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Filamentous inclusions in the matrix of mitochondria from human livers
J. ULTRASTRUCTURE RESEARCH
11,525-544 (1964)
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Filamentous Inclusions in the M a t r i x of Mitochondria from Human Livers ENRICO MUGNAINI1
Institute of Anatomy, University of Bergen, Norway, and Instituto di Patologia Medica & Centro di Microscopia Elettronica dell'Universitgl di Pisa, Italy Received May 26, 1964 Special paracrystalline inclusions, consisting of regularly arranged filaments 60 ~ thick, were found in the matrix of liver cell mitochondria from two human cases. The mitochondria often had very large dimensions and contained many cristae and dense granules in a matrix of normal electron density. A similar complex of features has been described previously by other workers in several pathological conditions, but the paracrystalline inclusions were said to consist of lamellae. In the cases studied here, the number of mitochondria and of lipofuscin granules was higher than normal. The findings are briefly discussed. It seems likely that the mitochondrial changes represent an unspecific reaction to injury. In liver cells f r o m patients suffering from cholestasis, Ekholm and Edlung (2) described a complex of unusual structural features in the mitochondria. Certain paracrystalline inclusions consisting of alternating dark and light lines were seen in the mitochondrial matrix. These lines were parallel and straight. The mitochondria sometimes atfained dimensions so large that they were termed "giant." J6z~quel (7) made similar observations of paracrystalline mitochondrial inclusions in cases of cholestasis and virus hepatitis and described the inclusions as consisting of parallel lamellae, comparable to myelin figures. She also noted that the mitochondria with these inclusions contained a large number of cristae and of dense granules. Similar findings were later made in liver cells in other pathological conditions (see 21 for references). Jdz6quel and Albot (8) observed them in the liver of a healthy individual as well. Lamellar (16) or myelin structure (5) has been claimed also by other workers to characterize these mitochondrial inclusions, and a "myelin degeneration" has been supposed to explain their genesis (7, 21, 23). During the examination of needle biopsy material from two individuals, one clinically appearing to be healthy and the other suffering from alcoholic dyspepsia, 1 This work was supported in part by U.S. Public Health Service Grant NB 02215-05. This aid is gratefully acknowledged.
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I have f o u n d in liver m i t o c h o n d r i a a complex of structural peculiarities similar to that as m e n t i o n e d above. However, certain i m p o r t a n t differences from earlier descriptions were noted, o n which a report will be given here. It should be stressed that the material studied is n o t suited for analysis of the f u n c t i o n a l or pathological conditions i n which the observed structures occur; this problem, therefore, is considered to lie outside the scope of the present paper. MATERIAL A N D METHODS The material consists of liver specimens from two individuals. Case 1: A 20-year-old man in good health. A routine clinical examination as well as liver function tests showed normal conditions. A biopsy was performed, with his permission, to obtain a normal material. Case 2: A 58-year-old man who had suffered from alcoholic dyspepsia for more than 3 years. X-ray examination disclosed a hyperthophic gastritis. All the tests revealed deficient liver function. After 3 days on an adjusted hospital diet, the two subjects, in the morning and fasting, were submitted to needle biopsy of the liver (13). For electron microscopy, the pieces were immediately fixed in Palade fixative (17) for 90 minutes and then dehydrated and embedded in n-butyl methacrylate. Ultrathin sections were cut with a Porter-Blum or with an LKB ultramicrotome, and stained with lead (9). Micrographs were taken with a Philips EM 100 or with a Siemens Elmiskop 1 b electron microscope. A portion of each biopsy was reserved for routine light microscopy examination. RESULTS A N D COMMENTS Mitochondria
The electron microscopical observations o n the c h o n d r i o m e were similar in the two cases; they will therefore be dealt with together. M i t o c h o n d r i a with u n u s u a l features were seen in several, b u t n o t in all, hepatocytes (cf. Figs. 1 a n d 2). Owing to the m e t h o d of biopsy, I have n o t been able to determine
Key to abbreviations be bile canaliculus ga Golgi apparatus ls lysosome bm bile material gl glycogen particles mb microbody E C N endothelial cell nucleus gm giant mitochondrion N nucleus of liver cell erg ergastoplasm Im limiting membrane ne nucleolus fv fat droplet (fat vacuole) lp lipofuscin granule rf reticular fibrils For the significance of arrows, see text to the separate figures. All the pictures shown in this paper were taken with a Siemens Elmiskop 1. Figs. 6, 13, and 19 are from case 2; all the others are from case 1.
FIG. 1. Portions of three liver cells which contain usual mitochondria. In the cell to the right, glycogen particles (gl) occupy a large cytoplasmic area, while in cells to the left they are distributed among other structures. Arrows indicate junctions between adjoining liver cells. × 27,500.
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the p o s i t i o n within the h e p a t i c lobule of cells c o n t a i n i n g these m i t o c h o n d r i a . F o r this reason, a n d since it is k n o w n t h a t the m i t o c h o n d r i a l p o p u l a t i o n varies in cells f r o m the different l o b u l a r zones, no q u a n t i t a t i v e d e t e r m i n a t i o n of the n u m b e r of m i t o c h o n d r i a p e r unit a r e a of c y t o p l a s m a n d of the v o l u m e ratio of m i t o c h o n d r i a t o c y t o p l a s m has been a t t e m p t e d on the basis of these m i c r o g r a p h s . Yet, the n u m b e r a n d the c o n c e n t r a t i o n of m i t o c h o n d r i a a p p e a r e d to be higher t h a n n o r m a l in m a n y cells with the characteristic changes here u n d e r c o n s i d e r a t i o n (Fig. 3). F u r t h e r m o r e , giant m i t o c h o n d r i a of different shapes were seen r e m a r k a b l y often in this m a t e r i a l (Figs. 4-6, 9, 13, 19, a n d 21). I n this c o n n e c t i o n it is n o t e d that a giant m i t o c h o n d r i o n a b o u t 4 # in diameter, if spherical, will have a v o l u m e of 18/z a, 180 times t h a t of a m i t o c h o n d r i o n 0.7/~ in diameter. I n single m i c r o g r a p h s , up to 5 m i t o c h o n d r i a with d i a m e t e r s exceeding 2 # were seen within single cells. Occurrence of n u m e r o u s cristae a n d dense granules was also a c o m m o n o b s e r v a t i o n (Figs. 5, 6, and 9). Such features have been described a n d discussed b y earlier a u t h o r s a n d will, therefore, n o t be extensively dealt w i t h here. The present descript i o n will essentially be limited to p r e v i o u s l y u n d e t e c t e d details a n d to features imm e d i a t e l y r e l a t e d to them. Paracrystalline inclusions were present in m i t o c h o n d r i a of u s u a l dimensions a s well as of g i a n t forms. W i t h o u t exception they o c c u r r e d in the matrix, i.e., never inside a crista, a n d generally close to a r o w of t u b u l a r cristae o r to the i n t e r n a l limiting m e m b r a n e (Fig. 4). The matrix, even in giant m i t o c h o n d r i a , displayed a n o r m a l electron density, a difference from t h a t seen in simple m i t o c h o n d r i a l swelling (18, 20). In several m i c r o g r a p h s the p a r a c r y s t a l l i n e inclusions h a d the a p p e a r a n c e of groups of straight, parallel, electron dense lines, regularly spaced. M i c r o g r a p h s showing m i t o c h o n d r i a l inclusions of identical a p p e a r a n c e have a p p e a r e d in several papers. B o t h the thickness of the d a r k lines a n d the distance between t h e m m e a s u r e d 1 1 Measurements on paracrystalline inclusions by different workers gave much the same result, but some differences are to be noted. Ekhohn and Edlung (2) found that "the thickness of the dark and light lines is about the same and varies between 50 and 100 A". Jdzdquel (7)reported that "the osmiophilic parallel lamellae" were about 40 A thick and the clear spaces 70-100 A, but in some inclusions the values were reduced to half. However, all measurements up to now have been on methacrylate-embedded material, which is known to undergo changes under the electron beam. Fro. 2. Portions of four liver ceils (one trinucleated). The arrows point toward mitochondria containing paracrystalline inclusions. Note the lipofuscin granules (some of which are labeled 1t)) and the numerous microbodies (unlabeled). × 3900. FIG. 3. Survey of portions of three liver cells. Note the high concentration of mitochondria, x 8600. F i t . 4. Very elongated mitochondria (arrows) with paracrystal line inclusions in the matrix. Elongated giant mitochondria (see also Fig. 12) generally were parallel to the limiting membranes of adjoining liver ceils, whereas globose or irregularly shaped giant forms occurred mainly in the central region of the cells (cf. Figs. 5, 6, and 16)). brn, bile material (?); ls, lysosomes. × 22,000. F~G. 5. Globose giant mitochondrion lying in the central region of the cell. × 26,000. FI6. 6. Paranuclear giant mitochondrion having a bizarre shape. Note the numerous cristae, dense granules, and paracrystalline inclusion. × 28,000.
.> z
C~ Z
m
C,
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FIG. 7. Mitochondrion, the shape of which seems to be determined by a paracrystalline inclusion consisting of parallel straight dense lines about 60/~ thick and spaced 60/~ apart × 50,0C0. FIG. 8. Mitochondrion of curious configuration with three paracrystalline inclusions oriented along the sides, x 38,300.
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F~o. 9. G i a n t m i t o c h o n d r i o n with several paracrystalline inclusions constituted either by d a r k lines, by granules (in t h e inset at higher magnification), or by short rods. x 55,000. I n s e t : x 120,000. 35 - 641838 J . Ultrastructure Research
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Fio. 10. Relatively thick section in which filaments overlie crista membranes at lm, limiting membrane. The inset is from a very thin section and shows that filamentous inclusions and mitochondrial membranes are in close proximity, x 30,000. Inset: 116,000. Fla. 11. Mitochondrion with circularly arranged cristae (arrows). x 40,000.
about 60 A, and the length of the lines sometimes amounted to 2 #. In elongated mitochondria, groups of lines were generally oriented parallel to the long axis of the organelle (Figs. 4 and 10). Occasionally, these inclusions even seemed to determine the shape of the mitochondrion, as shown in Figs. 7 and 8. The observations reported so far are in agreement with the descriptions by earlier authors and with the interpretation of the inclusions as consisting of lamellae. Also, inclusions displaying dark lines did not change in appearance when traced through serial sections, as expected from a lamellar structure.
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FIG. 12. Giant mitochondrion with two parts interconnected by a very thin bridge (arrow), which was present only in three consecutive serial sections. × 24,500. Fla. 13. Mitochondrion with paracrystalline inclusion and showing a voluminous projection. Note the packed cristae in the connecting region. × 38,000.
However, additional observations were made in this material which led to a rejection of this concept of the architecture of the inclusions. Most important, many inclusions were clearly seen to consist of groups of granules or of very short r o d s - as j udged from single micrographs (Fig. 9). Furthermore, close examination of several voluminous, globose mitochondria (some exceeding 5 # in diameter) in which inclusions were generally unoriented, revealed up to 30 inclusions within each mitochondrion, each being constituted either by dark lines, by short rods, or by granules. These three forms occurred with approximately the same frequency (Fig. 9). This proves that the paracrystalline inclusions are made up of parallel rows of filaments about 60 A thick and spaced at 60 A. They vary in appearance depending on the plane of sectioning. The unusual mitochondrial features that accompany the presence of oaracrystalline inclusions in this material, are similar to those reported in previous papers on the
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liver cells in other conditions. Therefore, it is very likely that we are dealing with the kind of mitochondrial inclusions described earlier. As an attempt to explain why filaments have been seen in the present material but not in the micrographs of previous authors, 1 it is suggested that in earlier studies only the more conspicuous longitudinal sections of the inclusions were selected, not cross sections. It seems less likely that the material which here occurs as filaments may, under other circumstances, give rise to parallel lamellae. Inclusions have earlier been described to be in "continuity" with crista membranes (7) or to "traverse" them (23). I have seen similar phenomena in relatively thick sections (Fig. 10). However, in extremely thin sections prepared with exceptional care, overlapping with cristae was minimal or absent (Figs. 9 and 10, inset). This fits well with the interpretation of the dark lines as filaments instead of lamellae, since the slender filaments may cover other structures even within one section. Filaments were observed in a distance of 50-80 ~ from cristal membranes in several micrographs (Fig. 10, inset). Whether there is any relation between these inclusions and the special particles attached to mitochondrial membranes and described by Fernfindez-Morfin (5) should be investigated with adequate techniques. In the cells with changed mitochondria, cristae sometimes presented a curious circular arrangement, the significance of which remains unclear (Fig. 11). In other mitochondria, some of which were of normal dimensions, numerous elongated cristae were seen, a feature which is unusual for liver mitochondria. It is a n open question whether a giant mitochondrion arises by "hypertrophy" from a single smaller mitochondrion or by fusion of several mitochondria. It may be mentioned that in several micrographs giant mitochondria were seen to present very thin interconnecting bridges (Fig. 12), or bud-like projections (Figs. 8 and 13). A large number of mitochondria showed other kinds of irregular configuration (Fig. 17 and others). Moreover, mitochondria of giant or normal size, were often seen partially (Figs. 14 and 15), or completely (Fig. 16) divided by a wall continuous with the inner membrane. These mitochondria thus resembled the dividing mitochondria found in rat liver by Fawcett (4). Since it is well known from motion pictures that mitochondria constantly change their shape, branching, dividing, and 1 High numbers of cristae and paracrystalline inclusions, which were perhaps filamentous, have been observed by Napolitano and Fawcett (15) in mitochondria of brown adipose tissue from newborn mice and rats. FIG. 14. Mitochondrion partially divided by membranes (continuous with the inner membrane of the limiting envelope). Note also the bud-like projection. × 41,500. FIG. 15. Another incomplete septum in a mitochondrion with paracrystalline inclusion. At arrow, lateral opening in the septum. × 33,000. F~G. 16. Mitochondrion completely divided into compartments by membranes. × 47,000. Fro. 17. Mitochondria connected by attenuated portions (arrows). ×21,000.
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FILAMENTOUSINCLUSIONS IN LIVER MITOCHONDRIA
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FIG. 21. Paracrystalline cytoplasmic regions of different shape and size (arrows). Note the numerous microbodies (unlabeled) and the two giant mitochondria (gin). x 21,700. rejoining, it is n o t possible to give a precise i n t e r p r e t a t i o n of every electron micrograph of m i t o c h o n d r i o n . However, the observations j u s t m e n t i o n e d m a y be t a k e n as a suggestion of an exceptionally high level of m i t o c h o n d r i a l activity. Some m i t o c h o n d r i a showed dilated intracristal spaces (Figs. 1 a n d 18) which c o n t a i n e d a n ill-defined material occasionally giving the impression of being helical filaments (Fig. 18, inset). These observations were too restricted to prove a possible identity with the intracristal helical filaments recently described by the a u t h o r (14)
FIG. 18. Mitochondrion of normal size with a dilated intracristal space containing some material difficult to define. The inset shows a detail at a higher magnification. The arrow points to a formation which gives the impression of being a helical filament, x 65,000. Inset: x 153,500. Fro. 19. Giant mitochondrion with dilated intracristal spaces (arrows). x 36,800. Fro. 20. Two typical centrioles (arrows) in the pericanalicular region, near the nucleus, x 47,500.
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in astrocytic m i t o c h o n d r i a from the rat corpus striatum. A few other m i t o c h o n d r i a w i t h o u t inclusions were r o u n d e d a n d displayed a clear matrix a n d peripheral cristae as i n the state of " c l o u d y swelling" (19, 20). This finding was interpreted as a sign of m i t o c h o n d r i a l disturbance.
General cytology A few additional remarks should be made on the appearance of the biopsy material studied in order to give a general idea of the tissue in which the special mitochondria occurred. In these cases, as in needle biopsies in general, the specimens were too small to permit any complete evaluation of the state of the organ from which they were taken.
Case 1 Light microscopy. The tissue architecture was well preserved. Hepatocytes generally were of normal appearance, rich in material with the characteristics of glycogen, but poor in fat vacuoles. Especially in the centrolobular zone many pigment granules were present, an unexpected finding in a young individual. Several binucleated and some trinucleated cells were observed. Some interlobular septa with many nuclei were present. Ultrastructure. The lumen of the sinusoids was often occluded by leucocytes and sometimes also contained debris of hepatic cells. In the cytoplasm of hepatocytes many heterogeneous dense bodies were seen, similar to those described by Essner and Novikoff (3) as lipofuscin (Figs. 2-4) as well as some lysosomes and numerous microbodies. Near the bile canaliculi several vacuoles with a heterogeneous content (bile material?) were present (Fig. 4). The presence of numerous glycogen particles was confirmed. In some cells they were dispersed between other cytoplasmic structures, while in others they occupied large cytoplasmic zones (Fig. 1). In some cells centrioles were observed in the pericanalicular zone (Fig. 20). In several micrographs of those cells containing unusual mitochondria, some cytoplasmic regions not limited by membranes were seen, which had a paracrystalline appearance. These formations occurred often near fat droplets or the cell nucleus, and several microbodies were always present in their proximity. The cytoplasmic inclusions were variable in shape and size (Fig. 21), the largest ones being up to 2/~ in diameter. Their appearance varied (cf. Figs. 22-24), probably depending on the plane of sectioning. In several instances rows of granules or groups of parallel lines 60 ~ thick, were seen, suggesting a filamentous structure (Figs. 24 and 25). It may be supposed, therefore, that some relationship exists between cytoplasmic and intramitochondrial paracrystalline inclusions. With respect to this, however, no further information was obtainable from the micrographs at hand. To the author'S knowledge similar formations have not previously been described in human liver cells.
FIGs. 22, 23, and 24. Three cytoplasmic inclusions differing in appearance, probably owing to the different planes of sectioning. In Fig. 22 parallel lines are seen, whereas in Fig. 24 several rows of granules, 60/~ in diameter, are distinct and suggest a filamentous structure. Within such cytoplasmic inclusions, zones with glycogen particles (gl) and ground substance are often seen. The arrow points out the region enlarged in Fig. 25. Fig. 22: × 59,000. Fig. 23: x 67,000. Fig. 24: x 59,000. FIG. 25. Enlargement of the region pointed out by arrow in Fig. 24. Parallel rows of granules are seen, several of which are clearly separated from each other (arrows). x 132,000.
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Case 2 Light microscopy. The hepatic architecture was still discernible. Liver cells were often swollen with compression of sinusoids. Some cells showed karyolysis. Fat vacuoles and areas with glycogen were seen in many ceils. Pigment granules were particularly numerous. Some groups of hepatocytes with voluminous, and well stained nuclei surrounded by a homogeneous cytoplasm (signs of regenerative processes) were observed. Ultrastructure. Intralobular bundles of reticular fibrils were very often encountered. Lipofuscin granules were extraordinarily numerous. Several fat droplets (or fat vacuoles) were seen, as well as lysosomes, some of which had a granular content resembling ferritin. Glycogen particles were numerous in all cells. Ergastoplasm was often localized to circumscribed cytoplasmic areas where mitochondria and free ribosomes were likewise present, in close association with the ergastoplasmic membranes.
CONCLUSIONS "Paracrystalline" inclusions seen in the mitochondrial matrix in this material clearly consisted of regularly arranged filaments. Many observed features suggested an unusual rate of chondriome activity. Even without statistical evidence, the impression was obtained that the number of mitochondria as well as the total mitochondrial mass was increased in many of the liver cells with the unusual mitochondria. Presumably all these mitochondrial changes form a part of pathological processes. In both of the two cases here studied, some signs of hepatic injury were present. In the first individual they were evident merely at the ultrastructural level, whereas in the second they were documented already by clinical examination and by liver function tests. It is recalled that an increase in number of cristae and an occurrence of giant mitochondria have frequently been observed in experimentally induced liver lesions (11, 18, 22, 24). In the human liver similar mitochondrial changes have been observed in several conditions (21). Therefore, it seems likely that such changes represent a n unspecific reaction of the ehondriome to cell injury, probably as a consequence of interference with some basic enzymatic activity. So far, the formation of paracrystalline inclusions has not been reported in liver mitochondria from laboratory animals. Carruthers and Steiner (1) showed an increase in size and a probable rise in the total number of mitochondria following 4-5 weeks of biliary obstruction in rats. No real "myelin figures" were seen, but packed elongated cristae occurred in some of the mitochondria. Mazzanti and Mugnaini (12) obtained the same results in rats about 5 weeks after bile duct ligature. The paracrystalline inclusions may thus be specific to the human being. This suggests the possibility of qualitative or quantitative species differences, which should be remembered in correlating findings in humans with experimental data from laboratory animals. The significance of the paracrystalline inclusions must at present remain
FILAMENTOUS INCLUSIONS IN LIVER MITOCHONDRIA
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u n k n o w n . 1 H i s t o c h e m i c a l a n d b i o c h e m i c a l studies o n livers shown to c o n t a i n such inclusions s h o u l d be carried out. T h e y m a y p r o v i d e interesting correlations w i t h these peculiar u l t r a s t r u c t u r a l features. The author is particularly indebted to Dr. Theodor W. Blackstad and to Professor Wilhelm H a r k m a r k for the critical revision of the manuscript.
ADDENDUM After the manuscript had been completed for publication, we received the April number of the American Journal of Pathology, where Drs. D. J. Svoboda and R. T. Manning, showed giant mitochondria of human liver cells containing paracrystalline inclusions which in transverse section appeared to be made up of parallel dots. This confirms the observations presented in this paper. Some pictures with the same details have been published also in the atlas by H. David, "Submikroskopische O r t h o - u n d Pathomorphologie der Leber" (Akademie Verlag, Berlin, 1964).
REFERENCES 1. CARRUTHERS,J. S. and STEINER,J. W., Gastroenterology 42, 419 (1962). 2. EKHOLM, R. and EDLUNG, Y., Intern. Kongr. Elektronenmikroskopie 4 Berlin 1958, Verhandl., Vol. 2, p. 273. Springer, Berlin, 1960. 3. ESSNER, E. and NOVlKOFF, A. B., J. Ultrastruct. Res. 3, 374 (1960). 4. FAWCETT, D. W., J. Natl. Cancer Inst. 15, Suppl., 1475 (1955). 5. FERN.g,NDEz-MORAN, H., in Symp. Intern. Soc. Cell Biol. 1, 411 (1962). 6. HOLLE, G., in MARTINI, G. A. (Ed.), Aktuelle Probleme der Hepatologie, p. 8. Thieme, Stuttgart, 1962. 7. JI~ZEQUEL,A. M., J. Ultrastruct. Res. 2, 210 (1959). 8. JI~ZEQUEL,A. M. and ALBOT, G., Semaine Hop. Paris 38, 523 (1962). 9. KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). 10. LUFT, R., IKKOS, D., PALMIERI, G., ERNSTER, L. and AFZELIUS, B., d. Clin. Invest. 41, 1776 (1962). 11. LUSE, S. A., BURCH, H. B. and HUNTER, F. E. JR., Proc. Intern. Congr. Electron Microscopy, 5th, 1962, Vol. 2, p. VV-5. Academic Press, New York, 1962. 12. MAZZANTI, L. and MUGNAINI, E., unpublished observations. 13. MENGHINr, G. and ORLANDI, F., La Puntura Biopsia Epatica. I. Pensiero Scientifico, Roma, 1957. 14. MUGNAINI, E., d. Cell. Biol., in press. 1 It should be mentioned here that a large number of mitochondria containing very numerous, often tubular, cristae and paracrystalline inclusions (the latter, however, were compared with "a pile of board"), have been described by Luft et al. (10) also in human skeletal muscle cells in a pathological condition. These authors in parallel biochemical studies were able to demonstrate a loosely coupled state of oxidative phosphorylation, and they concluded that the observed features "may be the result of a compensatory mechanism by which the muscle cell attempts to overcome the handicap imposed by the decreased biochemical efficiency of these mitochondria."
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15. NAPOLITANO,L. and FAWCETT,D. W., Y. Biophys. Biochem. Cytol. 4, 685 (1958). 16. NOVIKOFF,A. B., in BRACHET,J. and MIRSKY, A. E. (Eds.), The Cell, Vol. II, p. 299. Academic Press, New York, 1961. 17. PALADE,G. E., J. Exptl. Med. 95, 285 (1952). 18. REYNOLDS,E. S., ar. Cell. Biol. 19, 139 (1963). 19. ROUILLER,C., Ann. Anat. Pathol. 2, 548 (1957). 20. ROUmLER, C. and GANSLER,H., Syrup. Fine Structure Ceils, p. 82. Noordhoff, Groningen, 1954. 21. ROUmL~R, C. and J~z~QtmL, A. M., in ROUlLLER, C. (Ed.), The Liver, Vol. I, p. 195. Academic Press, New York, 1963. 22. SVOBODA,D. J. and HmGINSON, J., Am. J. Pathol. 43, 477 (1963). 23. TmERY, J. P. and CAROLbJ., Rev. lntern. Hepatol. 12, 207 (1962). 24. WILSON,J. W. and LEDuc, E. H., J. CellBiol. 16, 281 (1963).
11,525-544 (1964)
525
Filamentous Inclusions in the M a t r i x of Mitochondria from Human Livers ENRICO MUGNAINI1
Institute of Anatomy, University of Bergen, Norway, and Instituto di Patologia Medica & Centro di Microscopia Elettronica dell'Universitgl di Pisa, Italy Received May 26, 1964 Special paracrystalline inclusions, consisting of regularly arranged filaments 60 ~ thick, were found in the matrix of liver cell mitochondria from two human cases. The mitochondria often had very large dimensions and contained many cristae and dense granules in a matrix of normal electron density. A similar complex of features has been described previously by other workers in several pathological conditions, but the paracrystalline inclusions were said to consist of lamellae. In the cases studied here, the number of mitochondria and of lipofuscin granules was higher than normal. The findings are briefly discussed. It seems likely that the mitochondrial changes represent an unspecific reaction to injury. In liver cells f r o m patients suffering from cholestasis, Ekholm and Edlung (2) described a complex of unusual structural features in the mitochondria. Certain paracrystalline inclusions consisting of alternating dark and light lines were seen in the mitochondrial matrix. These lines were parallel and straight. The mitochondria sometimes atfained dimensions so large that they were termed "giant." J6z~quel (7) made similar observations of paracrystalline mitochondrial inclusions in cases of cholestasis and virus hepatitis and described the inclusions as consisting of parallel lamellae, comparable to myelin figures. She also noted that the mitochondria with these inclusions contained a large number of cristae and of dense granules. Similar findings were later made in liver cells in other pathological conditions (see 21 for references). Jdz6quel and Albot (8) observed them in the liver of a healthy individual as well. Lamellar (16) or myelin structure (5) has been claimed also by other workers to characterize these mitochondrial inclusions, and a "myelin degeneration" has been supposed to explain their genesis (7, 21, 23). During the examination of needle biopsy material from two individuals, one clinically appearing to be healthy and the other suffering from alcoholic dyspepsia, 1 This work was supported in part by U.S. Public Health Service Grant NB 02215-05. This aid is gratefully acknowledged.
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I have f o u n d in liver m i t o c h o n d r i a a complex of structural peculiarities similar to that as m e n t i o n e d above. However, certain i m p o r t a n t differences from earlier descriptions were noted, o n which a report will be given here. It should be stressed that the material studied is n o t suited for analysis of the f u n c t i o n a l or pathological conditions i n which the observed structures occur; this problem, therefore, is considered to lie outside the scope of the present paper. MATERIAL A N D METHODS The material consists of liver specimens from two individuals. Case 1: A 20-year-old man in good health. A routine clinical examination as well as liver function tests showed normal conditions. A biopsy was performed, with his permission, to obtain a normal material. Case 2: A 58-year-old man who had suffered from alcoholic dyspepsia for more than 3 years. X-ray examination disclosed a hyperthophic gastritis. All the tests revealed deficient liver function. After 3 days on an adjusted hospital diet, the two subjects, in the morning and fasting, were submitted to needle biopsy of the liver (13). For electron microscopy, the pieces were immediately fixed in Palade fixative (17) for 90 minutes and then dehydrated and embedded in n-butyl methacrylate. Ultrathin sections were cut with a Porter-Blum or with an LKB ultramicrotome, and stained with lead (9). Micrographs were taken with a Philips EM 100 or with a Siemens Elmiskop 1 b electron microscope. A portion of each biopsy was reserved for routine light microscopy examination. RESULTS A N D COMMENTS Mitochondria
The electron microscopical observations o n the c h o n d r i o m e were similar in the two cases; they will therefore be dealt with together. M i t o c h o n d r i a with u n u s u a l features were seen in several, b u t n o t in all, hepatocytes (cf. Figs. 1 a n d 2). Owing to the m e t h o d of biopsy, I have n o t been able to determine
Key to abbreviations be bile canaliculus ga Golgi apparatus ls lysosome bm bile material gl glycogen particles mb microbody E C N endothelial cell nucleus gm giant mitochondrion N nucleus of liver cell erg ergastoplasm Im limiting membrane ne nucleolus fv fat droplet (fat vacuole) lp lipofuscin granule rf reticular fibrils For the significance of arrows, see text to the separate figures. All the pictures shown in this paper were taken with a Siemens Elmiskop 1. Figs. 6, 13, and 19 are from case 2; all the others are from case 1.
FIG. 1. Portions of three liver cells which contain usual mitochondria. In the cell to the right, glycogen particles (gl) occupy a large cytoplasmic area, while in cells to the left they are distributed among other structures. Arrows indicate junctions between adjoining liver cells. × 27,500.
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the p o s i t i o n within the h e p a t i c lobule of cells c o n t a i n i n g these m i t o c h o n d r i a . F o r this reason, a n d since it is k n o w n t h a t the m i t o c h o n d r i a l p o p u l a t i o n varies in cells f r o m the different l o b u l a r zones, no q u a n t i t a t i v e d e t e r m i n a t i o n of the n u m b e r of m i t o c h o n d r i a p e r unit a r e a of c y t o p l a s m a n d of the v o l u m e ratio of m i t o c h o n d r i a t o c y t o p l a s m has been a t t e m p t e d on the basis of these m i c r o g r a p h s . Yet, the n u m b e r a n d the c o n c e n t r a t i o n of m i t o c h o n d r i a a p p e a r e d to be higher t h a n n o r m a l in m a n y cells with the characteristic changes here u n d e r c o n s i d e r a t i o n (Fig. 3). F u r t h e r m o r e , giant m i t o c h o n d r i a of different shapes were seen r e m a r k a b l y often in this m a t e r i a l (Figs. 4-6, 9, 13, 19, a n d 21). I n this c o n n e c t i o n it is n o t e d that a giant m i t o c h o n d r i o n a b o u t 4 # in diameter, if spherical, will have a v o l u m e of 18/z a, 180 times t h a t of a m i t o c h o n d r i o n 0.7/~ in diameter. I n single m i c r o g r a p h s , up to 5 m i t o c h o n d r i a with d i a m e t e r s exceeding 2 # were seen within single cells. Occurrence of n u m e r o u s cristae a n d dense granules was also a c o m m o n o b s e r v a t i o n (Figs. 5, 6, and 9). Such features have been described a n d discussed b y earlier a u t h o r s a n d will, therefore, n o t be extensively dealt w i t h here. The present descript i o n will essentially be limited to p r e v i o u s l y u n d e t e c t e d details a n d to features imm e d i a t e l y r e l a t e d to them. Paracrystalline inclusions were present in m i t o c h o n d r i a of u s u a l dimensions a s well as of g i a n t forms. W i t h o u t exception they o c c u r r e d in the matrix, i.e., never inside a crista, a n d generally close to a r o w of t u b u l a r cristae o r to the i n t e r n a l limiting m e m b r a n e (Fig. 4). The matrix, even in giant m i t o c h o n d r i a , displayed a n o r m a l electron density, a difference from t h a t seen in simple m i t o c h o n d r i a l swelling (18, 20). In several m i c r o g r a p h s the p a r a c r y s t a l l i n e inclusions h a d the a p p e a r a n c e of groups of straight, parallel, electron dense lines, regularly spaced. M i c r o g r a p h s showing m i t o c h o n d r i a l inclusions of identical a p p e a r a n c e have a p p e a r e d in several papers. B o t h the thickness of the d a r k lines a n d the distance between t h e m m e a s u r e d 1 1 Measurements on paracrystalline inclusions by different workers gave much the same result, but some differences are to be noted. Ekhohn and Edlung (2) found that "the thickness of the dark and light lines is about the same and varies between 50 and 100 A". Jdzdquel (7)reported that "the osmiophilic parallel lamellae" were about 40 A thick and the clear spaces 70-100 A, but in some inclusions the values were reduced to half. However, all measurements up to now have been on methacrylate-embedded material, which is known to undergo changes under the electron beam. Fro. 2. Portions of four liver ceils (one trinucleated). The arrows point toward mitochondria containing paracrystalline inclusions. Note the lipofuscin granules (some of which are labeled 1t)) and the numerous microbodies (unlabeled). × 3900. FIG. 3. Survey of portions of three liver cells. Note the high concentration of mitochondria, x 8600. F i t . 4. Very elongated mitochondria (arrows) with paracrystal line inclusions in the matrix. Elongated giant mitochondria (see also Fig. 12) generally were parallel to the limiting membranes of adjoining liver ceils, whereas globose or irregularly shaped giant forms occurred mainly in the central region of the cells (cf. Figs. 5, 6, and 16)). brn, bile material (?); ls, lysosomes. × 22,000. F~G. 5. Globose giant mitochondrion lying in the central region of the cell. × 26,000. FI6. 6. Paranuclear giant mitochondrion having a bizarre shape. Note the numerous cristae, dense granules, and paracrystalline inclusion. × 28,000.
.> z
C~ Z
m
C,
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FIG. 7. Mitochondrion, the shape of which seems to be determined by a paracrystalline inclusion consisting of parallel straight dense lines about 60/~ thick and spaced 60/~ apart × 50,0C0. FIG. 8. Mitochondrion of curious configuration with three paracrystalline inclusions oriented along the sides, x 38,300.
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F~o. 9. G i a n t m i t o c h o n d r i o n with several paracrystalline inclusions constituted either by d a r k lines, by granules (in t h e inset at higher magnification), or by short rods. x 55,000. I n s e t : x 120,000. 35 - 641838 J . Ultrastructure Research
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Fio. 10. Relatively thick section in which filaments overlie crista membranes at lm, limiting membrane. The inset is from a very thin section and shows that filamentous inclusions and mitochondrial membranes are in close proximity, x 30,000. Inset: 116,000. Fla. 11. Mitochondrion with circularly arranged cristae (arrows). x 40,000.
about 60 A, and the length of the lines sometimes amounted to 2 #. In elongated mitochondria, groups of lines were generally oriented parallel to the long axis of the organelle (Figs. 4 and 10). Occasionally, these inclusions even seemed to determine the shape of the mitochondrion, as shown in Figs. 7 and 8. The observations reported so far are in agreement with the descriptions by earlier authors and with the interpretation of the inclusions as consisting of lamellae. Also, inclusions displaying dark lines did not change in appearance when traced through serial sections, as expected from a lamellar structure.
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FIG. 12. Giant mitochondrion with two parts interconnected by a very thin bridge (arrow), which was present only in three consecutive serial sections. × 24,500. Fla. 13. Mitochondrion with paracrystalline inclusion and showing a voluminous projection. Note the packed cristae in the connecting region. × 38,000.
However, additional observations were made in this material which led to a rejection of this concept of the architecture of the inclusions. Most important, many inclusions were clearly seen to consist of groups of granules or of very short r o d s - as j udged from single micrographs (Fig. 9). Furthermore, close examination of several voluminous, globose mitochondria (some exceeding 5 # in diameter) in which inclusions were generally unoriented, revealed up to 30 inclusions within each mitochondrion, each being constituted either by dark lines, by short rods, or by granules. These three forms occurred with approximately the same frequency (Fig. 9). This proves that the paracrystalline inclusions are made up of parallel rows of filaments about 60 A thick and spaced at 60 A. They vary in appearance depending on the plane of sectioning. The unusual mitochondrial features that accompany the presence of oaracrystalline inclusions in this material, are similar to those reported in previous papers on the
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liver cells in other conditions. Therefore, it is very likely that we are dealing with the kind of mitochondrial inclusions described earlier. As an attempt to explain why filaments have been seen in the present material but not in the micrographs of previous authors, 1 it is suggested that in earlier studies only the more conspicuous longitudinal sections of the inclusions were selected, not cross sections. It seems less likely that the material which here occurs as filaments may, under other circumstances, give rise to parallel lamellae. Inclusions have earlier been described to be in "continuity" with crista membranes (7) or to "traverse" them (23). I have seen similar phenomena in relatively thick sections (Fig. 10). However, in extremely thin sections prepared with exceptional care, overlapping with cristae was minimal or absent (Figs. 9 and 10, inset). This fits well with the interpretation of the dark lines as filaments instead of lamellae, since the slender filaments may cover other structures even within one section. Filaments were observed in a distance of 50-80 ~ from cristal membranes in several micrographs (Fig. 10, inset). Whether there is any relation between these inclusions and the special particles attached to mitochondrial membranes and described by Fernfindez-Morfin (5) should be investigated with adequate techniques. In the cells with changed mitochondria, cristae sometimes presented a curious circular arrangement, the significance of which remains unclear (Fig. 11). In other mitochondria, some of which were of normal dimensions, numerous elongated cristae were seen, a feature which is unusual for liver mitochondria. It is a n open question whether a giant mitochondrion arises by "hypertrophy" from a single smaller mitochondrion or by fusion of several mitochondria. It may be mentioned that in several micrographs giant mitochondria were seen to present very thin interconnecting bridges (Fig. 12), or bud-like projections (Figs. 8 and 13). A large number of mitochondria showed other kinds of irregular configuration (Fig. 17 and others). Moreover, mitochondria of giant or normal size, were often seen partially (Figs. 14 and 15), or completely (Fig. 16) divided by a wall continuous with the inner membrane. These mitochondria thus resembled the dividing mitochondria found in rat liver by Fawcett (4). Since it is well known from motion pictures that mitochondria constantly change their shape, branching, dividing, and 1 High numbers of cristae and paracrystalline inclusions, which were perhaps filamentous, have been observed by Napolitano and Fawcett (15) in mitochondria of brown adipose tissue from newborn mice and rats. FIG. 14. Mitochondrion partially divided by membranes (continuous with the inner membrane of the limiting envelope). Note also the bud-like projection. × 41,500. FIG. 15. Another incomplete septum in a mitochondrion with paracrystalline inclusion. At arrow, lateral opening in the septum. × 33,000. F~G. 16. Mitochondrion completely divided into compartments by membranes. × 47,000. Fro. 17. Mitochondria connected by attenuated portions (arrows). ×21,000.
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FIG. 21. Paracrystalline cytoplasmic regions of different shape and size (arrows). Note the numerous microbodies (unlabeled) and the two giant mitochondria (gin). x 21,700. rejoining, it is n o t possible to give a precise i n t e r p r e t a t i o n of every electron micrograph of m i t o c h o n d r i o n . However, the observations j u s t m e n t i o n e d m a y be t a k e n as a suggestion of an exceptionally high level of m i t o c h o n d r i a l activity. Some m i t o c h o n d r i a showed dilated intracristal spaces (Figs. 1 a n d 18) which c o n t a i n e d a n ill-defined material occasionally giving the impression of being helical filaments (Fig. 18, inset). These observations were too restricted to prove a possible identity with the intracristal helical filaments recently described by the a u t h o r (14)
FIG. 18. Mitochondrion of normal size with a dilated intracristal space containing some material difficult to define. The inset shows a detail at a higher magnification. The arrow points to a formation which gives the impression of being a helical filament, x 65,000. Inset: x 153,500. Fro. 19. Giant mitochondrion with dilated intracristal spaces (arrows). x 36,800. Fro. 20. Two typical centrioles (arrows) in the pericanalicular region, near the nucleus, x 47,500.
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in astrocytic m i t o c h o n d r i a from the rat corpus striatum. A few other m i t o c h o n d r i a w i t h o u t inclusions were r o u n d e d a n d displayed a clear matrix a n d peripheral cristae as i n the state of " c l o u d y swelling" (19, 20). This finding was interpreted as a sign of m i t o c h o n d r i a l disturbance.
General cytology A few additional remarks should be made on the appearance of the biopsy material studied in order to give a general idea of the tissue in which the special mitochondria occurred. In these cases, as in needle biopsies in general, the specimens were too small to permit any complete evaluation of the state of the organ from which they were taken.
Case 1 Light microscopy. The tissue architecture was well preserved. Hepatocytes generally were of normal appearance, rich in material with the characteristics of glycogen, but poor in fat vacuoles. Especially in the centrolobular zone many pigment granules were present, an unexpected finding in a young individual. Several binucleated and some trinucleated cells were observed. Some interlobular septa with many nuclei were present. Ultrastructure. The lumen of the sinusoids was often occluded by leucocytes and sometimes also contained debris of hepatic cells. In the cytoplasm of hepatocytes many heterogeneous dense bodies were seen, similar to those described by Essner and Novikoff (3) as lipofuscin (Figs. 2-4) as well as some lysosomes and numerous microbodies. Near the bile canaliculi several vacuoles with a heterogeneous content (bile material?) were present (Fig. 4). The presence of numerous glycogen particles was confirmed. In some cells they were dispersed between other cytoplasmic structures, while in others they occupied large cytoplasmic zones (Fig. 1). In some cells centrioles were observed in the pericanalicular zone (Fig. 20). In several micrographs of those cells containing unusual mitochondria, some cytoplasmic regions not limited by membranes were seen, which had a paracrystalline appearance. These formations occurred often near fat droplets or the cell nucleus, and several microbodies were always present in their proximity. The cytoplasmic inclusions were variable in shape and size (Fig. 21), the largest ones being up to 2/~ in diameter. Their appearance varied (cf. Figs. 22-24), probably depending on the plane of sectioning. In several instances rows of granules or groups of parallel lines 60 ~ thick, were seen, suggesting a filamentous structure (Figs. 24 and 25). It may be supposed, therefore, that some relationship exists between cytoplasmic and intramitochondrial paracrystalline inclusions. With respect to this, however, no further information was obtainable from the micrographs at hand. To the author'S knowledge similar formations have not previously been described in human liver cells.
FIGs. 22, 23, and 24. Three cytoplasmic inclusions differing in appearance, probably owing to the different planes of sectioning. In Fig. 22 parallel lines are seen, whereas in Fig. 24 several rows of granules, 60/~ in diameter, are distinct and suggest a filamentous structure. Within such cytoplasmic inclusions, zones with glycogen particles (gl) and ground substance are often seen. The arrow points out the region enlarged in Fig. 25. Fig. 22: × 59,000. Fig. 23: x 67,000. Fig. 24: x 59,000. FIG. 25. Enlargement of the region pointed out by arrow in Fig. 24. Parallel rows of granules are seen, several of which are clearly separated from each other (arrows). x 132,000.
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Case 2 Light microscopy. The hepatic architecture was still discernible. Liver cells were often swollen with compression of sinusoids. Some cells showed karyolysis. Fat vacuoles and areas with glycogen were seen in many ceils. Pigment granules were particularly numerous. Some groups of hepatocytes with voluminous, and well stained nuclei surrounded by a homogeneous cytoplasm (signs of regenerative processes) were observed. Ultrastructure. Intralobular bundles of reticular fibrils were very often encountered. Lipofuscin granules were extraordinarily numerous. Several fat droplets (or fat vacuoles) were seen, as well as lysosomes, some of which had a granular content resembling ferritin. Glycogen particles were numerous in all cells. Ergastoplasm was often localized to circumscribed cytoplasmic areas where mitochondria and free ribosomes were likewise present, in close association with the ergastoplasmic membranes.
CONCLUSIONS "Paracrystalline" inclusions seen in the mitochondrial matrix in this material clearly consisted of regularly arranged filaments. Many observed features suggested an unusual rate of chondriome activity. Even without statistical evidence, the impression was obtained that the number of mitochondria as well as the total mitochondrial mass was increased in many of the liver cells with the unusual mitochondria. Presumably all these mitochondrial changes form a part of pathological processes. In both of the two cases here studied, some signs of hepatic injury were present. In the first individual they were evident merely at the ultrastructural level, whereas in the second they were documented already by clinical examination and by liver function tests. It is recalled that an increase in number of cristae and an occurrence of giant mitochondria have frequently been observed in experimentally induced liver lesions (11, 18, 22, 24). In the human liver similar mitochondrial changes have been observed in several conditions (21). Therefore, it seems likely that such changes represent a n unspecific reaction of the ehondriome to cell injury, probably as a consequence of interference with some basic enzymatic activity. So far, the formation of paracrystalline inclusions has not been reported in liver mitochondria from laboratory animals. Carruthers and Steiner (1) showed an increase in size and a probable rise in the total number of mitochondria following 4-5 weeks of biliary obstruction in rats. No real "myelin figures" were seen, but packed elongated cristae occurred in some of the mitochondria. Mazzanti and Mugnaini (12) obtained the same results in rats about 5 weeks after bile duct ligature. The paracrystalline inclusions may thus be specific to the human being. This suggests the possibility of qualitative or quantitative species differences, which should be remembered in correlating findings in humans with experimental data from laboratory animals. The significance of the paracrystalline inclusions must at present remain
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u n k n o w n . 1 H i s t o c h e m i c a l a n d b i o c h e m i c a l studies o n livers shown to c o n t a i n such inclusions s h o u l d be carried out. T h e y m a y p r o v i d e interesting correlations w i t h these peculiar u l t r a s t r u c t u r a l features. The author is particularly indebted to Dr. Theodor W. Blackstad and to Professor Wilhelm H a r k m a r k for the critical revision of the manuscript.
ADDENDUM After the manuscript had been completed for publication, we received the April number of the American Journal of Pathology, where Drs. D. J. Svoboda and R. T. Manning, showed giant mitochondria of human liver cells containing paracrystalline inclusions which in transverse section appeared to be made up of parallel dots. This confirms the observations presented in this paper. Some pictures with the same details have been published also in the atlas by H. David, "Submikroskopische O r t h o - u n d Pathomorphologie der Leber" (Akademie Verlag, Berlin, 1964).
REFERENCES 1. CARRUTHERS,J. S. and STEINER,J. W., Gastroenterology 42, 419 (1962). 2. EKHOLM, R. and EDLUNG, Y., Intern. Kongr. Elektronenmikroskopie 4 Berlin 1958, Verhandl., Vol. 2, p. 273. Springer, Berlin, 1960. 3. ESSNER, E. and NOVlKOFF, A. B., J. Ultrastruct. Res. 3, 374 (1960). 4. FAWCETT, D. W., J. Natl. Cancer Inst. 15, Suppl., 1475 (1955). 5. FERN.g,NDEz-MORAN, H., in Symp. Intern. Soc. Cell Biol. 1, 411 (1962). 6. HOLLE, G., in MARTINI, G. A. (Ed.), Aktuelle Probleme der Hepatologie, p. 8. Thieme, Stuttgart, 1962. 7. JI~ZEQUEL,A. M., J. Ultrastruct. Res. 2, 210 (1959). 8. JI~ZEQUEL,A. M. and ALBOT, G., Semaine Hop. Paris 38, 523 (1962). 9. KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). 10. LUFT, R., IKKOS, D., PALMIERI, G., ERNSTER, L. and AFZELIUS, B., d. Clin. Invest. 41, 1776 (1962). 11. LUSE, S. A., BURCH, H. B. and HUNTER, F. E. JR., Proc. Intern. Congr. Electron Microscopy, 5th, 1962, Vol. 2, p. VV-5. Academic Press, New York, 1962. 12. MAZZANTI, L. and MUGNAINI, E., unpublished observations. 13. MENGHINr, G. and ORLANDI, F., La Puntura Biopsia Epatica. I. Pensiero Scientifico, Roma, 1957. 14. MUGNAINI, E., d. Cell. Biol., in press. 1 It should be mentioned here that a large number of mitochondria containing very numerous, often tubular, cristae and paracrystalline inclusions (the latter, however, were compared with "a pile of board"), have been described by Luft et al. (10) also in human skeletal muscle cells in a pathological condition. These authors in parallel biochemical studies were able to demonstrate a loosely coupled state of oxidative phosphorylation, and they concluded that the observed features "may be the result of a compensatory mechanism by which the muscle cell attempts to overcome the handicap imposed by the decreased biochemical efficiency of these mitochondria."
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15. NAPOLITANO,L. and FAWCETT,D. W., Y. Biophys. Biochem. Cytol. 4, 685 (1958). 16. NOVIKOFF,A. B., in BRACHET,J. and MIRSKY, A. E. (Eds.), The Cell, Vol. II, p. 299. Academic Press, New York, 1961. 17. PALADE,G. E., J. Exptl. Med. 95, 285 (1952). 18. REYNOLDS,E. S., ar. Cell. Biol. 19, 139 (1963). 19. ROUILLER,C., Ann. Anat. Pathol. 2, 548 (1957). 20. ROUmLER, C. and GANSLER,H., Syrup. Fine Structure Ceils, p. 82. Noordhoff, Groningen, 1954. 21. ROUmL~R, C. and J~z~QtmL, A. M., in ROUlLLER, C. (Ed.), The Liver, Vol. I, p. 195. Academic Press, New York, 1963. 22. SVOBODA,D. J. and HmGINSON, J., Am. J. Pathol. 43, 477 (1963). 23. TmERY, J. P. and CAROLbJ., Rev. lntern. Hepatol. 12, 207 (1962). 24. WILSON,J. W. and LEDuc, E. H., J. CellBiol. 16, 281 (1963).