ELECTRON MICROSCOPE OBSERVATIONS ON INTRACELLULAR VIRUS-LIKE PARTICLES ASSOCIATED WITH THE CELLS OF THE LUCKÉ RENAL ADENOCARCINOMA

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mµ) having a thick capsule and a dense inner body (35 to 40 mµ) that is eccentrically placed within the central cavity (70 to 80 mµ). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.     

ELECTRON MICROSCOPIC STUDY OF THE CYTOPATHOLOGY OF ECHO VIRUS INFECTION IN CULTIVATED CELLS

The cultivated monkey kidney cell is subject to changes when infected with ECHO viruses 6, 9, and 19. The electron microscope reveals three stages of infection: (a) initial stage. The nucleus appears granular with chromatin condensation on the nuclear envelope. The cytoplasm contains electron transparent vesicles and vacuoles forming nests. (b) Intermediate stage. The nucleus seems to diminish, appearing more pycnotic and displaced toward the periphery. The cytoplasm is filled with electron transparent vacuoles and vesicles, and dense masses as well as some spiral bodies are seen. The mitochondria retain their shape. Dense particles are seen, which are possibly of viral nature. (c) Final stage. The nucleus is contracted to a narrow strip close to the cellular membrane or is completely destroyed. The cytoplasm shows no apparent changes. Crystals are frequently observed in cells infected with ECHO viruses 6 and 19, consisting of dense particles with an average diameter of 14.4 mµ ranging from approximately 13.2 to 15.6 mµ for ECHO virus 6, and 14.5 mµ ranging from approximately 12.5 to 16.5 mµ for ECHO virus 19. These particles are clustered in hexagonal packages forming angles of 75° and 105°. The particles in most crystals are arranged in rows separated by a constant distance, the latter varying from one crystal to another and being approximately 1.5 and 2.5 times the distance between particles. Other particles were observed which, however, are not considered to be of viral nature.     

THE ULTRASTRUCTURE OF GLIOSOMES IN THE BRAINS OF AMPHIBIA

Small fragments of superficial neuropil and fragments of deeper layers from various regions of the brains of Xenopus laevis Daud. and Rana esculenta L. were fixed in buffered osmium tetroxide, embedded in Vestopal W or methacrylate, and studied with the electron microscope. The glial fibers and their meningeal end-feet contain numerous large mitochondrion-like dense bodies for which the term "gliosome" has been adopted. Gliosomes have a specific and constant structure characterized by the presence of a row of peripheral and circular canaliculi and an electron-opaque fibrous or finely granular matrix. Also, another less frequently found type of gliosome is present which contains regular lamellar structures. The gliosomes vary considerably in size and may be very large, up to 9 µ in length. Numerous and various intermediate forms between mitochondria and gliosomes can be seen. Gliosomes are largest and most numerous in the distal portions of the glial fibers and in the meningeal end-feet.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

CHANGES IN THE ORGANIZATION OF TUBULIN DURING MEIOSIS IN THE EGGS OF THE SURF CLAM, SPISULA SOLIDISSIMA

Polymerized tubulin can be stabilized in Kane's spindle isolation medium (HGL solution), isolated by differential centrifugation and then assayed by colchicine binding activity. In the eggs of the surf clam, Spisula solidissima, the level of particulate tubulin undergoes a series of specific changes during first meiotic division. In either unactivated ("interphase") eggs or metaphase eggs the amount of particulate tubulin was about 13% of the total at 23°C. The amount of particulate tubulin decreased shortly after activation, reaching a minimum value at about 5 min, the time of nuclear membrane breakdown. The particulate tubulin concentration then rose, reaching a maximum at metaphase, and then decreased again during anaphase, reaching a minimum at first polar body formation. In HGL homogenates of unactivated eggs a structure is present which has been shown to contain the interphase particulate tubulin (IPT). This structure consists essentially of a 10–20 µ granular sphere attached to a membranous material which is probably part of the egg cortex. These particles are absent at the time of nuclear membrane breakdown, when the level of particulate tubulin is minimal and when the first signs of spindle formation are visible. Electron microscopy of these particles by negative staining indicates that they are composed of microtubules associated with a granular matrix which may be a polymorphic aggregate of tubulin.     

MORPHOLOGY OF THE OMMATIDIA OF THE COMPOUND EYE OF LIMULUS

The sensory portion of the ommatidium of the compound eye of Limulus has been studied with the electron microscope. In axial longitudinal section the rhabdom appears to be made up of small polygons, and in transverse section the rhabdom appears as a banded structure of dark lines. Thus in three dimensions the rhabdom resembles a honeycomb composed of tubular units, the long axes of which lie in transverse planes and are oriented perpendicular to the retinula cell's contours. The tubular units, which are about 140 mµ in diameter in Limulus (70 mµ in diameter in the spider and Scutigera), are microvilli of the borders of the retinula cells. The walls of these microvilli are continuous with fine linear structures (membranes) in the cytoplasm of the retinula cells. In transverse sections of the ommatidium oval bodies interpreted as mitochondria are observed in an annular zone at the tips of the rhabdom's rays. These mitochondria, which are 2 to 10 µ in diameter, are crowded with irregular closed outlines about 100 mµ in diameter. Possible functions of components of the ommatidium are discussed.     

ANALYTICAL DIFFUSION OF INFLUENZA VIRUS AND MOUSE ENCEPHALOMYELITIS VIRUS

Analytical diffusion has been applied to a study of influenza A virus in mouse lung, influenza A virus in the extra-embryonic fluids of the chick, and mouse encephalomyelitis virus in mouse brain. The results from influenza in mouse lung suggested that about 99 per cent of the infectivity was present in particles 200 mµ in diameter, and 1 per cent in particles 6 mµ in diameter. The results from influenza in extra-embryonic fluids indicated that the preparation was inhomogeneous and that the smallest virus units were about 6 mµ in diameter. The results from mouse encephalomyelitis virus indicated that the preparation was also inhomogeneous, with 10 per cent of the infectivity in particles about 15 mµ in diameter. It has been suggested that in virus preparations normal colloidal particles can act as carriers of much smaller virus units.     

ELECTRON MICROSCOPE STUDIES OF RABIES VIRUS IN MOUSE BRAIN

The cells of brains of 2- and 3-day old mice infected with street rabies virus were examined in the electron microscope. It was observed that characteristic rod-like or elongated particles were found within a "matrix" in the cytoplasm of nerve cells and of astrocytes. These rod-like particles can be separated into two types, on the basis of slightly different morphological features. One particle is 110 to 120 mµ wide and has double-membraned coats; the other is 120 to 130 mµ wide and is covered by a single limiting membrane. The former is closely associated with the endoplasmic reticulum. The biological relationship between the two types is unknown, but both types of particles are considered to be street rabies viruses because of their structural features. It is believed that segmentation and branching of elongated particles may play a role in virus multiplication. Negri bodies appear as dense round bodies containing various coarse structures but no virus particles.     

An Electron Microscope Study of the Rat Ovum

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 µ. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mµ in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations.     

THE PHAGOCYTOSIS OF SOLID PARTICLES : II. CARBON.

1. By measurements of the diameter and velocity of leucocytes and of the particles in two carbon suspensions, the relative rates of ingestion of the two suspensions by the leucocytes are predicted and the predictions verified experimentally. 2. The results indicate that 4.7µ particles of carbon are ingested as readily as 3.2µ particles. The more rapid apparent rate of ingestion of the 4.7µ particles is due to their greater availability rather than the greater capability of the leucocytes. 3. There is almost no phagocytosis of carbon in absence of serum or in heated serum. 4. The clumping of unwashed leucocytes is accllerated by serum and by the ingestion of carbon. 5. The available evidence indicates that the phagocytosis of bacteria does not follow the law for a monomolecular reaction, possibly because of the toxic effect upon the leucocytes of bacterial extracts.     

AN ELECTRON MICROSCOPE STUDY OF THE CYTOLOGY OF THE PROTOZOAN EUPLOTES PATELLA

1. Structurally the "sensory bristles" in Euplotes patella are typical cilia, but no ciliary rootlets connect their bases. 2. The "neuromotor fibrils" are composed of filaments 21 mµ in diameter. At the point of junction of the filaments with the peripheral ciliary fibrils a granular structure 65 to 90 mµ in diameter is seen which has dense central and peripheral zones separated by a less dense layer. Information on the interconnection of organelles is expanded. 3. A system of subpellicular fibrils is described. The external fibrillar system described by others could not be found. 4. The motorium is shown to be a mass of intertwining rootlet filaments. 5. The micronucleus is shown to have a spongy, dense material in a less dense material, all of which is surrounded by a double-layered membrane. 6. The double-layered macronuclear membrane contains annuli whose outside diameter is 70 mµ; the macronuclear bodies are sometimes closely applied to the membrane. In the macronuclear reorganization bands, the solution plane is a fine network, while the reconstruction plane is devoid of structure at the level of resolution observed. 7. The mitochondria are composed of tubules, only occasionally oriented, usually embedded in a surrounding material of lower density. 8. Microbodies whose diameters are 250 to 350 mµ are frequently observed in close association with mitochondrial surfaces. 9. The food vacuoles, contractile vacuoles, and ciliary vacuoles are bounded by single-layered membranes. In the food vacuoles, the bacteria are surrounded by membranes individually or in small groups. 10. Cytoplasmic rods localized in the oral region, and cytoplasmic granules dispersed at random, are described. No typical ergastoplasm, endoplasmic reticulum, or Golgi material was observed.     

THE FINE STRUCTURE OF THE AXON AND GROWTH CONE OF THE DORSAL ROOT NEUROBLAST OF THE RABBIT EMBRYO

The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed. Large numbers of microtubules, channels of agranular reticulum, and clusters of ribosomes are found throughout the length of the early axons. The filopodia of the growth cone appear as long thin processes or as broad flanges of cytoplasm having a finely filamentous matrix material and occasionally small ovoid or elongate vesicles. At first the varicosity is a small expansion of cytoplasm, usually containing channels of agranular reticulum and a few other organelles. The widely dilated cisternae of agranular reticulum frequently found within the growth cone probably correspond to the pinocytotic vacuoles seen in neurites in tissue culture. The varicosities enlarge to form bulbous masses of cytoplasm, which may measure up to 5 µ in width and 13 µ in length. They contain channels of agranular reticulum, microtubules, neurofilaments, mitochondria, heterogeneous dense bodies, and a few clusters of ribosomes. Large ovoid mitochondria having ribonucleoprotein particles in their matrix are common. Dense membrane specializations are found at the basal surface of the neuro-epithelial cell close to the area where the early neurites first enter the neural tube.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT LIVER

A preliminary electron microscope study has revealed the presence in lysosome-rich fractions, isolated from rat liver, of hitherto undescribed cytoplasmic particles, called "dense bodies." Approximately 0.37 µ in length, the dense bodies often possess an internal cavity and external membrane. They contain many electron-dense granules 55 to 77 A, or less, in diameter. Such dense bodies are also visible in electron micrographs of parenchymatous cells in liver sections. The correlations between dense bodies and lysosomes are listed, but until pure preparations are available it is not possible to assert that dense bodies and lysosomes are identical.     

ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

ROLE OF THE SARCOPLASMIC RETICULUM IN GLYCOGEN METABOLISM : Binding of Phosphorylase, Phosphorylase Kinase, and Primer Complexes to the Sarcovesicles of Rabbit Skeletal Muscle

Sarcoplasmic vesicles and β-glycogen particles 30–40 mµ in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mµ, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10–20 mµ in diameter attached to the outer surface of the membranes. These particles disappear after α-amylase treatment. Incubation of the sarcovesicular fraction with ¹⁴C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

INTERMITTENT STIMULATION BY LIGHT : V. THE RELATION BETWEEN INTENSITY AND CRITICAL FREQUENCY FOR DIFFERENT PARTS OF THE SPECTRUM

1. An optical system is described which furnishes large flickering fields whose brightness, even when reduced with monochromatic filters, is capable of covering the complete range of the relation between critical frequency and intensity. 2. For a centrally located test field of 19° diameter, with light from different parts of the spectrum, the data divide into a low intensity section identified with rod function, and a high intensity section identified with cone function. The transition between the two sections is marked by an inflection point which is sharp, except for 450 and 490 mµ where, though clearly present, it is somewhat rounded. 3. The intensity range covered by the flicker function is smallest in the red, and increases steadily as the wave-length decreases. The increase is due entirely to the extent of the low intensity, rod section which is smallest (non-existent for S. S.) in the red and largest in the violet. The high intensity cone portion for all colors is in the same position on the intensity axis, and the only effect of decreasing wave-length is to shift the rod section to lower intensities without changing its shape. 4. The measurements are faithfully described by two similar equations, one for the rods and one for the cones, both equations being derived from the general stationary state equation already used for various visual functions.     

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from Bacillus licheniformis, N-acetyl-d-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to N-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.     

CHONDROGENESIS, STUDIED WITH THE ELECTRON MICROSCOPE

The role of the cells in the fabrication of a connective tissue matrix, and the structural modifications which accompany cytodifferentiation have been investigated in developing epiphyseal cartilage of fetal rat by means of electron microscopy. Differentiation of the prechondral mesenchymal cells to chondroblasts is marked by the acquisition of an extensive endoplasmic reticulum, enlargement and concentration of the Golgi apparatus, the appearance of membrane-bounded cytoplasmic inclusions, and the formation of specialized foci of increased density in the cell cortex. These modifications are related to the secretion of the cartilage matrix. The matrix of young hyaline cartilage consists of groups of relatively short, straight, banded collagen fibrils of 10 to 20 mµ and a dense granular component embedded in an amorphous ground substance of moderate electron density. It is postulated that the first phase of fibrillogenesis takes place at the cell cortex in dense bands or striae within the ectoplasm subjacent to the cell membrane. These can be resolved into sheaves of "primary" fibrils of about 7 to 10 mµ. They are supposedly shed (by excortication) into the matrix space between the separating chondroblasts, where they may serve as "cores" of the definitive matrix fibrils. The diameter of the fibrils may subsequently increase up to threefold, presumably by incorporation of "soluble" or tropocollagen units from the ground substance. The chondroblast also discharges into the matrix the electrondense amorphous or granular contents of vesicles derived from the Golgi apparatus, and the mixed contents of large vacuoles or blebs bounded by distinctive double membranes. Small vesicles with amorphous homogeneous contents of moderate density are expelled in toto from the chondroblasts. In their subsequent evolution to chondrocytes, both nucleus and cytoplasm of the chondroblasts undergo striking condensation. Those moving toward the osteogenic plate accumulate increasingly large stores of glycogen. In the chondrocyte, the enlarged fused Golgi vesicles with dense contents, massed in the juxtanuclear zone, are the most prominent feature of the cytoplasm. Many of these make their way to the surface to discharge their contents. The hypertrophied chondrocytes of the epiphyseal plate ultimately yield up their entire contents to the matrix.     

THE USE OF SHADOW-CASTING TECHNIQUE FOR MEASUREMENT OF THE WIDTH OF ELONGATED PARTICLES

A method is described for the estimation of the true width of fibrillar or rod-like structures from electron micrographs of metal-shadowed preparations. The method is based on variations in the image width as a function of the angle (β) between the long axis of the fibril and the direction of the shadow in the plane of the preparation. The image width when β = 0° practically represents the real width of the elongated particle but is often indistinguishable from the background. The fibril image width is conveniently measured at β values between 15° and 90°. The true width is obtained by plotting the image width versus sin β and extrapolating to β = 0°. Latex spheres are sprayed with the fibrils or rods to indicate the direction of shadow. Tobacco mosaic virus (TMV) was used as a model structure because of its known constant diameter of 150 A (5). The width (in the case of TMV equal to the diameter) found by the present method was 150 A ± 8 A.