bluuebunny/crossref_metadata_2025 huggingface dataset:
QUANTITATIVE CHARACTERIZATION OF DENSE BODY, AUTOPHAGIC VACUOLE, AND ACID PHOSPHATASE-BEARING PARTICLE POPULATIONS DURING THE EARLY PHASES OF GLUCAGON-INDUCED AUTOPHAGY IN RAT LIVER
Authors: Deter Russell L.
Published: May 2004

Quantitative characterization of dense body, autophagic vacuole and acid phosphatase-bearing particle populations of rat liver have been made at 10 min intervals during the first 50 min following the intraperitoneal administration of glucagon. Beginning 10 to 20 min postinjection, increases in the number of autophagic vacuoles and in the osmotic sensitivity of acid phosphatase-bearing particles were observed, associated with a progressive disappearance of dense bodies. These changes appeared to reach a maximum 50 min after treatment. The average volume of autophagic vacuoles was found to be 440–870% greater than that of normal dense bodies during this time period. No consistent change in total acid phosphatase activity was noted. A detailed study of autophagic vacuole profile populations revealed the presence of five different types of profiles, two of which, types I and II, accounted for 76.3–94.4% of the profiles examined. Type I profiles primarily contained elements of the endoplasmic reticulum, free ribosomes, and ground cytoplasm. Type II profiles had mitochondrial profiles as their principal constituent, but endoplasmic reticulum and free ribosomes were also seen. At all time points type I profiles predominated, comprising 55–69% of the profiles found. Both profile types were bounded by single and double limiting membranes, the former being predominate. A time-dependent change in the ratio of single to double membrane-limited profiles could not be demonstrated. Morphometric parameters derived from profile size distributions indicated that the number of types I and II autophagic vacuoles increased with time, the rate being greater for the type II particle, except between 40 and 50 min. The average volume of the type II autophagic vacuole was consistently greater than that of the type I.
CONDENSING VACUOLE CONVERSION AND ZYMOGEN GRANULE DISCHARGE IN PANCREATIC EXOCRINE CELLS: METABOLIC STUDIES
Authors: Jamieson James D. , Palade George E.
Published: May 2004

We have examined, in the pancreatic exocrine cell, the metabolic requirements for the conversion of condensing vacuoles into zymogen granules and for the discharge of the contents of zymogen granules. To study condensing vacuole conversion, we pulse labeled guinea pig pancreatic slices for 4 min with leucine-3H and incubated them in chase medium for 20 min to allow labeled proteins to reach condensing vacuoles. Glycolytic and respiratory inhibitors were then added and incubation continued for 60 min to enable labeled proteins to reach granules in control slices. Electron microscope radioautography of cells or of zymogen granule pellets from treated slices showed that a large proportion of prelabeled condensing vacuoles underwent conversion in the presence of the combined inhibitors. Osmotic fragility studies on zymogen granule suspensions suggest that condensation may result from the aggregation of secretory proteins in an osmotically inactive form. Discharge was studied using an in vitro radioassay based on the finding that prelabeled zymogen granules can be induced to release their labeled contents to the incubation medium by carbamylcholine or pancreozymin. Induced discharge is not affected if protein synthesis is blocked by cycloheximide for up to 2 hr, but is strictly dependent on respiration. The data indicate that transport and discharge do not require the pari passu synthesis of secretory or nonsecretory proteins (e.g. membrane proteins), suggesting that the cell may reutilize its membranes during the secretory process. The energy requirements for zymogen discharge may be related to the fusion-fission of the granule membrane with the apical plasmalemma.
STUDIES ON SEEDS
Authors: Allen C. Freeman , Good Pearl , Mollenhauer Hilton H. , Totten Clara
Published: May 2004

Lipid content has been determined for two types of lipid-rich vesicles isolated from bush bean cotyledon at 24 hr of germination. The larger, nonassociating vesicles are four to six times richer in triglyceride than the smaller vesicles which associate strongly among themselves, as well as with smooth membranes in the cell. The larger vesicles contain about 640 µmoles of phospholipid per gram of protein, while the smaller vesicles have only one-half to two-thirds as much phospholipid per gram of protein. The ratio of individual phospholipids in both kinds of vesicles is close to 20% phosphatidylethanolamine, 60% phosphatidylcholine, and 20% phosphatidylinositol. The fatty acid composition of all phospholipids is similar, and quite different from that of triglyceride, which contains twice as much linolenic acid and less than one-fourth as much palmitic acid. Pea cotyledon has quantitatively the same lipid content as bean cotyledon.
ELECTRON MICROSCOPE OBSERVATIONS ON FORM CHANGES IN PHOTORECEPTOR OUTER SEGMENTS AND THEIR SACCULES IN RESPONSE TO OSMOTIC STRESS
Authors: Cohen Adolph I.
Published: May 2004

Isolated retinas of rats or frogs were incubated in various salt or sucrose solutions over a wide range of osmolarities and then fixed in 1% osmium tetroxide solutions of matching osmolarities. Light and electron microscope observations were concentrated on the outer segments of the intact photoreceptors. These became globular and of increasing size with increasing hypoosmolarity and irregularly linear and condensed in hyperosmotic solutions. Isoosmotic incubations of rat retinas in solutions containing potassium as the only cation also produced swelling of the outer segments when chloride or acetate was present; but swelling was less when the cation was sodium and it was not seen with either cation when the anion was methylsulfate. The effects of various metabolic and membrane poisons are also reported. The behavior of the saccules within the outer segments was equivocal. While there was a tendency toward more saccules with wider lumina with hypoosmolarity, most of the saccules were not swollen. Surprisingly, intrasaccular space was consistently enlarged in rat retinas exposed to hyperosmotic sucrose but not to salt. The saccules or their derivatives within swollen outer segments tend to maintain their intersaccule spacing and approximation to the cell membrane. It was also noted that the ciliary connectives resist swelling and that retinal Müller cells swell readily.
THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS
Authors: Shortman Ken , Williams Neil , Jackson Heather , Russell Pamela , Byrt Pauline , Diener E.
Published: May 2004

Four separate effects can be demonstrated when lymphoid cell suspensions are passed through columns of siliconed glass beads. (a) A temperature-dependent "active adherence" of phagocytic cells, such as macrophages and polymorphs. (b) A temperature-independent and selective trapping by "physical adherence" of particular classes of lymphoid cells, including certain antibody-forming cells. (c) A "size-filtration" effect that traps larger cells, but only becomes significant with beads below 100 µ in diameter. (d) A selective retention of damaged cells, which occurs with all columns under all conditions tested. An active adherence column technique has been developed to separate phagocytes from lymphocytes while minimizing selection within the lymphocyte population by physical adherence or size filtration. In less than 10 min at 37°C it reproducibly produces a preparation of mouse spleen lymphocytes >500-fold depleted of active macrophages, and approximately 50-fold depleted of active polymorphs, with good over-all cell recoveries and cell viability. The lymphocyte fraction appears fully active in its ability to initiate immune responses to at least two different antigens, but is changed in over-all composition and selectively depleted in certain classes of antibody-forming cells.
THE OSMOTIC BEHAVIOR OF ROD PHOTORECEPTOR OUTER SEGMENT DISCS
Authors: Heller Joram , Ostwald Thomas J. , Bok Dean
Published: May 2004

The permeability properties of frog rod photoreceptor outer segment discs were investigated in preparations of purified, dark-adapted, outer segment fragments by the techniques of direct volume measurement and electron microscopy. Outer segment discs were found to swell and contract reversibly in response to changes in the osmotic pressure of the bathing medium in accordance with the Boyle-van't Hoff law. By use of the criterion of reversible osmotic swelling, the disc membrane is impermeable to Na+, K+, Mg+2, Ca+2, Cl-, and (PO4)-3 ions, whereas it is freely permeable to ammonium acetate. The disc membrane is impermeable to sucrose, although its osmotic behavior towards this substance is different from its behavior towards impermeable ions. Electron microscopy showed that the osmotic effects on the rod outer segment fragments represent changes in the intradiscal volume. Fixation with glutaraldehyde did not abolish the permeability properties of the disc membrane, and fixed membranes were still capable of osmotic volume changes. It is concluded from this study that the frog's rod photoreceptor outer segment discs are free-floating membranous organelles with an inside space separate and distinct from the photoreceptor intracellular space.
COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG
Authors: Meldolesi J. , Jamieson J. D. , Palade G. E.
Published: May 2004

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ∼20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.
EXPERIMENTAL ALTERATION OF COUPLING RESISTANCE AT AN ELECTROTONIC SYNAPSE
Authors: Asada Y. , Bennett M. V. L.
Published: May 2004

Adjacent segments of the septate axon of the crayfish Procambarus are electrotonically coupled by junction located in the septa between them (see Pappas et al. 1970. J. Cell Biol. 49:173). The coupling resistance at the septa was changed by several experimental treatments. Mechanical injury to an axon increased coupling resistance (more than 7-fold); no recovery of coupling resistance was observed, although the resting potential and resistance of the injured axon could return to near normal levels. Immersion in salines with Na propionate substituted for NaCl increased coupling resistance (mean: 6.1-fold). On return of the preparation to normal saline, coupling resistance recovered virtually completely. Immersion in low Ca++ solutions moderately increased coupling resistance (3.5-fold or less), but return to normal saline was followed by large increases in coupling resistance (5–100-fold). 60 nM Ca++ is near the maximum concentration that leads to increased coupling resistance on return to normal saline. Large increases in coupling resistance are associated with separation of junctional membranes (Pappas et al. 1970. Ibid.); calculations show that the separated membranes greatly increase in resistance. Increase in coupling resistance is probably an important response to injury. Mechanisms underlying changes reported here may be relevant to normal physiological processes of coupling and decoupling.
LOCALIZATION OF THE SITES OF γ-AMINOBUTYRIC ACID (GABA) UPTAKE IN LOBSTER NERVE-MUSCLE PREPARATIONS
Authors: Orkand Paula M. , Kravitz Edward A.
Published: May 2004

The principal sites of γ-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.
A PATTERN OF EPIDERMAL CELL MIGRATION DURING WOUND HEALING
Authors: Krawczyk Walter S.
Published: May 2004

Epidermal repair during wound healing is under investigation at both the light and electron microscopic levels. Suction-induced subepidermal blisters have been employed to produce two complementary model wound healing systems. These two model systems are: (a) intact subepidermal blisters, and (b) opened subepidermal blisters (the blister roof was removed immediately after induction, leaving an open wound). From these studies a pattern of movement for epidermal cells in wound healing is proposed. This pattern of movement is the same for both model systems. Epidermal cells appear to move by rolling or sliding over one another. Fine fibers oriented in the cortical cytoplasm may play an important role in the movement of these epidermal cells. Also instrumental in mediating this movement are intercellular junctions (desmosomes) and a firm attachment to a substrate through hemidesmosomes. In the intact subepidermal blisters hemidesmosomal attachment is made to a continuous and homogeneous substrate, the retained basal lamina. In the opened subepidermal blisters contact of epidermal cells is made to a discontinuous substrate composed of sporadic areas of fibrin and underlying mesenchymal cells.
DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES
Authors: Leskes A. , Siekevitz P. , Palade G. E.
Published: May 2004

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.
DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES
Authors: Leskes A. , Siekevitz P. , Palade G. E.
Published: May 2004

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.
THE RENEWAL OF ROD AND CONE OUTER SEGMENTS IN THE RHESUS MONKEY
Authors: Young Richard W.
Published: May 2004

The renewal of retinal rod and cone outer segments has been studied by radioautography in rhesus monkeys examined 2 and 4 days after injection of leucine-3H. The cell outer segment consists of a stack of photosensitive, membranous discs. In both rods and cones some of the newly formed (radioactive) protein became distributed throughout the outer segment. Furthermore, in rods (but not in cones), there was a transverse band of concentrated radioactive protein slightly above the outer segment base 2 days after injection. This was due to the formation of new discs, into which labeled protein had been incorporated. At 4 days, these radioactive discs were located farther from the outer segment base. Repeated assembly of new discs had displaced them away from the basal assembly site and along the outer segment. Measurements of the displacement rate indicated that each retinal rod produces 80–90 discs per day, and that the entire complement of outer segment discs is replaced every 9–13 days. To compensate for the continual formation of new discs, groups of old discs are intermittently shed from the apical end of the cell and phagocytized by the pigment epithelium. Each pigment epithelial cell engulfs and destroys about 2000–4000 rod outer segment discs daily. The similarity between visual cells in the rhesus monkey and those in man suggests that the same renewal processes occur in the human retina.
STUDIES ON ISOLATED AGGREGATING OLIGORIBONUCLEOPROTEINS OF THE EPIDERMIS WITH HISTOCHEMICAL AND MORPHOLOGICAL CHARACTERISTICS OF KERATOHYALIN
Authors: Ugel Arthur R.
Published: May 2004

Histochemical and ultrastructural studies demonstrate that keratohyalin can be mobilized from fresh specimens of cattle hoof epidermis by 1.0 M potassium phosphate buffer (pH 7.0). Macroaggregates with histochemical characteristics identical to those of in situ keratohyalin granules (staining by Harris' hematoxylin, Congo red, diazotized sulfanilic acid, sodium alizarin sulfonate, toluidine blue, methyl green-pyronin, and acridine orange) and with similar morphological characteristics at the ultrastructural level are formed upon dialyzing the extracted keratohyalin against distilled water. Staining by basic dyes (toluidine blue, methyl green-pyronin, and acridine orange) is abolished by treating either in situ keratohyalin granules or isolated macroaggregates with ribonuclease. Electrophoresis of isolated macroaggregates on polyacrylamide gels in the presence of sodium decylsulfate results in the fractionation of a 13 member oligomeric series of ribonucleoproteins and two non-homologous species of ribonucleoproteins. The oligomeric series can be purified by isolating "stacked" oligomers on low concentration (3%) polyacrylamide gels. Fractionated oligomers on polyacrylamide gels and aggregates formed from purified ribonucleoproteins demonstrate histochemical characteristics identical to those of in situ keratohyalin granules. Aggregates formed from denatured ribonucleoproteins are highly disordered and are markedly different from in situ keratohyalin granules or nondenatured isolated macroaggregates at the ultrastructural level, possibly due to irreversible denaturation of the oligomers by sodium decylsulfate.
THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES
Authors: Bone Q. , Denton E. J.
Published: May 2004

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.
CORRELATION BETWEEN PINOCYTOSIS AND HYDROOSMOSIS INDUCED BY NEUROHYPOPHYSEAL HORMONES AND MEDIATED BY ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE
Authors: Masur Sandra K. , Holtzman Eric , Schwartz Irving L. , Walter Roderich
Published: May 2004

The isolated urinary bladder of the toad responds to neurohypophyseal hormone with a net increase of water transport from the mucosal to the serosal solution in the presence of an osmotic gradient. This response is mediated intracellularly by cyclic 3',5'-adenosine monophosphate (AMP). The present study demonstrates that hydroosmotically active substances such as oxytocin, dibutyryl cyclic 3',5'-AMP, and theophylline, but not hydroosmotically inactive substances, induce the uptake of horseradish peroxidase from the mucosal solution. Peroxidase taken up by the mucosal cells is demonstrable in small tubules and vesicles, and eventually accumulates in lysosomes. The uptake of peroxidase from the serosal solution into similar bodies in the mucosal cells is not hormone-dependent. It is also shown that peroxidase does not penetrate the tight junction from either the mucosal or serosal solution. These results extend previous findings which implicated the apical membrane of the mucosal epithelium as the site affected by neurohypophyseal hormones. A mechanism based on secretory phenomena is proposed as a framework for future investigations of apical membrane permeability changes and pinocytosis.
ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS
Authors: Yamada Kenneth M. , Spooner Brian S. , Wessells Norman K.
Published: May 2004

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.
ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS
Authors: Lee Se Yong , Krsmanovic Velibor , Brawerman George
Published: May 2004

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.
IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS
Authors: Bernhard W. , Viron Annie
Published: May 2004

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.
SITES OF CYTOPLASMIC RIBONUCLEOPROTEIN-FILAMENT ASSEMBLY IN RELATION TO HELICAL BODY FORMATION IN AXENIC TROPHOZOITES OF ENTAMOEBA HISTOLYTICA
Authors: Kress Yvonne , Wittner Murray , Rosenbaum Robert M.
Published: May 2004

Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-3H and cytidine-3H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.
ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE
Authors: Noel J. S. , Dewey W. C. , Abel J. H. , Thompson R. P.
Published: May 2004

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G1. During very early G1, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G1, a granular component appeared which was intimately associated with the fibrous material. By the middle of G1, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G1 to the middle of S primarily due to the accumulation of the granular component. During the G2 period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).
ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER
Authors: Cheetham R. D. , Morré D. James , Pannek Carol , Friend Daniel S.
Published: May 2004

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.
NUCLEAR-CYTOPLASMIC RELATIONS IN THE MITOSIS OF SEA URCHIN EGGS
Authors: Rustad Ronald C. , Yuyama Shuhei , Rustad Lynne C.
Published: May 2004

Sea urchin eggs were cut into halves. The nucleate and anucleate halves and whole eggs were irradiated with γ-rays and then fertilized with normal sperm. The first mitosis of the diploid half-egg was more delayed than the division of the whole egg. There was a small, but highly significant, delay of the mitosis of the haploid half-egg, thus demonstrating cytoplasmic sensitivity to ionizing radiation. Since the sensitivity of nucleate cells is influenced by cytoplasmic volume, the problem of the role of cytoplasm in repair is considered in relation to these data and other reports in the literature.
A Cytochemical Study on the Pancreas of the Guinea Pig
Authors: Siekevitz Philip , Palade George E.
Published: May 2004

Five ribonucleoprotein (RNP) fractions were isolated from the postmitochondrial supernatant of the pancreas of the guinea pig. Two were obtained from the microsomes which, by deoxycholate (DOC) treatment, were subdivided into a DOC-soluble and a DOC-insoluble fraction. The latter was taken to represent attached RNP particles. Two other fractions obtained from the microsomal supernatant supposedly represent free RNP particles existing as such in the cytoplasm, while a third fraction resisted sedimentation for 20 hours at 105,000 g and is considered to be a soluble nucleoprotein. These fractions exhibited different RNA/protein ratios and also different RNA turnover patterns, as determined after in vivo labelling with adenine-8-C14. However, little discernible differences could be detected in the nucleotide composition of the RNA moieties of these RNP fractions.

Amino acid-"activating" enzymes were found to occur in the fraction containing the soluble nucleoproteins.

The discussion focuses on the relationships between these fractions and protein synthesis in the pancreas, using data given in this and a previous paper, and data contained in the literature.
Fine Structure of Bacillus subtilis
Authors: Tokuyasu Kiyoteru , Yamada Eichi
Published: May 2004

The sporulation process in Bacillus subtilis has been studied principally with KMnO4 fixation, but also, for the purpose of comparison, with OsO4 and mixtures of both fixatives.

At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex.

The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail.

Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.
Abnormal Intranuclear and Cytoplasmic Formations Associated with a Chemically Induced, Transplantable Chicken Sarcoma
Authors: Binggeli Marlene Friedlaender
Published: May 2004

Thirty GRCH/15 tumors (a 1, 2, 5, 6-dibenzanthracene-induced chicken sarcoma) were examined in the light and the electron microscope.

Associated with the sarcoma were two types of abnormal intranuclear lesions, one in the form of a vacuole, the other as an aggregate containing glycogen. In the electron microscope, one type of lesion observed showed an organized microfibrillar structure.

Abnormal cytoplasmic formations occurred as massed clusters of thread-like or tubular material, which gave rise to small bodies with concentric shell structure; similar bodies were found associated with vacuoles.
Cell Fractionation of Anterior Pituitary Glands from Beef and Pig
Authors: Labella Frank S. , Brown J. H. U.
Published: May 2004

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.
Cytological Studies of the Nematocysts of Hydra
Authors: Chapman George B. , Tilney Lewis G.
Published: May 2004

Entire hydras or tentacles were fixed in OsO4 or in KMnO4 and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope.

In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix.

The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed.

Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.
Correlation of Physical and Biological Properties of Mouse Mammary Tumor Agent
Authors: Moore Dan H. , Lasfargues E. Y. , Murray Margaret R. , Haagensen Cushman D. , Pollard E. C.
Published: May 2004

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 mµ). 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 mµ. 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 mµ in diameter and a small agent 20 to 30 mµ in diameter or possibly smaller. Furthermore, the presence in the milk of an inhibitor 40 to 60 mµ 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.
Production of the Milk Agent in Cultures of Mouse Mammary Carcinoma
Authors: Lasfargues E. Y. , Moore Dan H. , Murray Margaret R. , Haagensen Cushman D. , Pollard E. C.
Published: May 2004

Thin sections of tissue cultures grown from tumors of the RIII high-breast-cancer strain mice were studied in the electron microscope. These tissues contain an abundance of particles whose morphology is consistent with biophysical measurement of the milk agent. These particles, found only extracellularly in our cultures, are formed at the cell membrane. The process of formation, as reconstructed from sections, appears to include a thickening and protrusion of the cell membrane which then evolves gradually into a dense sphere and separates from the cell in much the same manner as does influenza virus. The contents of the newly formed body are later rearranged to form a nucleoid within a membranous sac.
The Swelling of Rat Liver Mitochondria by Thyroxine and its Reversal
Authors: Lehninger Albert L. , Ray Betty Lou , Schneider Marion
Published: May 2004

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20°.

Aging of mitochondria at 0° causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine.

The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn++, and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane.

Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.
The Electron Microscopy of the Left Colleterial Gland of the Cockroach
Authors: Mercer E. H. , Brunet P. C. J.
Published: May 2004

A study has been made of the cells of the left colleterial gland of the cockroach, Periplaneta americana (L.), using the electron microscope, and the results compared with previous histological and histochemical studies.

The colleterial gland consists of an arborescent bunch of long tubules composed mainly of the cells which secrete the structural protein of the egg case ("type 4 cells"). Other types of cells: chitinogenic cells and "type 2 and 3 cells" each with a different cytology are described.

The type 4 cells, which form the structural protein, reveal a cytological pattern very similar to that described for mammalian cells in a state of active protein synthesis. There is an elaborate development of particle-studded membranes in the cytoplasm. Smaller, rounded agranular vesicles also occur.

The free secretory surface of the secreting cells forms the "end-apparatus" of the light microscopists. The invaginated surface is cast into numerous long narrow processes usually radially arranged and directed into a funnel-like formation derived from the thin intima lining the lumen of the gland (Text-fig. 2). The secretion in the form of small balls may be seen in the cavity of the end-apparatus and sometimes in the narrow processes.

The small chitinogenic cells, lying between the protein-forming cells and the thin intima which they secrete, have a different cytology perhaps related to the fact that they form a polysaccharide rather than a protein. There is a very poor development of the particle-studded membranes of the type found in protein-forming cells.

The type 2 cells, supposed to form an oxidase, have an end-apparatus that is similar to, but more complex than, those of the type 4 cells and their cytoplasm is almost completely filled with mitochondria. There is some evidence that mitochondria play a part in forming the oxidase and pass into the tubules of the end-apparatus.

Type 3 cells resemble both types 2 and 4 and are probably a transient intermediate form.