By A. Pullman (auth.), J. W. Gorrod, O. Albano, E. Ferrari, S. Papa (eds.)
cytochemical concepts (ICC) which supply an invaluable adjunct to investigations by means of immunoblotting. a specific good thing about a cytochemical method of the investiga tion of mitochondrial issues is that it permits the mosaic distribution of sure of those defects to be detected, while the tissue homogeniza tion inquisitive about traditional enzyme assays or immunoblotting precludes this. an extra good thing about MEA or ICC is that simply small quantities of tissue are wanted, that is very important considering that the various affected sufferers are babies or babies. the most goal of this communica tion is to stipulate ways that those strategies can be utilized within the analysis and extra research of mitochondrial problems. Reference might be made not just to these events during which MEA and ICC provide merits over regular enzyme asays and immunoblotting but additionally to contexts during which the opposite applies. four. 2 fabrics and techniques Muscle biopsies for cytochemical research have been snap-frozen utilizing isopentane cooled to - 150°C in liquid nitrogen. Samples have been saved in heat-sealed polythene packets within the vapour section of liquid nitrogen packing containers. four. 2. 1 Microphotometric enzyme assays Frozen sections eight Jlm thick have been lower utilizing a Reichert-J ung Frigocut cryostat microtome built with motor-driven slicing motion to keep up maximal reproducibility of part thickness. Sections have been picked up on microscope slides and air-dried for 15 min at room temperature.
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1980) Mech. , 12, 375-93. T. , 12, 94-100. , Herbert, j. el al. (1987) Gene, 55, 205-17. A. 1 INTRODUCTION The initial detection of mitochondrial abnormalities in human muscle biopsies has always relied heavily on the use of cytochemical techniques. , 1972). , 1985). , 1983). In modern diagnostic practice the traditional role of cytochemistry in preliminary screening protocols has been extended to include techniques which have as their specific aim the investigation of individual respiratory complexes in situ.
1989). In this case no morphological evidence of an underlying respiratory chain defect was found on routine cytochemical screening. Immunocytochemical investigation using subunit-specific and holocomplex antisera demonstrated a significant decrease in immunoreactivity in tissue sections which was particularly marked in the case of the nFeS protein (Fig. 4). 4 (a) Complex III immunolabelling (nFeS protein) in skeletal muscle from a normal child aged 20 months. (b) Severe decrease in immunolabelling in muscle of infant with complex III deficiency.
Oligomycin ~_ _'--_---'_ _ 4 [F&] 2 4 [OSCp] /oIg Fo-polypeptides·mg protein- 1 Fig. 10 ATPase activity in trypsinized USMP supplemented with purified Fl' For trypsin (50 ~g/mg USMP protein) digestion and other conditions see Guerrieri et at. (1989), Houstek et a/ (1988) and Zanotti et at. (1988). , 1988). 0-0, control USMP supplemented with PVP protein. , trypsinized USMP reconstituted with F1 in the presence of PVP (3 ~g/mg particle protein) and OSCP (at the concentrations reported in the figure).
Molecular Basis of Neurological Disorders and Their Treatment by A. Pullman (auth.), J. W. Gorrod, O. Albano, E. Ferrari, S. Papa (eds.)