Ectopic expression of the human adenine nucleotide translocase, isoform 3 (ANT-3). Characterization of ligand binding properties.

The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Overexpression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.

Use of gloves in the community: why, when, which and how?

The use of gloves has become routine since the early 1980s. It is recognized that they have an important part to play in the protection of both staff and patient/clients in the prevention of cross-infection. However, as choices relating to glove usage have increased over the years, health-care staff need to adopt a risk assessment approach, considering why, when, which and how gloves should be used. This article outlines the different types of gloves available and when they should be used.

Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line.

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.