The Journal of Biophysical and Biochemical Cytology最新文献

A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0-2 degrees C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.

De-embedded ultrathin sections of ethanol-fixed Tipula Iridescent Virus particles were incubated with pepsin at pH 1.8, trypsin at pH 7.7, and DNase at pH 7.7. The outer shell of the particles, but not an inner core, was removed by the action of pepsin. Conversely, the inner core, but not the outer shell, was removed by the action of trypsin and DNase in combination, but not by either enzyme acting alone. These results are taken to mean that the outer shell of the particles is protein in nature and the inner core is nucleoprotein. Whole virus particles were also exposed to the same 3 enzymes. Trypsin and/or DNase had no effect on the whole particles, while pepsin at pH 1.8 digested away the outer shell of the particles and released an intact core, resistant to pepsin. The protein nature of the digested outer shells and the nucleoprotein nature of the released cores were confirmed by ultraviolet absorption spectra. Chemical analyses showed that the cores contain 89 per cent of the whole virus phosphorus but only 35 per cent of the nitrogen, while the outer shells contain only 5 per cent of the phosphorus but 63 per cent of the nitrogen. On the basis of nitrogen: phosphorus ratios the composition of the cores is estimated to be about 30 per cent DNA and 60 to 65 per cent protein.

The embryo of a tarsonemid mite was found to be suitable for in vivo observations of muscle development by polarization microscopy. The four dorsal muscles of the metapodosoma each contain three sarcomeres, the anterior two of which can be seen clearly. These sarcomeres can be identified and followed during much of their development. Sarcomeres are about 2.5 micra long when first detected and increase in length until they are about 10 micra long. The change in length is associated with a slow, approximately constant rate of increase in the length of the A region, and an initially slow then much more rapid increase in the length of the I band. Preceding the period when the I band elongates rapidly there is an increase in the diameter of the muscle fibers and an increase in the retardation of the A band. A, I, Z, and H bands are visible during most of these changes. The change in A band length has been interpreted in terms of the growth of the A filaments which have been observed by electron microscopy in muscles of other animals. It is suggested that the exceptionally long sarcomeres in this mite result from the early fixing of the number of sarcomeres in a given muscle fiber.

A description of the fine structure of the yolk of the unincubated hen's egg has been provided, which will serve as a basis for further studies on yolk digestion. The gross components of the yolk (that is, free-floating lipid drops, yellow and white yolk spheres together with their enclosed lipid subdroplets, and aqueous protein fluid) could be recognized by phase contrast and low power electron microscopy. The majority of the lipid drops, whether free floating or enclosed within yolk spheres, were composed of particles about 30 to 60 A in diameter. The protein component of the yolk was found to consist of round profiles about 250 A in diameter. The surfaces of the yolk spheres were of three types, and it is difficult to decide which represents the true structure although reasons are given for believing that yolk spheres are not normally enclosed by membranes identical with cell membranes.

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 micro still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.

Discrete localization of stain in pericanalicular granules was found in 10 micro frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 micro thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 micro slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 micro frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.

In the fungus Neurospora crassa, hexagonal crystalline inclusions have been observed with both the light and electron microscopes. These crystals have been enriched by differential centrifugation and found to be identical with ergosterol by the criteria of ultraviolet spectral analysis and cytochemical analysis. Observations have been made on the distribution and fine structure of the crystalline bodies in various wild type and mutant strains of N. crassa.

Chick myofibrils in different states of contraction were treated with fluorescein-labeled antibodies. The rabbit antibodies were prepared against chick myosin, light and heavy meromyosins, and actin. For any one state of contraction, a single myofibril was photographed through the phase contrast microscope, stained with one of the antisera, and photographed through the fluorescence microscope. The cytological changes in the sarcomeres accompanying contraction as observed under phase were correlated with changes in the distribution of the precipitated antibodies as observed under the fluorescence microscope. The changing patterns observed through the fluorescence microscope were compared with those predicted by the sliding filament model of contraction.