CENTRIFUGAL,BIOCHEMICAL, AND ELECTRON MICROSCOPIC ANALYSIS OF CYTOPLASMIC PARTICULATES IN LIVER HOMOGENATES

A combined centrifugal, biochemical, and electron microscopic study of the cytoplasmic particulates present in 0.88 M sucrose homogenates of rat liver has been carried out. Size distribution analyses of particles containing pentose nucleic acid (PNA) and exhibiting several types of enzymatic activity revealed three major size groups within the range of particle radius between 10 and 500 mµ. A different array of biochemical properties was associated with each size group. The largest particles, with an average radius (assuming spherical shape) in the region of 220 to 260 mµ, contained all of the succinic dehydrogenase activity of the cytoplasmic extract, 29 per cent of the diphosphopyridine nucleotide (DPN)-cytochrome c reductase activity, and minor amounts of PNA and acid phosphatase activity. Cytologically, this group of particles was identified with the mitochondria. All of the uricase activity, 58 per cent of the acid phosphatase activity, and 26 per cent of the PNA was apparently associated with a second size group of particles (average radius 120 mµ) which were tentatively identified by electron microscopy with vesicular structures derived from the ergastoplasm of the intact cell. The third particle group demonstrated by centrifugation exhibited a major size distribution peak at 25 mµ and a second smaller peak at 55 mµ. Over 50 per cent of the total cytoplasmic PNA and DPN-cytochrome c reductase activity was associated with particles in this size group. Electron microscopy revealed a morphologically heterogeneous population of particles within this size range.     

Correlation of physical and biological properties of mouse mammary tumor agent

Biophysical procedures have been used to determine the size and structure of the biologically active agent responsible for the transmission, through milk, of mouse mammary adenocarcinoma. Filtration of milk from RIII high-breast-cancer mice through gradocol membranes with decreasing pore sizes indicated that a minimum of activity passed through intermediate pore sizes (100 to 160 mmicro). Filtrates through smaller pores were significantly active. Milk treated with small doses of deuteron irradiation produced more tumors than the control, unirradiated milk; larger doses indicated a particle size much less than 100 mmicro. Free diffusion experiments indicated that the activity was associated with particles of two different sizes. Altogether the data denoted the presence of a large agent about 100 mmicro in diameter and a small agent 20 to 30 mmicro in diameter or possibly smaller. Furthermore, the presence in the milk of an inhibitor 40 to 60 mmicro is indicated by the results of all three approaches. The complex nature of the milk agent disclosed by the physical measurements agrees with the picture of one of the structures revealed by electron microscopy as well as with seemingly conflicting measurements reported in the literature. The large agent defined by these indirect methods corresponds to the whole particle seen in the electron microscope and the small agent corresponds to its internal core or nucleoid. It is suggested that the nucleoid is essentially a nucleic acid which may, in the absence of the "inhibitor," retain its activity after being stripped of its outer membrane or sac.     

Pancreatic microsomes; an integrated morphological and biochemical study

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, ( approximately 15 mmicro), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mmicro), limited by a thin membrane (7 mmicro) which bears small ( approximately 15 mmicro) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules ( approximately 150 mmicro) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small ( approximately 15 mmicro), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.     

Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, _w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mµ in diameter. The 46 and 28 S subunits also appeared spheroidal.     

Thin sections. I. A study of section thickness and physical distortion produced during microtomy

Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mmicro; silver, 60 to 90 mmicro; gold, 90 to 150 mmicro; purple, 150 to 190 mmicro; blue, 190 to 240 mmicro; green, 240 to 280 mmicro; and yellow, 280 to 320 mmicro. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed.     

Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies

This study on human neutrophils was conducted to measure the kinetics of degranulation of the different cytoplasmic granules into phagocytic vacuoles, and to relate the timing of these events to the burst of respiration that accompanies phagocytosis by these cells. Purified neutrophils were incubated with latex particles opsonized with human immunoglobulin (Ig)G, and phagocytosis was stopped at timed intervals. The cells were examined by electron microscopy to document the sequence of degranulation of the cytoplasmic granules. The azurophil granules and lyosomes were identified by histochemical staining for peroxidase and acid phosphatase, respectively. Phagocytic vacuoles were separated from cell homogenates by floatation on sucrose gradients and assayed for contained lactoferrin, myeloperoxidase, and acid hydrolases. The conclusions drawn from the biochemical and morphological studies were in agreement and indicated: particle uptake and vacuole closure can be completed within 20 s; both the specific and azurophil granules fuse with the phagocytic vacuole much earlier than is generally appreciated, with half-saturation times of 39 s (99% confidence limits, 15-72); oxygen consumption has kinetics similar to those of the fusion of these granules with the phagosome; degranulation of the acid hydrolases beta- glucuronidase, N-acetyl-beta-glucosaminidase (biochemical assays), and acid phosphatase (biochemical assay and electron microscopic cytochemistry) have kinetics of degranulation that are similar to each other but totally different from and much slower than that of myeloperoxidase with half-saturation times of between 354 and 682 s (99% confidence limits, 246-883). This suggests that the acid hydrolases are not co-located with myeloperoxidase in the azurophil granule but are contained in distinct lysosomes, or "tertiary granules".     

CYTOPLASMIC PARTICLES AND AMINOACYL TRANSFERASE I ACTIVITY

It has been possible to show by electron microscopy of samples selected from sucrose gradients that particles of specific size and shape are present in supernatant fluids derived from nucleated animal and plant cells, but not in extracts from Escherichia coli. Aminoacyl transferase I activity in these same gradients sediments in two peaks representing material of approximately 5–7S and 18–20S. A rectangular particle, 100 x 145 A in size, sediments at 19S and coincides with the second peak of transferase I activity. The possibility that the rectangular particle may be a "carrier" particle associated with transferase I is discussed.     

Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.     

The size and number of intramembrane particles in cells of the photosynthetic bacterium Rhodopseudomonas capsulata studied by freeze-fracture electron microscopy.

By freeze-fracture electron microscopy, particles have been observed on the protoplasmic leaflet (PF face) of cytoplasmic and intracytoplasmic membranes of the photosynthetic bacterium Rhodopseudomonas capsulata. The particles are present under all culture conditions of chemotrophically and phototrophically grown cells. However, the number of particles per microM2 increased significantly when the formation of the photosynthetic apparatus in the membrane is induced. Intracytoplasmic membranes, where the bulk of photosynthetic activity is localized, always have a higher density of particles than cytoplasmic membranes. Under all conditions particles with a diameter of 9.5 nm dominate. The frequency of particles with diameters greater or smaller than 9.5 nm changed with culture conditions. A comparison of biochemical and electron microscopic data have lead us to the conclusion that the particles, formed under conditions which allow the synthesis of the photosynthetic apparatus, are composed of photochemical reaction centers and antenna light-harvesting bacteriochlorophyll I (B 875)-protein complexes. The total molecular weight of these particles is calculated to be 500,000.     

STUDIES ON FORMALIN FIXATION FOR ELECTRON MICROSCOPY AND CYTOCHEMICAL STAINING PURPOSES

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°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.     

Calf thymus composition: a comparison of differential centrifugation and chemical fractionation procedures

The extraction behavior of thymus and the composition of fractions prepared from this organ has been studied. Sequential extraction methods using 0.15 M NaCl followed by water gave information with respect to the weight fraction of cytoplasmic and nuclear constituents. Lipide, nucleic acid, and electrophoretic analysis of the extracts provided additional information. A less complex electrophoretic pattern was obtained from subsequent extracts in the sequence. Sucrose and saline dispersates obtained from tissue fragmented with either the Potter-Elvehjem homogenizer or in a Waring blendor were fractionated, using standard differential sedimentation methods. The fractions obtained by means of four different dispersion procedures were compared in terms of yield, chemical analysis, and electrophoretic composition. The quantity of material in thymus having the sedimentation characteristics of liver mitochondrial and microsomal fractions was remarkably small. Both the suspension medium employed and the method used to bring about a disruption of the cells in the tissue affected the yield of "particulate" material. The components present in the later extracts in the sequence, E(4) to E(7), in the case of sequential extraction study resembled with respect to chemical composition and electrophoretic characteristics, the microsome fraction prepared by differential sedimentation methods. About 76 per cent of the PNA in the tissue appeared to be in the cytoplasm. The remaining 24 per cent PNA was found in the nucleus and accounted for 1.7 per cent of nucleus on a dry weight basis. From 75 to 88 per cent of cytoplasmic PNA was extracted from the tissue and 76 to 94 per cent of the PNA in the extract was found in the final supernatant solutions, depending upon the dispersion methods and suspension medium used in the extraction procedure. The composition of the final supernatant fractions using differential sedimentation methods were comparable in terms of electrophoretic properties, protein concentration, nucleic acid content, and fractionation behavior to saline extracts E(1) to E(3), of thymus used in earlier studies.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

STUDIES ON ISOLATED CELL COMPONENTS : XVI. The Distribution of Acid Phenyl Phosphatase Activities in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.     

Subcellular distribution of hydrolases in Naegleria fowleri

The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) alpha-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.     

Analog modeling of glucagon-induced autophagy in rat liver. II. Evaluation of iron labeling as a means for identifying telolysosome, autophagosome and autolysosome populations.

To evaluate the potential usefulness of iron labeling as a means for identifying the telolysome, autophagosome and autolysosome populations of rat liver, animals treated with Jectofer (iron-citric acid-sorbitol complex), or with Jectofer followed by glucagon, have been studied with a variety of biochemical and morphological methods. Differential centrifugation studies of liver homogenates revealed that the sedimentation velocity and mechanical fragility of acid phosphatase bearing particles increase with the duration of Jectofer treatment and that iron accumulates in the mitochondrial and nuclear fractions. Rate sedimentation studies confirmed the change in sedimentation velocity, which was shown to be due in part to a marked increase in particle density. Quantitative morphological analysis of liver M + L and N + M + L fractions revealed a nearly complete absence of pericanalicular dense bodies after 6–7 days of Jectofer treatment. In these fractions a new type of particle containing fine electron dense granules was seen. The mean volume of these particles was decreased and their number increased when compared to dense bodies but the general morphology and overall size distribution of the two particle classes were similar. In animals given both Jectofer and glucagon, autophagic vacuole formation was similar to that found in animals receiving only glucagon. However, the increase in osmotic fragility of acid phosphatase bearing particles usually seen after glucagon administration occurred at a significantly slower rate. Examination of paniculate fractions revealed the presence of autophagic vacuoles with (autolysosomes) and without (autophagosomes) fine dense granules. The number of autolysosomes and their relative proportion in the autophagic vacuole population were correlated with an increase in the osmotic fragility of the acid phosphatase bearing particles in the same fraction. Organelle degeneration was observed more frequently in autolysosome profiles. These results support the contention that iron labeling can be used to separate the principal particle populations participating in the autophagic response induced by glucagon.     

Subcellular Distribution of Hydrolases in Naegleria fowleri1

The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) a-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.     

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 mmicro) having a thick capsule and a dense inner body (35 to 40 mmicro) that is eccentrically placed within the central cavity (70 to 80 mmicro). 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.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.