Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of 14C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.
In a review of the fine structure of striated muscle , Barer (1948) writes: "Relatively little interest seems to have been taken in the problems of muscle structure in the years between 1900 and 1930. The old observers had attacked the problem with such energy and in so many ways that there seemed relatively little left to do." While it is true that the present century inherited from the last many basic concepts of muscle structure recognized today as valid, the study of this subject by no means flagged, since, along with the foundations provided by Bowman, K611iker, and others, there came a welter of controversy and confusion. Many such controversies stemmed from the varied interpretation of techniques of observation , especially of staining methods, but of perhaps greater importance was the conceptual confusion arising out of the comparison between muscle and other cells. Terms which now have a rather precise structural connotation-Golgi apparatus, reticulum or ergastoplasm, sarcosome or mitochondrion-have all had a long history, but for each term that has survived, a score have passed out of use. Consider, for example, the term mitochondrion. Cowdry, in 1921, listed thirty other names which he recognized to be synonymous , in use before the general term was introduced by Benda in 1897, and Cowdry completed this list with twenty-three further synonyms and seventeen terms used to suggest origin, behavior, and function, coined after the acceptance of the mitochondrion as a universal cell organelle. It is essential that, in attempting to trace the development of concepts of muscle structure, parallel work on other tissues be considered, because such a comparison was inherent in the original observations and descriptions of those who first investigated the organization of cells. As Bennett (1955, 1958) points out, we owe many of the current basic concepts of muscle structure to the work of Bowman, Brficke, K611iker, and others, but only quite recent work has confirmed the essential validity of their ideas, and at the turn of the century there was scarcely a single aspect of muscle organization that was not entirely controversial. We shall not review or even mention all these controversies, but rather shall trace the origin and ultimate resolution of just one, concerning the nature of the sarcoplasm, and show that workers in the early years of this century by no means ignored or abandoned the unfinished lines of approach inherited from the last. By …
Several months following parenteral injections of saccharated iron oxide into DBA/2J mice, granules rich in iron were found in nuclei of scattered parenchymal liver cells as well as in the cytoplasm. As seen in the light microscope, the intranuclear granules were brown; most of them measured between 0.5 µ and 1 µ in cross-section. They gave positive Prussian blue tests, and were not selectively stainable with pyronine. Electron micrographs of the granules showed closely packed aggregates of ferritin molecules, occasionally in paracrystalline order. The intranuclear collections were often surrounded by bands of material of moderate opacity. Scattered ferritin molecules and collections of such molecules were also present in the cytoplasm of many liver cells, but there seemed to be no quantitative relationship between intranuclear and cytoplasmic ferritin. Liver cells from untreated control mice failed to reveal intranuclear deposits of ferritin. Although the site of origin of the intranuclear aggregates of ferritin is unknown, the findings suggest the possibility that under suitable circumstances ferritin synthesis may take place within nuclei of liver cells—perhaps induced by the presence of colloidal iron.
In connection with the potential development of automatic two-wavelength microspectrophotometry, a new version of the two-wavelength method has been formulated. Unlike its predecessors, the Ornstein and Patau versions, the new method varies the area of the photometric field seeking to maximize a relationship between distributional errors at the two wavelengths. Stating this distributional error relationship in conventional photometric terms, the conditions at the maximum are defined by taking the first derivative with respect to field size and setting it equal to zero. This operation supplies two equations; one relates the transmittances at the two wavelengths, and a second states the relative amount of chromophore in the field in terms of transmittance at one wavelength. With the first equation to drive a servomechanism which sets the appropriate field size, the desired answer can then be obtained directly and continuously from the second equation. The result is identical in theory with those of the earlier methods, but the technique is more suitable for electronic computing.
The torpedine electric fish Narcine brasiliensis has two morphologically distinct electric organs (main and accessory) which also differ with respect to a number of electrophysiological properties. The fine structure of the electroplaques of these organs has been examined by electron microscopy and by a histochemical method for localizing esterase activity with a high degree of resolution. In both kinds of electroplaques the innervated surface (ventral in those of the main organ, dorsal in those of the accessory) is the only site of esterase activity. The latter is further confined to the regions of synaptic contact between vesicle-containing axon terminals and the electroplaque membrane. The synaptic apparatus is similar to, but less elaborate than, that of neuromuscular junctions. The axon terminals and electroplaque membranes are free of connective tissue envelopments. The membrane of the uninnervated surfaces forms a continuum with a dense canalicular network which penetrates deeply into the 7 micro thick electroplaques of the main organ. The canalicular network has about the same thickness in the 20 micro electroplaques of the accessory organ. Except for this difference, the two kinds of cells appear to have the same fine structure. This finding is discussed in relation to the electrophysiological data on functional differences.
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.
A study has been made of developmental variations in anthers of Lilium longiflorum with respect to the deamination of deoxycytidine and 5-methyldeoxycytidine. Although a periodicity of enzymatic activities surrounding the mitotic cycle has been found, the over-all evidence is against the significance of such periodicity to chromosome duplication. On the basis of evidence obtained a distinction has been drawn between enzyme systems functioning in a broad supporting role and those immediately tied to morphogenetic events.
The light and, to a lesser extent, the dark cells of the cortical collecting tubules in mouse kidney contain a great number of granules which according to histochemical tests are composed of phospholipids and proteins. These granules are bounded by a triple-layered membrane measuring approximately 75 A across, and contain one or several crystals with a hexagonal or square lattice. These crystals are built up of rod-shaped units, which appear dense after osmium fixation, measure about 48 A in diameter, and are separated by a light interspace of similar dimensions. The mean center-to-center distance of the rods is about 96 A. The structure is explained as a lipoprotein crystallized within a membrane-bounded vacuole. No relationship between these granules and mitochondria was found. The physiological significance of the granules remains unknown.
As electron microscopic techniques have improved, the fine structure of bacterial cells has come to be understood more clearly. There are, however, many details yet to be elucidated. I propose to report here some observations on the plasma membrane of Staphylococcus aureus, using sectioning techniques. Bradfield (1956) has already described some structural features of Staphylococcus aureus using the thin sectioning method.
Cyanidium caldarium is a thermophilic, acidophilic alga with a cell d iameter of ca. 3 #. It has generally been assigned to the Cyanophyta (blue-green algae) bu t on the basis of morphology, p igment content, and life history, Hirose (2) suggested tha t it should be assigned to the R h o d o p h y t a (red algae). Cyanidium grows, respires, and photosynthesizes optimally at pH 2.0 to 3.0, and at 55°C. (1). Because of its small size, disputed taxonomic position, and remarkable physiological characteristics, the fine structure of this organism is of part icular interest. The cells were grown in a mineral nu t r i en t med ium conta ining 2 per cent glucose at pH 2.0 (liquid) or 5.8 (agar slants) at a l ight intensity of 150 to 250 foot-candles. Cells were fixed for 30 minutes in an unbuffered solution of 5 per cent K M N O 4 at room temperature . Fixation was also a t tempted with a variety of procedures employing buffered osmium tetroxide. The material was dehydra ted in alcohol at room tempera ture and embedded in an Araldite mixture containing 5 per cent plasticizer. The plasticizer was added to permit centrifugation of the cells after Araldite
An electron microscopic survey has been made of the gastric parietal or oxyntic cell of the human, cat, beaver, dog, hamster, rat, mouse, and bat, and of the corresponding cell type in two species of frog, two species of toad, and the horned lizard. A feature consistently found in the parietal cells of the mammals or their equivalent in the lower vertebrates is the agranular endoplasmic reticulum, which takes the form of branching and anastomosing small tubules approximately 200 to 500 A in diameter, sometimes expanded into flattened cisternae. In mammalian parietal cells this form of the endoplasmic reticulum is found only in limited amounts, but in the corresponding secretory cells of the amphibia and reptilia the tubular agranular reticulum is abundant. It is believed to comprise a more or less continuous system of channels, but owing to their tortuous course only short profiles are seen in thin sections. Immediately subjacent to the plasmalemma at the free surface, the cytoplasm is relatively free of organelles but is occasionally traversed by the agranular reticulum, which appears to be continuous at some points with the cell surface. The possible participation of the agranular endoplasmic reticulum in hydrochloric acid secretion is discussed.
The effect of tricyano-amino-propene, a dimer of malononitrile, on the base composition of the RNA in isolated Deiters' nerve cells and their oligodendroglial cells has been studied using a microelectrophoretic method. Tri-a-p in a dose of 20 mg/kg has the effect of increasing the RNA and protein content per nerve cell by 25 per cent and decreasing the glia RNA by 45 per cent. The RNA base composition of the nerve cells from the control animals differs from that of their glial cells. The guanine of the nerve cell is significantly higher than that of the glia, but the content of cytosine is higher in the glia than in the RNA of nerve cell. The cytosine of nerve cells decreased significantly after tri-a-p administration. In the glial cells the cytosine showed a 20 per cent increase, and the guanine a 25 per cent decrease. Tri-a-p sharpened the difference in RNA composition already existing between the control nerve cells and their glial cells by almost 300 per cent for the guanine and by 400 per cent for the cytosine. The chemical and functional relationship between the nerve cell and its oligodendroglial cells is discussed.
A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.
In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg(++)-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca(++) and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.
A simple procedure has been established for controlling and measuring the pH of media in which the bicarbonate-carbonic acid system is the predominant buffer. The HCO(-) (3) concentration was maintained at 22.5 mM and the H(2)CO(3) concentration was varied by equilibrating the media with 0.5 to 40 per cent CO(2) in air. The curve relating extracellular pH to 3 day cell growth was similar for glass-attached HeLa and Chang liver cells. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range. Comparable pH growth curves were also obtained with newly isolated cells from rat liver and skeletal muscle. It was shown that the effect of pH on growth was independent of the CO(2) concentration and that the essential nutrients in the medium were stable over the pH range studied. Although alkalosis depressed the 3 day cell population, cells exposed to a pH of 8.0 to 8.2 grew at the maximal rate for the first 12 to 24 hours. Growth then ceased abruptly and the cells entered a steady state with respect to net protein synthesis. This was followed by cytoplasmic retraction and cell death. Increasing the concentrations of calcium or magnesium in the medium failed to prevent the effects of alkalosis. Moreover, the increase in CO(-) (3) concentration of the media and the concomitant decrease in Ca(++) ion concentration that occur at high pH were eliminated as determining factors in the growth failure and death. While acidosis had a less pronounced effect on the 3 day cell population, its effect on the growth rate was immediate. The increase in cell generation time was proportional to the H(+) ion concentration. In each of the cell lines studied, acidosis was accompanied by a striking increase in the number of cytoplasmic perinuclear granules. These granules which stain supravitally with Janus green are extracted from fixed cells with lipid solvents. They maintain their identity in cell homogenates and may be isolated from the other subcellular structures by differential centrifugation; at 100,000 g they form a distinct layer at the top of the supernatant fraction. On the basis of their physical and chemical properties, these granules have been called lipid-rich particles. The accumulation of lipid-rich particles in acidosis was independent of the growth rate and the CO(2) concentration.
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 µ 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.
Light blue fluorescent globules accumulate in the cells of the anterior region of the fatbody of Drosophila larvae near the time of pupation. This fluorescent material appears in the Ore-R wild type strain as well as mutant strains in which the synthesis of both the red and brown eye pigments is affected. The vermilion mutant, which is characterized by the absence of the brown pigment component in the eye, was the only strain among those examined which did not develop the light blue fluorescent globules. Utilizing chromatographic techniques together with the information gained by examination of the mutant strains, the fluorescent material has been identified as kynurenine. Of particular interest is the manner of appearance of the fluorescent material in the vicinity of the nuclear membrane of the fat cells.
Electron micrographs of ribosomal RNA from Escherichia coli, microsomal RNA from calf, rat, and chick liver, Bacillus cereus RNA and E. coli soluble RNA are presented. Filaments of about 10 A in diameter could be observed in preparations obtained from aqueous solutions of high molecular weight RNA. When ammonium acetate solutions were used a tendency for coiling and aggregation was observed. E. coli soluble RNA appears as small, sometimes elongated particles the smallest diameter being of about 10 A.
Osmium-fixed, undecalcified, callus tissue from healing fractures of rat tibias was sectioned with a diamond knife for study with the electron microscope. Large multinucleated cells were found adjacent to bone. A characteristic labyrinthine infolded border was consistently seen in parts of the cells close to the bone surface. The innermost parts of this "ruffled border" gave rise to vacuoles. The bone surface was always disrupted under the "ruffled border" of the cells. Needle-like crystals were seen at the osseous fringe, within folds in the ruffled border as well as within vacuoles deeper in the cells. Collagen fibers denuded of crystals were never observed. Mitochondria, containing clusters of fine granules, were abundant. The part of the cell away from bone contained rough endoplasmic reticulum and the cell membrane was thrown into irregular microvilli. These observations are discussed in relation to current concepts of osteoclastic resorption of bone.
