2 G. D. Ludwig and F. W. Struthers, "Considerations underlying the use of ultrasound to detect gallstones and foreign bodies in ACOUSTIC V'ELOCITY AND IMPEDANCE OF TISSUES 863

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A brief survey is given of the calibration methods for hydrophones in the ultrasonic frequency range. The methods presently used in the Physikalisch-Technische Bundesanstalt (PTB), Braunschweig, West Germany, for hydrophone calibrations in the frequency range from 1 to 15 MHz are the primary subject of concern. These methods are the two-transducer reciprocity method for the calibration at discrete frequencies, the time-delay-spectrometry substitution method for quasifrequency continuous calibrations, and the two-transducer reciprocity method with time-delay spectrometry, also for quasi-frequency continuous calibration. Compared with the calibration at discrete frequencies, the expenditure of time for a calibration is considerably reduced in the case of the last-mentioned method. The influencing parameters which affect the evaluation of the measurement uncertainty are briefly discussed for the calibration methods applied at the PTB.

Outer and inner membranes of Neurospora crassa mitochondria were separated by the combined swelling, shrinking, sonication procedure. Membranes were characterized by electron microscopy and by marker enzyme activities. A red carotenoid pigment was found to be concentrated in the outer membrane. The inner mitochondrial membrane was resolved into about 20 protein bands on polyacrylarnide gel electrophoresis, whereas the outer membrane shows essentially one single protein band. Only negligible incorporation of radioactive amino acids occurs into outer membrane when isolated mitochondria are synthesizing polypeptide chains. I n agreement with this observation labeling of outer membrane protein is almost entirely blocked, when whole Neurospora cells are incubated with radioactive amino acids in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Finally, the essential electrophoretic protein band from outer membrane does not become labeled when mitochondria are incubated with radioactive amino acids either in vitro or in vivo in the presence of cycloheximide. It is concluded that the vast majority, if not all, of the outer membrane protein is synthesized by the cytoplasmic system and that polypeptide chains formed by the mitochondrial ribosomes are integrated into the inner mitochondrial membrane.Two systems of protein synthesis, one mitochondrial (intrinsic) and the other extramitochondrial or cytoplasmic (extrinsic) contribute to mitochondrial biogenesis. It is generally accepted that both systems provide proteins to be integrated into the mitochondrial membranes, whereas the soluble matrix proteins of mitochondria are supplied by the extrinsic system [ 11. I n distinguishing between the biogenesis of mitochondrial outer and inner membranes, it was suggested that the outer membrane of rat liver mitochondria was derived from the endoplasmic reticulum and hence its proteins synthesized by the cytoplasmic system [2,3]. The latter suggestion was substantiated by the observation that isolated mitochondria from rat liver incorporate amino acids only into the inner membrane proteins [4 -61. However, these studies have the disadvantage of having been performed in a rather unphysiological system, in which a number of processes might be impaired, including the formation of complete polypeptide chains on and release from the mitochondrial ribosomes, combination of proteins with newly synthesized phospholipid molecules, as well as transport mechanisms. I n order to avoid these difficulties and to properly evaluate the results obtained in the system in vitro, a study was made with intact cells.Cycloheximide, a specific inhibitor of extramitochondrial protein synthesis, can be used to differentiate between the intrinsic and extrinsic systems [7,8,25]. I n the experiments reported here, the effect of this antibiotic on protein synthesis in whole Neurospora crassa cells was used to study biogenesis of outer and inner mitochondrial membranes. MATERIALS AND METHODSCultivation of Neurospora and Preparation o...

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