bluuebunny/crossref_metadata_2025 huggingface dataset:
PLASMA MEMBRANE AND INTERNALIZED IMMUNOGLOBULINS OF LYMPH NODE CELLS STUDIED WITH CONJUGATES OF ANTIBODY OR ITS FAB FRAGMENTS WITH HORSERADISH PEROXIDASE
Authors: Antoine Jean-Claude , Avrameas Stratis , Gonatas Nicholas K. , Stieber Anna , Gonatas Jacqueline O.
Published: May 2004

Normal rat and mouse lymphoid cells were incubated at 0°–4°C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5–30 min at 20° or 37°C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with 125I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([125I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins.

In the electron microscope, Fab-PO conjugates at 0°–4°C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20° or 37°C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0°–4°C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining.

Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37°C.

Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37°C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.
ENZYME LOCALIZATION IN THE ANAEROBIC MITOCHONDRIA OF ASCARIS LUMBRICOIDES
Authors: Rew Robert S. , Saz Howard J.
Published: May 2004

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.
INDUCTION OF ARYL HYDROCARBON (BENZO[a]PYRENE) HYDROXYLASE AND TYROSINE AMINOTRANSFERASE IN HEPATOMA CELLS IN CULTURE
Authors: Whitlock James P. , Miller Haruko , Gelboin Harry V.
Published: May 2004

In the Reuber (H35) hepatoma cell strain, microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase is induced 25-fold by the polycyclic hydrocarbon benz[a]anthracene but is not induced by the steroid hormone dexamethasone. Soluble tyrosine aminotransferase is induced sixfold by dexamethasone and twofold by benz[a]anthracene. Each enzyme requires similar inducer concentrations for induction, and their induction kinetics are similar. The induction of each enzyme requires RNA and protein synthesis; in each case the transcriptional and translational steps can occur independently. The two induction systems are differentially sensitive to inhibitors of macromolecular synthesis. Simultaneous exposure to both inducers produces increases in both enzyme activities that are greater than those produced by either inducer alone. Each inducer acts at a pretranslational level to produce this synergistic effect. The results suggest that the requirements for macromolecular synthesis are similar for the induction of each enzyme, but that the turnover of enzyme-specific macromolecules may differ for each.
BIOCHEMICAL AND MORPHOLOGICAL CHARACTERIZATION OF AZUROPHIL AND SPECIFIC GRANULES OF HUMAN NEUTROPHILIC POLYMORPHONUCLEAR LEUKOCYTES
Authors: Bretz Ursula , Baggiolini Marco
Published: May 2004

Postnuclear supernates from homogenates of purified neutrophil polymorphonuclear leukocytes (PMNs) from human blood were fractionated by zonal sedimentation and isopycnic equilibration in sucrose gradients. The fractions were characterized biochemically by measuring protein content and the activities of eight enzymes. Selected fractions were further analyzed by electron microscopy. In both centrifugation systems, azurophil and specific granules could be resolved almost completely. Azurophil granules sediment three to four times faster than the specifics and have an average density of 1.23. They contain all the peroxidase of the cells, large portions of four lysosomal hydrolases, and about half of the total lysozyme, and therefore appear to be, in biochemical terms, very similar to the azurophil granules of rabbit PMNs. The specific granules, which have an average density of 1.19, contain the remaining half of the lysozyme but appear to be free of the other components of the azurophil granules, and of alkaline phosphatase. Isopycnic equilibration disclosed a minor lysosomal population, which strongly overlaps the specific granules, and made possible the identification of a membrane-fraction which is characterized by the presence of the thiol-sensitive acid 4-nitrophenyl phosphatase and of alkaline phosphatase.
ENDOPLASMIC FILAMENTS GENERATE THE MOTIVE FORCE FOR ROTATIONAL STREAMING IN NITELLA
Authors: Allen Nina Strömgren
Published: May 2004

The streaming endoplasm of characean cells has been shown to contain previously unreported endoplasmic filaments along which bending waves are observed under the light microscope using special techniques. The bending waves are similar to those propagated along sperm tails causing propulsion of sperm. In Nitella there is reason to believe that nearly all of the filaments are anchored in the cortex and that their beating propels the endoplasm in which they are suspended. This hypothesis is supported by calculations in which typical and average wave parameters have been inserted into the classical hydrodynamic equations derived for sperm tail bending waves. These calculations come within an order of magnitude of predicting the velocity of streaming and they show that waves of the character described, propagated along an estimated 52 m of endoplasmic filaments per cell, must generate a total motive force per cell within less than an order of magnitude of the forces measured experimentally by others. If we assume that undulating filaments produce the force driving the endoplasm, then the method described for measuring the motive force could lead to a lower than actual value for the motive force, since both centrifugation and vacuolar perfusion would reverse the orientation of some filaments. Observations of the initiation of particle translation in association with the filaments suggest that particle transport and wave propagation, which occur at the same velocity, may both be dependent on the same process. The possibility that some form of contractility provides the motive force for filament flection and particle transport is discussed.
CYCLIC AMP MEDIATES THE CONCANAVALIN A AGGLUTINABILITY OF MOUSE FIBROBLASTS
Authors: Willingham Mark C. , Pastan Ira
Published: May 2004

We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMPtcs) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMPtcs cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt2cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMPtcs cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.
THE EFFECTS OF ISOPROPYL N-PHENYL CARBAMATE ON THE GREEN ALGA OEDOGONIUM CARDIACUM
Authors: Coss Ronald A. , Pickett-Heaps Jeremy D.
Published: May 2004

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2°C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10-4 M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-µm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.
PATTERN OF OSMIUM DEPOSITION IN THE PARIETAL CELLS OF THE STOMACH
Authors: Winborn William B. , Seelig Leonard L.
Published: May 2004

Parietal cells of the stomach of the hamster show extensive amounts of dense material in a variety of organelles after prolonged exposure to a solution of osmium tetroxide. Conspicuous amounts of reduced osmium compounds are evident within the granular endoplasmic reticulum, perinuclear cisterna, and vesicular elements of the Golgi complex. Dense material is also apparent within cristae of the mitochondria, the surface coat of the microvilli of the intracellular canaliculus, and vesicular elements of the multivesicular bodies. Multivesicular bodies, containing numerous small osmiophilic elements, are often seen surrounding and/or in close contact with mitochondria. The proximity of the multivesicular bodies to the mitochondria appears to be related to an autophagic process involving degradation of mitochondria. The distribution and intensity of the precipitates within the organelles of the parietal cells vary in different regions of the gastric glands. The findings of this study emphasize that cell structures other than the Golgi complex may contain large concentrations of reduced osmium compounds after prolonged exposure to a solution of osmium tetroxide.
ANALYTICAL FRACTIONATION OF HOMOGENATES FROM CULTURED RAT EMBRYO FIBROBLASTS
Authors: Tulkens Paul , Beaufay Henry , Trouet André
Published: May 2004

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-ß-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized.

In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.
IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION
Authors: Conkie D. , Affara N. , Harrison P. R. , Paul J. , Jones K.
Published: May 2004

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation.
PROTEIN DEGRADATION IN CULTURED CELLS
Authors: Wibo Maurice , Poole Brian
Published: May 2004

The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B1 but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B1.
ACROSOMAL DISRUPTION IN SPERM
Authors: Friend Daniel S. , Rudolf Irene
Published: May 2004

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.
THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES
Authors: Nir Izhak , Hall Michael O.
Published: May 2004

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO4 with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO4. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.
THREE-DIMENSIONAL ULTRASTRUCTURE OF THE CRAYFISH NEUROMUSCULAR APPARATUS
Authors: Jahromi S. S. , Atwood H. L.
Published: May 2004

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.
MEMBRANE DIFFERENTIATIONS IN FREEZE-FRACTURED MAMMALIAN SPERM
Authors: Friend Daniel S. , Fawcett Don W.
Published: May 2004

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas.

The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles.

The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6–8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.
STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE
Authors: Mareš V. , Schultze B. , Maurer W.
Published: May 2004

Neurons of the mouse were labeled with [3H]thymidine during their prenatal period of proliferation. The 3H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the 3H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [3H]ß self-absorption in the material of the sections.

Injection of a high dose of [3H]thymidine into young adult mice did not result in 3H labeling of either Purkinje or other neurons in other brain regions.

The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.
THE REGENERATION OF CILIA IN PARTIALLY DECILIATED TETRAHYMENA
Authors: Rannestad John
Published: May 2004

Partial deciliation of Tetrahymena resulted in cells losing 75% of their cilia, with the balance being paralyzed. The paralyzed cilia are resorbed in the first 20 min after partial deciliation, and regeneration of cilia begins before resorption is completed. Inhibition of protein synthesis with cycloheximide does not inhibit ciliary resorption or regeneration, whereas vinblastine sulfate inhibits regeneration but not resorption. Inhibition of regeneration occurs in completely deciliated cells when they are treated with cyclohexmimide or vinblastine sulfate. It is concluded that the resorbing cilia contribute materials which allow regeneration to occur in the absence of protein synthesis.

The volume of cilia regenerated in the presence of cycloheximide in partially deciliated cells is greater than the ciliary volume which is resorbed. This suggests the Tetrahymena cells have a pool of ciliary precursors. This pool does not contribute materials for regeneration in completely deciliated cells which are treated with cycloheximide. It is concluded that resorbing cilia in partially deciliated cells contribute materials which potentiate assembly of cilia from the pool of precursors.
SOME BIOCHEMICAL PROPERTIES OF CHINESE HAMSTER CELLS SENSITIVE AND RESISTANT TO ACTINOMYCIN D
Authors: Peterson Robert H. F. , O'Neil Judith A. , Biedler June L.
Published: May 2004

A graded series of drug-resistant Chinese hamster sublines has been examined for biochemical changes accompanying resistance to actinomycin D. The most highly resistant subline, DC-3F/AD X, is maintained at 10 µg/ml of the antibiotic. It was shown that over 250 times more actinomycin D is required to inhibit RNA synthesis in this subline than in the parental DC-3F line. The DC-3F/AD X subline was also shown to have a somewhat reduced capacity to transport uridine as compared to parental cells. Sensitive cells took up over 50 times more tritiated antibiotic than the most resistant cells, as determined in a 1-h assay. Uptake of actinomycin D was shown to be temperature-dependent in both resistant and sensitive cells and was not influenced by various metabolic inhibitors. Resistance could not be explained by a rapid uptake and release of the antibiotic, as demonstrated in efflux experiments, or by its metabolism. In addition, highly resistant cells which are cross-resistant to puromycin were shown to have a reduced capacity to take up labeled puromycin. These studies provide further evidence indicating that the mechanism of resistance to actinomycin D is reduced permeability to drug and suggesting that cell membrane alteration accounts for resistance to both actinomycin D and puromycin.
THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS
Authors: Kedinger Claude , Simard Rene
Published: May 2004

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.
THE α-GLYCEROPHOSPHATE CYCLE IN DROSOPHILA MELANOGASTER
Authors: O'Brien Stephen J. , Shimada Yoshio
Published: May 2004

"Null" mutations previously isolated at the αGpdh-1 locus of Drosophila melanogaster, because of disruption of the energy-producing α-glycerophosphate cycle, severely restrict the flight ability and relative viability of affected individuals. Two "null" alleles, αGpdh-1BO-1-4, and αGpdh-1BO-1-5, when made hemizygous with a deficiency of the αGpdh-1 locus, Df(2L)GdhA, were rendered homozygous by recombination with and selective elimination of the Df(2L)GdhA chromosome. After over 25 generations, a homozygous αGpdh-1BO-1-4 stock regained the ability to fly despite the continued absence of measurable αGPDH activity. Inter se heterozygotes of three noncomplementing αGpdh-1 "null" alleles and the "adapted" αGpdh-1BO-1-4 homozygotes were examined for metabolic enzymatic activities related to the energy-producing and pyridine nucleotide-regulating functions of the α-glycerophosphate cycle in Drosophila. The enzyme functions tested included glyceraldehyde-3-phosphate dehydrogenase, cytoplasmic and soluble malate dehydrogenase, lactate dehydrogenase, mitochondrial NADH oxidation, oxidative phosphorylation, and respiratory control with the substrates α-glycerophosphate, succinate, and pyruvate. These activities in any of the mutant genotypes in early adult life were indistinguishable from those in the wild type. There was, however, a premature deterioration and atrophy of the ultrastructural integrity of flight muscle sarcosomes observed by electron microscopy in the "null" mutants. These observations were correlated with a decrease in state 3 mitochondrial oxidation with α-glycerophosphate, succinate, and pyruvate, as well as with loss of respiratory control in adults as early as 2 wk after eclosion. Such observations, which normally are seen in aged dipterans, were accompanied by premature mortality of the mutant heterozygotes. The adapted αGpdh-1BO-1-4 was identical with wild type in each of the aging characters with the single exception of lowered rates of mitochondrial oxidative phosphorylation.
TRANSCRIPTION SITES IN SPREAD MEIOTIC PROPHASE CHROMOSOMES FROM MOUSE SPERMATOCYTES
Authors: Kierszenbaum A. L. , Tres Laura L.
Published: May 2004

Mouse spermatocytes at pachytene stage have been examined by whole-mount electron microscope techniques complemented with autoradiography as an approach for visualizing their transcriptive activity. Structural elements of meiotic bivalents, such as synaptonemal complexes and chromatin fibers, have been satisfactorily displayed in the total set of autosomal and sexual bivalents in single spermatocytes. Adequate preservation of the entire set of bivalents has provided a basis for recognition of sites where presumptive preribosomal RNA and heterogeneous nuclear RNA species are being transcribed at different segments of autosomal bivalents. Nucleoli attached to the basal knob region where nucleolar organizer cistrons are assumed to be located and ribonucleoprotein fibrils associated with distinct chromatin loops have been recognized. These structural findings have been correlated with display of [3H]uridine incorporation sites in thin-section and whole-mount electron microscopy autoradiographic preparations. A low transcriptive activity of the sexual bivalent contrasted with extensive gene expression in autosomal bivalents. Each sex chromosome shows a double axial core. A short region of pairing with a synaptonemal complex joins the two chromosomes at one end. We conclude that variations in the rate of RNA synthesis throughout meiotic prophase stages in the mouse are expressed as fluctuations in the amount and distribution of distinct RNA species at specific segments of the bivalents.
THE VISUALIZATION OF THE PHOTOSYNTHETIC COUPLING FACTOR IN EMBEDDED SPINACH CHLOROPLASTS
Authors: Oleszko Susan , Moudrianakis Evangelos N.
Published: May 2004

Spinach chloroplast lamellae were stained with aqueous uranyl acetate immediately after glutaraldehyde-osmium fixation but before dehydration and embedding. Under these conditions, the lamellae are shown in thin sections to have 95-Å x 115-Å coupling factor particles on their surfaces. The particles can be seen only on the matrix side of nonopposed thylakoids, and are shown to occur on both stromal and granal lamellae, regardless of the organization of the lamellae into stacks. It is estimated that, in native, fully coupled chloroplast lamellae, there is on the average one coupling factor for every 500 chlorophyll molecules. The morphological appearance of the particles is not affected by a variety of buffers, by changes in illumination or temperature, or by alterations in the energy state of the membranes during preparation.

The particles can be removed from the membranes with low concentrations of Na2EDTA, and the photophosphorylating activity of the membranes is concomitantly lost. Both the activity and the appearance of the particles can be restored to the membranes by rebinding EDTA-extracted coupling factors to the uncoupled membranes.
EFFECTS OF CYTOCHALASIN B ON THE RESPONSE OF TOAD URINARY BLADDER TO VASOPRESSIN
Authors: Davis Walter L. , Goodman David B. P. , Schuster Richard J. , Rasmussen Howard , Martin James H.
Published: May 2004

A combined physiological and morphological study of the effects of cytochalasin B (CB) on the toad urinary bladder has been carried out. CB inhibits the hydro-osmotic response to vasopressin without altering basal water permeability or diffusion, or the increase in 3H2O diffusion observed after hormone addition. Although CB increases [22Na]-, [36Cl]-, and [14C]urea fluxes, and decreases transepithelial potential, no alteration in basal short-circuit current, the vasopressin-induced increase in this parameter, or [14C]inulin permeability occurs. In the absence of hormone, CB does not markedly alter the structure of the toad bladder. However, in the presence of vasopressin, CB induces the formation of large intracellular vacuoles. These results suggest a possible coupling of solute and water movement across the tissue.
Quantitative studies of pinocytosis. I. Kinetics of uptake of (125I)polyvinylpyrrolidone by rat yolk sac cultured in vitro.
Authors: Williams K E , Kidston E M , Beck F , Lloyd J B
Published: May 2004

A method is described for the in vitro culture of 17.5-day rat visceral yolk sac. Tissue survival was good as judged by light and electron microscopy. The rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone by the tissue was constant both within and between experiments. Within the concentration range 0.15-24 mug/ml, the 125I-labeled polyvinylpyrrolidone neither stimulated nor inhibited pinocytosis. The system offers many advantages in the quantitative study of the physical basis of pinocytosis.
Growth of mammalian cells on substrates coated with cellular microexudates. I. Effect on cell growth at low population densities.
Authors: Weiss L , Poste G , MacKearnin A , Willett K
Published: May 2004

Mammalian and avian cells cultured on glass or plastic substrates produce microexudates of cellular macromolecules which remain bound to the substrate when the cells are detached. The gross macromolecular composition of microexudates from a range of diploid, heteroploid, and virus-transformed cells was determined with cells labeled with radioisotopes. Significant differences in the amounts of cellular glycoproteins, proteins, and RNA present in microexudates were found between different cell types and between cells of the same type at different stages of growth. Inoculation of cells onto substrates "coated" with microexudates altered their growth behavior. Microexudates from exponentially growing subconfluent homotypic and heterotypic cell populations enhanced the growth of mouse and chick embryo cells seeded at very low densities, but similar microexudates had no effect on the proliferation of cells seeded at higher densities. The enhanced growth of low-density cell populations seeded on microexudates was compared with the growth enhancement produced by feeder cell layers and conditioned medium.
Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.
Authors: Castle J D , Jamieson J D , Palade G E
Published: May 2004

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.
Ribosomal RNA metabolism during renal hypertrophy. Evidence of decreased degradation of newly synthesized ribosomal RNA.
Authors: Hill J M
Published: May 2004

The degradation rates of kidney rRNA labeled before UNI or sham are unchanged 5 days after the operations (t one-and-a half, 88 h). Therefore, there is no contribution from pre-existing rRNA to the increased amount of rRNA in the stimulated kidney. After labeling with L-(methyl-3H)methionine, the kinetics of incorporation into rRNA precursors, 10-60 min and at the postoperative times of 4, 16, 36, and 96 h. The specific activity of cytoplasmic rRNA after 1-h labeling with L-(methyl-3H)methionine increased occured at 4 or 96 h. Since (a) the rate of degradation of rRNA, (b) the kinetics of incorporation and processing of rRNA precursors, and (c) the rate of RNA synthesis appear unchanged after UNI, the accretion of rRNA must involve decreased degradation of newly synthesized rRNA.
Direct biochemical measurements of microtubule assembly and disassembly in Chinese hamster ovary cells. The effect of intercellular contact, cold, D2O, and N6,O2'-dibutyryl cyclic adenosine monophosphate.
Authors: Rubin R W , Weiss G D
Published: May 2004

A study was undertaken to develop a means of quantitating the amount of tubulin present as a soluble pool and as intact microtubules in cultured Chinese hamster ovary cells. A procedure was developed in which these cells grown on monolayer culture in Petri dishes were placed in a "microtubule stabilizing medium" (MTM) consisting of 50% glycerol, 10% dimethylsulfoxide and sodium phosphate magnesium buffer, as described previously by Filner and Behnke. These cells then were homogenized and the homogenate was spun in the ultracentrifuge. Colchicine binding activity was then determined in the supernates and the pellets. The values, when compared with total colchicine binding activity present in replicate homogenates, were used to determine the percentage of tubulin present as intact microtubules. A statistical analysis of thin sections of cells treated with MTM revealed no statistically significant difference between MTM-treated cells and untreated controls. It was further discovered that the relative amount of colchicine binding activity recovered in the high speed pellet varied dramatically, depending upon the cell number of the culture being studied. Preconfluent cultures showed very low colchicine binding activity averaging less than 5%, while confluent and postconfluent cultures often possessed as high as 25% of their total colchicine binding activity in pelletable material. Although cold and D2O treatment had little or no effect on these values, N6,O2'-dibutyryl cyclic adenosine monophosphate increased them. It is hoped that this study will serve as the basis for a reliable quantitative procedure for measuring microtubule polymerization and depolymerization in vivo.
The role of cortical orientation in the control of the direction of ciliary beat in Paramecium.
Authors: Tamm S L , Sonneborn T M , Dippell R V
Published: May 2004

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.
Detection by means of cell fusion of macromolecular synthesis involved in the reconstruction of the nuclear envelope in mitosis.
Authors: Obara Y , Weinfeld H , Sandberg A A
Published: May 2004

Using the cultured Chinese hamster cell line Don, G1 or S or a mixture of late-S/G2 cells were prepared by release from metaphase arrest. Metaphase (M) cells were also obtained by mitotic arrest of log-phase cultures with Colcemid and held in metaphase; such M cells remained untreated with any other compound and were termed standard M cells. When interphase (I) cells were fused at pH 8.0 and 37 degrees C with standard cells in the presence of Colcemid by means of UV-inactivated Sendai virus, binucleate interphase-metaphase (I-M) cells were obtained. In a given I-M cell there occurred within 30 min after fusion either prophasing of the I nucleus or formation of a nuclear envelope (NE) around the chromosomes. About 20% of early G1 cells, 35% of cells at the G1/S boundary, 50% of S cells, and 70% of late S/G2 cells could induce NE formation. If, before fusion, cycloheximide (CHE), an inhibitor of protein synthesis, was present during release from M arrest, the cells entered G1 but not S. About 20% of such early G1 cells, like the untreated early G1 cells, had the capacity to induce NE formation during subsequent fusion. If the cells were blocked in S with 5 mM thymidine (TdR), At least 80% of these cells could induce NE formation during subsequent fusion, but in the presence of both TdR and CHE only 35% could do so. It appeared, therefore, that protein synthesis in interphase was required for NE formation. Experiments with actinomycin D indicated that RNA synthesis was also necessary for acquisition of NE-inducing capacity. About 35% of G1 cells from confluent monolayers had the NE-inducing capacity, but prolonged exposure to CHE reduced their number to 8% . Removal of CHE restored the ability while the cells still remained in G1. This result indicated that continuing protein synthesis in the G1 cell was needed for NE formation subsequent to fusion. The fact that macromolecular synthesis must occur in the I cell before fusion if NE formation was to occur in the fused I-M cell lends further support to evidence adduced earlier that this phenomenon is a normal mitotic event. Prophasing of the I nucleus in I-M cells did not appear to be dependent on macromolecular synthesis in the I cell; earlier results from this laboratory showed, however, that protein synthesis in the prior G2 period of the M cell of the I-M pair was required for prophasing.
Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.
Authors: Hubbard A L , Cohn Z A
Published: May 2004

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.
Mitosis in the cellular slime mold Polysphondylium violaceum.
Authors: Roos U P
Published: May 2004

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.