investigated by enzyme linked immunosorbent assay. Using a western blot to detect oxidised proteins, one protein band, later identified as containing ankyrin, was found to be oxidised in patients but not controls and was reduced significantly by 60% in all patients after dialysis and by 20% after vitamin C treatment pre-dialysis. Ankyrin oxidation analysis may be useful in a stratified medicines approach as a possible marker to identify requirements for intervention in dialysis patients.

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

Characterisation of the plasma membrane subproteome of bloodstream form Trypanosoma brucei

Proteome analysis by conventional approaches is biased against hydrophobic membrane proteins, many of which are also of low abundance. We have isolated plasma membrane sheets from bloodstream forms of Trypanosoma brucei by subcellular fractionation, and then applied a battery of complementary protein separation and identification techniques to identify a large number of proteins in this fraction. The results of these analyses have been combined to generate a subproteome for the pellicular plasma membrane of bloodstream forms of T. brucei as well as a separate subproteome for the pellicular cytoskeleton. In parallel, we have used in silico approaches to predict the relative abundance of proteins potentially expressed by bloodstream form trypanosomes, and to identify likely polytopic membrane proteins, providing quality control for the experimentally defined plasma membrane subproteome. We show that the application of multiple high-resolution proteomic techniques to an enriched organelle fraction is a valuable approach for the characterisation of relatively intractable membrane proteomes. We present here the most complete analysis of a protozoan plasma membrane proteome to date and show the presence of a large number of integral membrane proteins, including 11 nucleoside/nucleobase transporters, 15 ion pumps and channels and a large number of adenylate cyclases hitherto listed as putative proteins.