bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei.
Authors: Herman R , Weymouth L , Penman S
Published: May 2004

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single-stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.
Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium
Authors: Greene LA
Published: May 2004

The PC12 clone is a noradrenergic cell line derived from a rat pheochromocytoma. In culture medium containing horse serum, PC12 cells undergo mitosis; when nerve growth factor (NGF) is included in the medium, the cells cease multiplication and extend neuritis. It is shown here: (a) that PC12 cells are not viable in serum-free medium. When serum is withdrawn, 90 percent of the cells die within 4-6 days and 99 percent by 2-3 wk. (b) If NGF is added at the time of serum withdrawal, the cells undergo one doubling and remain viable for at least 1 mo. (c) Addition of NGF to cultures after more than 2 days in serum-free conditions results in maintenance of surviving cells, but not in an increase in cell number. (d) NGD also induces neurite outgrowth from PC12 cells in serum-free medium. (e) NGF-treated cells exhibit much less cell-cell and neurite-neurite aggregation in the absence than in the presence of serum. (f) The apparent minimum level of 2.5S NGF required for PC12 survival and morphological differentiation in serum-free medium is about 10 ng/ml (approximately 0.4 nM). (g) Withdrawal of NGF in serum-free conditions results in degeneration of neurites and loss of cell viability. (h) Experiments with campotothecin demonstrate that the effects of NGF on survival and neurite outgrowth may be uncoupled and suggest that the survival effects are transcriptionally independent. The present results also suggest that PC12 cells have a requirement for NGF (similar to that of normal sympathetic neurons) and that serum may substitute for this requirement. In addition, the present system of maintaining a highly differentiated cell line in a chemically defined medium suggests certain experimental opportunities.
Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.
Authors: Hoffstein S , Weissmann G
Published: May 2004

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.
Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups.
Authors: Reinecke M , Walther C
Published: May 2004

Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4 degrees C in the dark, ZIO reacts at pH 4.2--4.0 fairly selectively with the matrix of synaptic vesicles. Approximately 53% of the vesicles are completely and 4% partially stained. The percentage of ZIO-positive vesicles is increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by approximately 52%; 2 min of rest restores this number of its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. 2 min after the end of stimulation, partially ZIO-positive vesicles are three times more frequent than before. With all experimental conditions, the average volume of vesicles was as follows: ZIO-negative less than partially ZIO-positive less than completely ZIO-positive. The average volume of ZIO-positive vesicles is almost unaffected by stimulation; that of ZIO-negative vesicles is decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. It is suggested (a) that ZIO demonstrates intravesicular protein(s) containing SH-groups and (b) that the completely ZIO-positive vesicles represent the mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.
Isolation and partial characterization of Drosophila myoblasts from primary cultures of embryonic cells.
Authors: Bernstein S I , Fyrberg E A , Donady J J
Published: May 2004

We describe a method for preparing highly enriched cultures of Drosophila myoblasts from a heterogeneous cell population derived from gastrulating embryos. Enriched cultures are prepared by plating this heterogeneous population of cells in medium from which much of the free calcium is chelated by ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). Adhesion of myoblasts to tissue culture plastic is better than that of other cell types when plated in this medium. Data concerning cell identity, timing of S phase, and fusion kinetics document the degree of enrichment for myogenic cells and illustrate their synchronous differentiation in vitro.
Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.
Authors: Rodriguez Boulan E , Kreibich G , Sabatini D D
Published: May 2004

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.
Histone localization in polytene chromosomes by immunofluorescence.
Authors: Kurth P D , Moudrianakis E N , Bustin M
Published: May 2004

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.
Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.
Authors: Verma D P , Kazazian V , Zogbi V , Bal A K
Published: May 2004

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.
Direct measurement of species-specific cohesion in cellular slime molds.
Authors: Springer W R , Barondes S H
Published: May 2004

Partially differentiated cells of two species of cellular slime molds, Dictyostelium discoideum and Dictyostelium purpureum, were labeled with isothiocyanate derivatives of fluorescent dyes. The labeled cells of each species segregated promptly when mixed and placed on moist filters. We determined whether cells studied at a time when they demonstrated this capacity to segregate showed a preferential adherence to cells of the same species. When labeled dissociated cells of each species were interacted with an unlabeled immobilized layer of cells of each species under appropriate conditions, binding was, in part, species-specific.
Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers.
Authors: Bunge R P , Bunge M B
Published: May 2004

Explants of fetal rat sensory ganglia, cultured under conditions allowing axon and Schwann cell outgrowth in the absence of fibroblasts, occasionally develop nerve fascicles that are partially suspended in culture medium above the collagen substrate. In these suspended regions, fascicles are abnormal in that Schwann cells are decreased in number, are confined to occasional clusters along the fascicle, provide ensheathment for only a few axons at the fascicle periphery, and do not form myelin. When these fascicles are presented with a substrate of reconstituted rat-tail collagen, Schwann cell numbers increase, ensheathment of small nerve fibers occurs normally, and larger axons are myelinated. We conclude that, for normal development, Schwann cells require contact with extracellular matrix as well as axons. The Schwann cell abnormalities in suspended fascicles are similar to those observed in nerve roots of dystrophic mice.
Ionic interactions between bovine chymotrypsinogen A and chondroitin sulfate A.B.C.. A possible model for molecular aggregation in zymogen granules.
Authors: Reggio H , Dagorn J C
Published: May 2004

The formation of large aggregates by ionic interactions between acidic glucosaminoglycans and cationic secretory proteins has been proposed as one of the critical steps in the concentration process in the condensing vacuoles of secretory cells. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A and chondroitin sulfate as a simplified model. Small amounts of chondroitin sulfate were found able to induce chymotrypsinogen precipitation. Like zymogen granules, the resulting aggregates were moderately sensitive to ionic strength and insensitive to osmolality. Moreover, their pH dependence was similar to that of isolated zymogen granules. When sulfated glucosaminoglycans isolated from the zymogen granules of the guinea pig pancreas were used instead of chondroitin sulfate, the same kind of interactions with chymotrypsinogen were obtained. Our data support the hypothesis that the strong ionic interactions between those sulfated glucosaminoglycans and cationic proteins could be responsible for the concentration process.
The effects of temperature and glucose on protein biosynthesis by immature (round) spermatids from rat testes.
Authors: Nakamura M , Romrell L J , Hall P F
Published: May 2004

A method is described for the preparation of highly purified fractions (greater than 80% pure) of immature spermatids (round, steps 1--8) from rat testes by centrifugal elutriation in sufficient yields for biochemical studies when four rat testes are used. Electron microscopy established the identity of the cells and demonstrated that the cell membrane is intact. Some cells develop nuclear and cytoplasmic vacuoles during the 2 h required for preparation. Immature spermatids prepared by this method use glucose with an increase in oxygen consumption, lactate production, and protein synthesis over control levels (no glucose). The testicular cell suspension from which spermatids are separated, like whole testis and spermatids themselves, show higher incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C and in the presence of glucose. A subcellular system prepared from immature spermatids with excess ATP shows greater incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C. This difference does not result from increased breakdown of protein. It is concluded that body temperature (38 degrees C) inhibits some aspect(s) of protein synthesis in addition to previously reported effects on amino acid transport and production of ATP (Means and Hall. 1969. Endocrinology. 84:285--297.).
Calcium control of ciliary arrest in mussel gill cells.
Authors: Walter M F , Satir P
Published: May 2004

After several hours in 20 mM sodium phosphate and 40 mM KCI (pH 7.4) or similar simple solutions, ciliated cells exfoliate en masse from stripped gill epithelium of freshwater mussels, e.g., Elliptio complanatus. Three types of ciliated cells--lateral (L), laterofrontal (LF), and frontal (F)--can be distiniguished and counted separately in the suspensions. About one-half of the cells of each type remain motile. Motility is unaffected by addition of 10(-5) M A23187 or 10(-2) M Ca+2 added separately, but when ionophore and Ca+2 are added together, ciliary beat is largely arrested. Treatment of the cells with Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) results in a total loss of motility as the ciliary membrane becomes disrupted. Such models can be reactivated by addition of ATP and Mg+2. All ciliated cell types are reactivated to about the same extent. At least 80% of the activity of the untreated preparation returns. Ca+2-EGTA buffers added to the reactivating solutions permit titration of free Ca+2 concentration vs. percent motility. Activity is unchanged for all cell types at Ca+2 less than 10(-7) M; at 10(-6) Ca+2, L cilia of all cell types are arrested differentially, whereas at Ca+2 greater than 10(-4) M most cilia of all cell types are arrested. We conclude: (a) that increasing cytoplasmic Ca+2 is directly responsible for ciliary arrest, (b) that the readily reversible physiological arrest response of the L cilia in the intact gill is caused by a rise in free Ca+2 in narrow limits from ca. 5 x 10(-7) M to ca. 8 x 10(-7) M, and (c) that the site which is sensitive to Ca+2 is part of the ciliary axoneme or the basal apparatus.
Compositional analysis of growing axons from rat sympathetic neurons.
Authors: Estridge M , Bunge R
Published: May 2004

We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described.
Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide.
Authors: Kelly P T , Cotman C W
Published: May 2004

The major proteins in isolated synaptic junctions (SJs) and postsynaptic densities (PSDs) have been compared to actin, tubulin, and the major neurofilament (NF) protein by two-dimensional gel electrophoresis and tryptic peptide map analysis. These studies show: (a) tubulin is present in SJ and PSD fractions and is identical to cytoplasmic tubulin, (b) actin in these fractions is very similar to the gamma- and beta-actin found predominantly in nonmuscle cells, and (c) the major PSD protein is distinct from all other known fibrous proteins.
The corpus luteum of the guinea pig. III. Cytochemical studies on the Golgi complex and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles.
Authors: Paavola L G
Published: May 2004

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double-walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum degeneration of guinea pig corpora lutea. Furthermore they suggest that in regressing luteal cells GERL is responsible for packaging acid hydrolases into lytic bodies.
Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.
Authors: Forest C L , Goodenough D A , Goodenough U W
Published: May 2004

The temperature-sensitive gametogenesis-defective mutant, gam-1 is sex-limited, expressed only in mating type minus (mt-), and can sexually agglutinate but not fuse at the restrictive temperature (35 degrees C) with gametes of wild type (wt) mt+. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gam-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35 degrees C, each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gam-1 flagella. The two mutant phenotypes displayed by gam-1 have been denoted gam-1-I and gam-1-II. An agglutination reaction involving gam-1-I cells does not result in activation of the wt mt+ mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-1-II cells, but because the gam-1 mt- mating sturcture still fails to activate, cell fusion is precluded. We conclude that the gam-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.
Growth-related fluctuation in messenger RNA utilization in animal cells.
Authors: Lee G T , Engelhardt D L
Published: May 2004

Monkey fibroblasts maintained in culture regulate their levels of intracellular protein throughout the growth cycle by means of variations in the rate of protein biosynthesis. Cytoplasmic mRNA in stationary phase cells was compared to that in exponential phase cells. In stationary phase cells 56% of the cytoplasmic polyadenylated RNA was found in the 40--90S postpolysomal region of sucrose sedimentation gradients, while only 23% was found in this region in exponential phase cells. Analysis of electron micrographs of sectioned exponential and stationary phase cells revealed that this shift in polyadenylated RNA location is accompanied by a loss of polysome-like aggregates of ribosomes. Most if not all of this species of postpolysomal polyadenylated RNA is not being translated by single ribosomes since no detectable amounts of nascent peptide were present in this region. This nonpolysomal polyadenylated RNA is comparable in size to polysomal polyadenylated RNA. The length of the 3'-poly(A) tract was also comparable for these two species. The extent of capping of poly(A)-containing molecules was also comparable for these two species. The template activity of nonpolysomal RNA in a wheat germ extract was comparable to that of polysomal RNA. The peptides produced by these two preparations were of a similar large size. Furthermore, most of the nonpolysomal polyadenylated RNA of stationary phase cells was driven into polysomes in the presence of a low dose of cycloheximide. Therefore, we conclude that the untranslated mRNA that accumulates in stationary phase cells is structurally intact, is fully capable of being translated, and is not being translated due to the operation of a translational initiation block.
Binding of cytochrome b5 to membranes of isolated subcellular organelles from rat liver.
Authors: Remacle J
Published: May 2004

The in vitro incorporation of a well-characterized integral protein cytochrome b5 into membranes of various subcellular organelles was investigated by biochemical and immunochemical methods. Microsomes, peroxisomes, and outer mitochondrial membranes, all containing endogenous cytochrome b5, incorporated large amounts of the hemoprotein in such a way that it was reducible by an inherent NADH cytochrome b5 reductase. Lysosomal membranes did not incorporate cytochrome b5. Inner mitochondrial and Golgi membranes, which do not naturally contain cytochrome b5, bound it in vitro but it was not reduced in the presence of NADH. These results show some discrepancies between the natural localization and the in vitro binding of cytochrome b5. They confirm one aspect of the fluid membrane theory and bring new elements to our understanding of the maintenance of the specific features of the membranes of subcellular organelles with respect to the cell dynamism.
Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.
Authors: Dessau W , Sasse J , Timpl R , Jilek F , von der Mark K
Published: May 2004

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.
Localized mast cell degranulation induced by concanavalin A-sepharose beads. Implications for the Ca2+ hypothesis of stimulus-secretion coupling.
Authors: Lawson D , Fewtrell C , Raff M C
Published: May 2004

Concanavalin A (Con A) covalently linked to Sepharose 4B beads induced localized degranulation of sensitized rat peritoneal mast cells in regions of contact between beads and cells. This degranulation was Ca2+ dependent and was not seen when sensitized mast cells bound to beads conjugated with a nonstimulating lectin, wheat germ agglutinin, or when unsensitized mast cells bound to Con A-Sepharose. The finding that sensitized mast cells which had adhered to Con A-Sepharose beads degranulated in regions of the cell away from the area of bead contact if exposed to soluble Con A excluded the possibility that the localized release was due to a redistribution of the IgE receptors or putative Ca2+ channels to the region of bead contact. The results suggest that, if an influx of Ca2+ is the mechanism for initiating mast cell degranulation, then the opening of Ca2+ channels in the plasma membrane of activated mast cells is a localized event and that Ca2+ acts locally within the cell to initiate exocytosis.
Distribution of an endogenous lectin in the developing chick optic tectum
Authors: Gremo F , Kobiler D , Barondes SH
Published: May 2004

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin.

No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.
Localization of intestinal sucrase-isomaltase complex on the microvillous membrane by electron microscopy using nonlabeled antibodies
Authors: Nishi Y , Takesue Y
Published: May 2004

Microvillous vesicles isolated from rabbit small intestine showed a trilaminar membrane with a rather smooth surface, which was apparently not affected by papain solubilizing sucrase-isomaltase complex or by trypsin unable to solubilize it. When microvilous vesicles or trysinized ones were incubated with immunoglobulin G against the sucrase-isomaltase complex or monovalent fragments therefrom, an apparently continuous electron-opaque layer approximately 180 A in width appeared around the external surface of vesicles. Such a layer was not formed on papainized vesicles. Microvillous vesicles and trypsinized ones negatively stained with phosphotungate showed a great number of particles protruding approximately 150 A from the membrane surface, but papainized vesicles did not. The particles existed close to one another and appeared to form a particulate layer 150 A in width on the surface. The antibodies, whether they were divalent or monovalent, increased the width of the layer to approximately 200 A and obscured the fine particulate structure of intact and trypsinized vesicles. Papainized vesicles retained their smooth surface upon interaction with antibodies. These results, together with those with the Triton-solubilized sucrase- isomaltase complex (Nishi and Takesue, 1978), J. Ultra-struct. Res., 62:1- 12), indicate not only that sucrase-isomaltase complexes are located close to one another on the membrane, but also that they or at least their protein portions protrude approximately 150 A from the surface of the trilaminar membrane.
Binding of [125I] wheat germ agglutinin to Chinese hamster ovary cells under conditions which affect the mobility of membrane components.
Authors: Stanley P , Carver J P
Published: May 2004

The binding of [125I]wheat germ agglutinin ([125I]WGA) of high specific activity to Chinese hamster ovary (CHO) cells has been examined over a millionfold range of WGA concentrations and correlated with the phenomena of agglutination and capping by WGA. Analysis of the binding data by the method of Scatchard gives a complex curve indicative of positive cooperativity amongst high-affinity binding sites. Binding assays performed under conditions which inhibit capping and/or agglutination, such as low temperature or glutaraldehyde fixation, give similarly complex binding curves. Thus, the gross mobility of WGA receptors in the membrane does not appear to be responsible for the cooperative binding of WGA to CHO cells.
Visualization by fluorescence of chloroplast DNA in higher plants by means of the DNA-specific probe 4'6-diamidino-2-phenylindole.
Authors: James T W , Jope C
Published: May 2004

The DNA in isolated chloroplasts was visualized by the fluorescent probe 4'6-diamidino-2-phenylindole (DAPI). When excited with light of 360 nm, the DNA-DAPI complex fluoresces brilliantly at 450 nm. Nuclei also fluoresce but their nucleoli do not. RNase and Pronase treatment of chloroplasts did not affect the fluorescence but both pre- and posttreatment of DAPI-stained chloroplasts with DNase specifically destroyed the fluorescence. DNA-DAPI complexes in the chloroplasts show up as bright dots. These are distributed uniformly within the chloroplast except for the outer margins. The fluorescent dots can be seen at different focal levels. The number of DNA dots is roughly proportional to chloroplast area which, in turn, is a function of leaf size. The number of fluorescent dots also gave the impression that large leaves with large chloroplasts contain more chloroplast DNA than nuclear DNA.
Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera.
Authors: Goodenough U W , Jurivich D
Published: May 2004

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.
Scalar constraints in Tetrahymena evolution. Quantitative basal body variations within and between species.
Authors: Nanney D L , Chen S S , Meyer E B
Published: May 2004

Tetrahymenas of 17 species of the T. pyriformis complex have been stained with protargol and analyzed for numbers of basal bodies in half cells just before cell division. At this stage, cells of all strains manifest considerable variation in numbers of basal bodies; the coefficient of variation (sigma/m) is usually between 0.05 and 0.10. Much of this variability is observed in cells in the same nutritional state, at the same stage of the growth cycle, and in the same part of the life cycle. The basal body variability may be related to the variation in macronuclear DNA content that results from the imprecise amitotic macronuclear division. With a few exceptions, strains of different species are difficult to distinguish on the basis of basal body numbers. The species means in the samples examined show a range only from 234 (T. furgasoni) to 481 (T. capricornis), about a twofold difference. This limited variation in the means suggests that these organisms are constrained within narrow limited by some scalar function of their organismic design, which prevents an evolutionary size dispersion--even though molecular scrambling has occurred in the complex at an appreciable rate for a very long evolutionary interval.
THE STRIATED MUSCULATURE OF BLOOD VESSELS
Authors: Karrer H. E.
Published: May 2004

The interconnections and the surfaces of the striated muscle cells which occur in thoracic and in lung veins of the mouse were studied with the electron microscope. The osmium-fixed tissues were embedded in methacrylate or in araldite and sectioned with a Porter-Blum microtome. Many preparations were stained before embedding with phosphotungstic acid or after sectioning with uranyl acetate. Typical intercalated discs are observed in this muscle. They are similar to the discs found in heart muscle. These intercalated discs represent boundaries between separate muscle cells. Along the discs, cells are joined in planes normal to their myofilaments. The same cells are also joined in planes parallel to the myofilaments by means of lateral interconnections. These lateral cell boundaries are in continuity with the intercalated discs. Three morphologically distinct parts occur within the lateral cell interconnections: One is characterized by small vesicles along the plasma membrane, the second part has the structure of desmosomes, and a third part represents an external compound membrane (formed by the two plasma membranes of the adjoining cells) and is termed "quintuple-layered cell interconnection." Small vesicles and plasma membrane enfoldings along the free surface of muscle cells are interpreted as products of a pinocytosis (phagocytosis) process. Some of them are seen to contain small membrane-bounded bodies or granules. The free cell surface shows a characteristic outer dense layer ("basement membrane") which accompanies the plasma membrane. The topographic relation of this dense layer with the plasma membrane seems to vary in different preparations. The significance of this variation is not well understood. On two occasions a typical arrangement o vesicles and tubules was observed at Z band levels, just beneath the plasma membrane. These structures are believed to represent endoplasmic reticulum. Their possible significance for the conduction of excitation is discussed.
ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES
Authors: Drochmans P.
Published: May 2004

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.
STUDIES ON THE ENDOPLASMIC RETICULUM
Authors: Porter Keith R. , Yamada Eichi
Published: May 2004

Pigment epithelial cells of the frog's retina have been examined by methods of electron microscopy with special attention focused on the fine structure of the endoplasmic reticulum and the myeloid bodies. These cells, as reported previously, send apical prolongations into the spaces between the rod outer segments, and within these extensions, pigment migrates in response to light stimulation. The cytoplasm of these cells is filled with a compact lattice of membrane-limited tubules, the surfaces of which are smooth or particle-free. In this respect, the endoplasmic reticulum here resembles that encountered in cells which produce lipid-rich secretions. The myeloid bodies comprise paired membranes arranged in stacks shaped like biconvex lenses. At their margins the membranes are continuous with elements of the ER and in consequence of this the myeloid body is referred to as a differentiation of the reticulum. The paired membranes resemble in their thickness and spacings those which make up the outer segments; they are therefore regarded as intracellular photoreceptors of possible significance in the activation of pigment migration and other physiologic functions of these cells. The fuscin granules are enclosed in membranes which are also continuous with those of the ER. The granules seem to move independently of the prolongations in which they are contained. The report also describes the fine structure of the terminal bar apparatus, the fibrous layer intervening between the epithelium and the choroid blood vessels, and comments on the functions of the organelles depicted.