bluuebunny/crossref_metadata_2025 huggingface dataset:
A THERMODYNAMIC ANALYSIS OF MITOTIC SPINDLE EQUILIBRIUM AT ACTIVE METAPHASE
Authors: Stephens R. E.
Published: May 2004

The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoué. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55–65 kcal/mol and +197–233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoué and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoué, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D2O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D2O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoué's earlier contention that D2O treatment can modify the available spindle pool.
ELECTRON MICROSCOPE AUTORADIOGRAPHY OF ERYTHROID CELLS USING RADIOACTIVE IRON
Authors: Parry D. M. , Blackett N. M.
Published: May 2004

The "circle analysis" method of Williams (1969) and a new improved method employing hypothetical grains described in the previous paper have been used to analyze the distribution of autoradiographic grains over erythroid bone marrow cells labeled with radioactive iron, 55Fe. The resolution obtainable with this isotope was determined by measuring the distribution of grains about a thin line source. This distribution was also used in calculation of the circle size for the Williams's analysis and the distances of hypothetical grains for the new method. The new method provides estimates for the amount of activity in the regions of condensed and extended nuclear chromatin and for the concentration of isotope at the junction between these two areas. The possible significance of activity in this junctional region is discussed.
THE ROLE OF THE GOLGI COMPLEX IN SULFATE METABOLISM
Authors: Young Richard W.
Published: May 2004

This investigation was designed to determine if sulfate metabolism is the function of a particular cell organelle, or whether the site of sulfation varies, depending upon the type of cell and the class of sulfated compound. Rats and mice were injected intravenously with inorganic sulfate labeled with 35S (H235SO4), and were then killed by vascular perfusion of fixative 5–30 min later. Several tissues were prepared for electron microscope autoradiography. 14 different types of specialized cells which incorporated the labeled sulfate were analyzed. In every case, the sulfate was initially detected in the smooth membranes and vesicles of the Golgi complex. Available evidence indicates that these cells were engaged in the synthesis of several different sulfated compounds, including mucopolysaccharides, glycoproteins, lipids, and steroids. These results lead to the generalization that the enzymes required for the transfer of inorganic sulfate to a variety of acceptor molecules are located in the Golgi complex.
FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS
Authors: Edidin Michael , Fambrough Douglas
Published: May 2004

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10-9 cm2 s-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.
DIMETHYL SULFOXIDE-INDUCED REVERSION OF SEVERAL FEATURES OF POLYOMA TRANSFORMED BABY HAMSTER KIDNEY CELLS (BHK-21)
Authors: Kisch Alexander L. , Kelley Robert O. , Crissman Harry , Paxton Linda
Published: May 2004

Cells of a polyoma virus transformed clonal line (Cl-I) of baby hamster kidney fibroblasts (BHK-21) were grown in medium containing 2 percent dimethylsulfoxide (DMSO). Unlike the untransformed BHK-21 cells, Cl-I cells adapted to replication in the presence of DMSO, and they exhibited a rapidly reversible phenotypic reversion of a number of properties characteristic of the transformed state. Restoration of density dependent growth inhibition with accumulation of cells in the G1 phase of the cell cycle occurred and was associated with restoration of contact dependent behavior and with reversion of histological and ultrastructural features towards those which characterize untransformed cells. Concomitantly, Cl-I cells grown in 2 percent DMSO lost the ability to form colonies in semisolid medium. The data presented suggest that DMSO alters the expression of cellular functions which were altered as a result of viral transformation and which may be involved in cell tumorigenicity.
THE ULTRASTRUCTURE OF Z DISKS FROM WHITE, INTERMEDIATE, AND RED FIBERS OF MAMMALIAN STRIATED MUSCLES
Authors: Rowe R. W. D.
Published: May 2004

The Z disk ultrastructure of white, intermediate, and red fibers from mammalian muscle is examined. Three models are proposed that explain the differences between the three types of Z disk. The three models are all based on the same concept, i.e., looping filaments from both sides of the Z disk. The differences between the models are in terms of the spatial relationships of adjacent loops within the Z disk. In the white fiber Z disk model all the loops from one side of the Z disk are on the same plane. In intermediate fibers there are two planes of loops from both sides of the Z disk, whereas in red fibers there are three planes of loops from both sides. The implications of these three structures are discussed in relation to known physiological differences between the fiber types.
OCTAHEDRAL CRYSTALS IN PHYCOMYCES. II
Authors: Ootaki T. , Wolken J. J.
Published: May 2004

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the ß-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265–285 nm, 350–380 nm, and 450–470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".
EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION
Authors: Heuser J. E. , Reese T. S.
Published: May 2004

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.
INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS
Authors: Rattner J. B. , Phillips Stephanie G.
Published: May 2004

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G1 nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G1 or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.
A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES
Authors: Tokuyasu K. T.
Published: May 2004

Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.
PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS
Authors: Rubin Robert W. , Cunningham William P.
Published: May 2004

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.
ENDOPLASMIC RETICULUM AS THE SITE OF LECITHIN FORMATION IN CASTOR BEAN ENDOSPERM
Authors: Lord J. M. , Kagawa T. , Moore T. S. , Beevers H.
Published: May 2004

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b5 and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm3 and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm3 and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.
THE CONTRACTILE PROCESS IN THE CILIATE, STENTOR COERULEUS
Authors: Huang B. , Pitelka D. R.
Published: May 2004

The structural basis for the function of microtubules and filaments in cell body contractility in the ciliate Stentor coeruleus was investigated. Cells in the extended state were obtained for ultrastructural analysis by treatment before fixation with a solution containing 10 mM EGTA, 50–80 mM Tris, 3 mM MgSO4, 7.5 mM NH4Cl, 10 mM phosphate buffer (pH 7.1). The response of Stentor to changes in the divalent cation concentrations in this solution suggests that Ca+2 and Mg+2 are physiologically important in the regulation of ciliate contractility. The generation of motive force for changes in cell length in Stentor resides in two distinct longitudinal cortical fiber systems, the km fibers and myonemes. Cyclic changes in cell length are associated with (a) the relative sliding of parallel, overlapping microtubule ribbons in the km fibers, and (b) a distinct alteration in the structure of the contractile filaments constituting the myonemes. The microtubule and filament systems are distinguished functionally as antagonistic contractile elements. The development of motive force for cell extension is accomplished by active microtubule-to-microtubule sliding generated by specific intertubule bridges. Evidence is presented which suggests that active shortening of contractile filaments, reflected in a reversible structural transformation of dense 4-nm filaments to tubular 10–12-nm filaments, provides the basis for rapid cell contraction.
CONDITIONAL MUTANTS IN CHLAMYDOMONAS REINHARDTII BLOCKED IN THE VEGETATIVE CELL CYCLE
Authors: Howell Stephen H. , Naliboff Jay A.
Published: May 2004

Conditional "cycle-blocked" (cb) mutants of Chlamydomonas reinhardtii have been detected and isolated. These mutants exhibit normal vegetative growth at permissive temperature but are unable to complete a cell cycle (or a specified number of cell cycles) at restrictive temperature. A simple technique has been devised to determine the cell cycle stage in each mutant when the defective gene product, which ultimately affects cell division, completes its function. This stage is called the "block point", and is determined by scoring the residual cell division in an exponentially growing population after shift to temperature restrictive conditions. In the cb mutants isolated so far, block points representing many stages throughout the cell cycle have been found. Two categories of cb mutants are described here: one set which prevents the subsequent cell division when the cell encounters the block point after a shift to restrictive temperature, and another set which permits an additional round of cell division after the block point is encountered. The general applicability of block point analysis to other cell systems is presented.
ULTRASTRUCTURAL CHANGES IN THE MITOCHONDRIA OF CEREBELLAR PURKINJE CELLS OF NERVOUS MUTANT MICE
Authors: Landis Story C.
Published: May 2004

The maturation of cerebellar Purkinje cells of normal and nervous (nr/nr) mutant mice has been studied by light and electron microscopy. In the mutant, 90% of Purkinje cells selectively degenerate between postnatal days 23 and 50. Losses are greater in lateral than medial regions. Other cerebellar neurons appear normal. The first morphological abnormality recognized is the presence of rounded mitochondria in perikarya of some Purkinje cells of the mutant at 9 days after birth. By 15 days, all nr/nr Purkinje cells contain spherical mitochondria and begin to deviate from the normal maturational sequence. Elaboration of the extensive dendritic tree halts midway and newly formed axon collateral fibers degenerate. In the perikaryon, the basal polysomal accumulation and climbing fiber-somatic spine synapses are sometimes abnormally retained. Cisternae of the Golgi apparatus and rough endoplasmic reticulum cease to form aligned stacks, and decrease in number, while polysomes dissociate into free ribosomes. These changes are progressive, culminating in cell death. Although every nr/nr Purkinje cell demonstrates spherical mitochondria, some cells survive the critical period, retain a near-normal complement of organelles, and reacquire normal-appearing mitochondria. The disorder appears intrinsic to Purkinje cells since all major classes of synapses were identified before cell death.
INHIBITION OF CELLULAR DIFFERENTIATION BY PHOSPHOLIPASE C
Authors: Nameroff Mark , Trotter John A. , Keller John M. , Munar Erlynda
Published: May 2004

In cell culture, a partially purified commercial preparation of phospholipase C (PLC) from Clostridium welchii inhibited fusion of myoblasts at concentrations of 12–50 µg per ml. At lower concentrations, PLC-treated cultures were indistinguishable from controls, and at concentrations above 100 µg per ml, PLC-treated cells detached from their substrates. The effect was reversible and fusion resumed approximately one cell cycle time after removal of the enzyme. Neither the percent of cells in the mitotic cycle nor the duration of the different phases of the cycle were altered by PLC at concentrations which inhibited fusion. Cell motility was not reduced by the enzyme. Unfused, PLC-treated myoblasts were virtually indistinguishable in ultrastructure from untreated cells just before fusion. In the presence of PLC, mononucleated myogenic cells did not synthesize thick (150 Å) filaments. Treatment of culture medium with insolubilized commercial PLC did not abolish the capacity of the medium to support myogenesis. Chondrocytes treated with PLC divided repeatedly but failed to synthesize metachromatic matrix and failed to incorporate labeled sulfate into chondroitin sulfate. PLC was further purified by chromatography on Sephadex G-100. The resulting preparation was free of detectable protease, yielded one band on SDS-acrylamide gel electrophoresis, and displayed all of the biological activities of the less pure material.
ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS
Authors: Klietmann Wolfgang , Sato Nobuhiro , Nass Margit M. K.
Published: May 2004

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a3 show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.
A TIME-SEQUENCE AUTORADIOGRAPHIC STUDY OF THE IN VIVO INCORPORATION OF [1, 2-3H]CHOLESTEROL INTO PERIPHERAL NERVE MYELIN
Authors: Rawlins Frank A.
Published: May 2004

A time-sequence study of the incorporation and distribution of cholesterol in peripheral nerve myelin was carried out by electron microscope autoradiography. [1,2-3H]Cholesterol was injected into 10-day old mice and the sciatic nerves were dissected out at 10, 20, 40, 60, 90, 120, and 180 min after the injection. 20 min after injection the higher densities of grains due to the presence of [3H]cholesterol were confined to the outer and inner edges of the myelin sheath. Practically no cholesterol was detected in the midzone of the myelin sheath. 1 ½ h after injection, cholesterol showed a wider distribution within the myelin sheath, the higher densities of grains occurring over the two peripheral myelin bands, each approximately 3,100 Å wide. Cholesterol was also present in the center of the myelin sheath but to a considerably lesser extent. 3 h after injection cholesterol appeared homogeneously distributed within the myelin sheath. Schwann cell and axon compartments were also labeled at each time interval studied beginning 20 min postinjection. These observations indicate that preformed cholesterol enters myelin first and almost simultaneously through the inner and outer edges of the sheath; only after 90 min does the density of labeled cholesterol in the central zone of myelin reach the same density as that in the outer and inner zones. These findings suggest that cholesterol used by the nerve fibers in the formation and maintenance of the myelin sheath enters the lamellae from the Schwann cell cytoplasm and from the axon. The possibility of a bidirectional movement of molecules, i.e. from the Schwann cell to the axon and from the axon to the Schwann cell through the myelin sheath, is noted. The results are discussed in the light of recent observations on the exchange, reutilization, and transaxonal movement of cholesterol.
REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA
Authors: Phillips David M. , Phillips Stephanie Gordon
Published: May 2004

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.
THE DISTRIBUTION, ULTRASTRUCTURE, AND CHEMISTRY OF MICROFILAMENTS IN CULTURED CHICK EMBRYO FIBROBLASTS
Authors: Perdue J. F.
Published: May 2004

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.
LOCALIZATION OF ENZYMES WITHIN MICROBODIES
Authors: Huang A. H. C. , Beevers H.
Published: May 2004

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm3 which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [14C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm3, whereas the original glyoxysomes appeared at density 1.24 g/cm3. Electron microscopy showed that the fraction at 1.21–1.22 g/cm3 was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.
ULTRASTRUCTURAL BASIS OF BIOCHEMICAL EFFECTS IN A SERIES OF LETHAL ALLELES IN THE MOUSE
Authors: Trigg Monica J. , Gluecksohn-Waelsch Salome
Published: May 2004

The fine structure of newborn and fetal mouse liver and of newborn kidney cells homozygous for any of three albino alleles known to have multiple biochemical effects was investigated. Electron microscope studies of mutant cells revealed dilation and vesiculation of the rough endoplasmic reticulum in parenchymal liver cells, as well as dilation and other anomalies of the Golgi apparatus. These abnormalities were observed in all newborn mutants but never in littermate controls. Although they were most pronounced in liver parenchymal cells, they were found also to a lesser degree in kidney cells, but they were absent altogether in other cell types of the mutant newborn. Homozygous fetuses showed similar anomalies in the liver at 19 days of gestational age. In one of the alleles studied, mutant liver parenchymal cells were found to be abnormal as early as the 18th day of gestation. There appears to be a striking parallelism between the biochemical defects and those of the cellular membranes in homozygous mutant newborn and fetuses. Although the specific nature of the mutational effect on membrane structure remains unknown, the results are compatible with the assumption that a mutationally caused defect in a membrane component interferes with a mechanism vital in the integration of morphological and biochemical differentiation.
STRUCTURE OF LIMULUS STRIATED MUSCLE
Authors: Dewey Maynard M. , Levine Rhea J. C. , Colflesh David E.
Published: May 2004

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 µm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 µm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.
THE STRUCTURE OF A SIMPLE Z LINE
Authors: Franzini-Armstrong Clara
Published: May 2004

The structural simplicity of the Z line in fish muscle fibers allows direct visualization of its basic geometry. Models which postulate termination of the I filaments at the edges of the Z line and the direct linkage of I filaments belonging to the two adjacent sarcomeres by Z filaments crossing the whole width of the Z line give the best fit to the electron micrographs. The structure of fish Z lines is not significantly altered by the use of different fixation procedures and by changes in sarcomere length.
THE MECHANISM OF ACTION OF COLCHICINE
Authors: Wilson Leslie , Meza Isaura
Published: May 2004

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 105 liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (Ki = 1.3 x 10-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.
SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES
Authors: Reaven Eve P. , Axline Stanton G.
Published: May 2004

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass.
COBALT AS A MITOCHONDRIAL DENSITY MARKER IN A STUDY OF CYTOPLASMIC EXCHANGE DURING MATING OF SCHIZOPHYLLUM COMMUNE
Authors: Watrud Lidia S. , Ellingboe Albert H.
Published: May 2004

A slow in vivo uptake of cobalt from a growth medium resulted in an increase in density of mitochondria of Schizophyllum commune. Differential labeling of donor and resident mycelia, and subsequent analysis of resident mycelia surrounding donor implants, detected cobalt-dense mitochondria and demonstrated exchange of mitochondria after hyphal fusion. Transfer of mitochondria occurred in fully compatible, common-A, and common-AB matings, but was not detected in common-B matings of the tetrapolar Basidiomycete S. commune.
NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS
Authors: Simmons T. , Heywood P. , Hodge L.
Published: May 2004

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G1) has been investigated in synchronized HeLa S3 cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G1 was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.
LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION
Authors: Bowers William E.
Published: May 2004

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.
THE ORGANIC-INORGANIC RELATIONSHIP IN CALCIFIED MITOCHONDRIA
Authors: Bonucci E. , Derenzini M. , Marinozzi V.
Published: May 2004

Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl4 intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca2+-binding sites in mitochondria and to the general problems of soft tissue calcification.
GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES
Authors: Ehrenreich J. H. , Bergeron J. J. M. , Siekevitz P. , Palade G. E.
Published: May 2004

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ∼90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ∼13 x 106 g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.
GLUTARALDEHYDE FIXATION OF ISOLATED EUCARYOTIC NUCLEI
Authors: Olins Donald E. , Wright Everline B.
Published: May 2004

Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, kapp: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)n molecules (where n = 2 to ∼8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.