bluuebunny/crossref_metadata_2025 huggingface dataset:
External fibronectin of cultured human fibroblasts is predominantly a matrix protein.
Authors: Hedman K , Vaheri A , Wartiovaara J
Published: May 2004

The distribution of a major glycoprotein (fibronectin) of human fibroblast cultures was studied in immunoelectron microscopy with peroxidase- or ferritin-labeled antibodies. External fibronectin was visualized in pericellular structures, in some areas on the growth substratum, and to a lesser degree in close association with the upper and lower surface membranes of the cell. The pericellular fibronectin-containing structures consisted of amorphous or vaguely fibrillar material forming strands or patches, 50-500 nm in diameter; the structures appeared to mediate distant cell-to-cell and cell-to-substrate contacts. When in close association with the plasma membrane, fibronectin markers were seen as discrete patches. The exact relationship between this form of fibronectin and the plasma membrane, however, remained open. Filamentous material was commonly seen in the cortical cytoplasm under patches of membrane-associated fibronectin. The distribution that we observed is consistent with the proposed roles of fibronectin in cell interactions with neighboring structures and with its presence in vivo as an extracellular glycoprotein in connective tissue matrix and basal laminae.
Meiosis in Coprinus. VIII. A time-course study of the fusion and division of the spindle pole body during meiosis.
Authors: Lu B C
Published: May 2004

The time-course study of meiosis in the fungus Coprinus cinereus (C. lagopus) by electron microscopy reveals that two monoglobular spindle pole bodies (SPB's) of prekaryogamy nuclei come together during karyogamy and are fused. The fusion SPB of postkaryogamy nucleus persists through zygotene and pachytene as evidenced by the presence of axial components and synaptonemal complexes. At early diplotene, the SPB divides. The divided SPB takes on a diglobular form, which grows in size to form two daughter SPB's. These separate and move to opposite poles at metaphase I.
Molecular and biological properties of a macrophage colony-stimulating factor from mouse yolk sacs
Authors: Johnson GR , Burgess AW
Published: May 2004

A colony-stimulating factor (M-CSF) has been partially purified and concentrated from mouse yolk sac-conditioned medium (YSCM). M-CSF appeared to preferentially stimulate CBA bone marrow granulocyte-macrophage progenitor cells (GM-CFC) to differentiate to form macrophage colonies in semisolid agar cultures. By comparison, colony-stimulating factor (GM-CSF) from mouse lung-conditioned medium (MLCM) stimulated the formation of granulocytic, mixed granulocytic-macrophage, and pure macrophage colonies. Mixing experiments indicated that both M-CSF and GM-CSF stimulated all of the GM-CFC but that the smaller CFC were more sensitive to GM-CSF and that the larger CFC were more sensitive to M-CSF. Almost all developing "clones" stimulated initially with M-CSF continued to develop when transferred to cultures containing GM-CSF. In the converse situation, only 50% of GM-CSF prestimulated "clones" survived when transferred to cultures containing M-CSF. All clones initially stimulated by M-CSF or transferred to cultures stimulated by M-CSF contained macrophages after 7 days of culture. These results suggest that there is a population of cells (GM-CFC) that are capable of differentiating to form both granulocytes and macrophages, but, once these cells are activated by a specific CSF (e.g. M-CSF), they are committed to a particular differentiation pathway. The pattern of CFC differentiation was not directly related to the rate of proliferation: cultures maximally stimulated by M-CSF produced mostly macrophage colonies, but the presence of small amounts of GM-CSF produced granulocytic cells in 30% of the colonies. Gel filtration, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and affinity chromatography with concanavalin A-Sepharose indicated that M-CSF from yolk sacs was a glycoprotein with an apparent molecular weight of 60,000. There was some heterogeneity of the carbohydrate portion of the molecule as evidenced by chromatography on concanavalin A-Sepharose.
Enrichment of spinal cord cell cultures with motoneurons
Authors: Berg DK , Fischbach GD
Published: May 2004

Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221-283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation.
High molecular weight MAPs are part of the mitotic spindle
Authors: Sherline P , Schiavone K
Published: May 2004

We have found that the microtubule-associated proteins of high molecular weight are located in the mitotic spindle. Indirect immunofluorescence studies reveal that the pattern of distribution of these proteins is similar to that described for tubulin and corresponds to the known phases of mitosis.
Sulfated compounds in the zymogen granules of the guinea pig pancreas
Authors: Reggio HA , Palade GE
Published: May 2004

Sulfate incorporation into the guinea pig pancreas was investigated by light (LM) and electron microscope (EM) autoradiography using a system of minilobules incubated in vitro for 60 min in Krebs-Ringer bicarbonate medium (KRB) containing 35SO4(-2). In acinar cells, examined by EM autoradiography, the label was found concentrated over Golgi elements (including condensing vacuoles) and zymogen granules. 35SO4(-2) was also incorporated by the epithelial cells of the entire pancreatic duct system, the incorporation being surprisingly high in the epithelium of the major ducts. In all ductal epithelia, autoradiographic grains appeared over the Golgi complex and the plasmalemma. Since a contribution of duct epithelium to the sulfated compounds found in the discharged secretion could not be ruled out, a purified zymogen granule fraction was used as a source material for the isolation of sulfated compounds of acinar origin. The presence of 35S-radioactivity in the zymogen granules and condensing vacuoles of this fraction was ascertained by autoradiography (of sectioned pellets). From a lysate of this zymogen granule fraction, a soluble sulfated compound of low isoelectric point and high molecular weight was isolated by gel filtration under conditions that allowed its satisfactory separation from the bulk of the secretory proteins.
Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane
Authors: Franke WW , Grund C , Schmid E , Mandelkow E
Published: May 2004

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.
Actin in xenopus oocytes: II. intracellular distribution and polymerizability
Authors: Merriam RW , Clark TG
Published: May 2004

The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions.

The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater.
Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes
Authors: Kreibich G , Ulrich BL , Sabatini DD
Published: May 2004

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.
Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates
Authors: Weibel ER , Paumgartner D
Published: May 2004

The basic stereological formulas for estimating volume (Vv) and surface (Sv) densities are strictly valid only for true infinitely thin sections; the use of "ultrathin" sections of finite thickness T introduces systematic errors, mostly in the sense of overestimation of the parameters. These errors depend on the size and shape of the structural elements and on T. Correction factors for this effect of T are derived by considering model structures that simulate the shape and arrangement of subcellular organelles: (a) spherical vesicles, (b) disks as models for rough endoplasmic reticulum (RER) cisternae, (c) cylindrical tublules as models for smooth endoplasmic reticulum (SER) tubules, microvilli, etc. For vesicles, a model of discrete convex spherical particles is assumed; the correction factors consider loss of caps due to grazing sections and size distribution of the vesicles. The disk and tubule models are used in connection with the new integral geometric formulas of R.E. Miles which consider random aggregates of "inter-penetrating" particles so that the resultant structure is non-convex and thus approximates in nature the networks characteristic of endoplasmic reticulum (ER). Some practical examples relative to liver cells show that the errors due to section thickness may be of the order of 20-40% or more. Computation formulas as well as graphs are given for the determination of the correction factors for Vv and Sv.
Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement
Authors: Ritter H , Inoué S , Kubai D
Published: May 2004

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally.
Enrichment and visualization of small replication units from cultured mammalian cells
Authors: Burks DJ , Stambrook PJ
Published: May 2004

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.
Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin
Authors: Gospodarowicz D , Brown KD , Birdwell CR , Zetter BR
Published: May 2004

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.
Actin filament destruction by osmium tetroxide
Authors: Maupin-Szamier P , Pollard TD
Published: May 2004

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.
Cation-induced attachment of ciliary dynein cross-bridges
Authors: Warner FD
Published: May 2004

Isolated, demembranated Unio gill cilia that have been activated and fixed for thin-section electron microscopy in the presence of 2 mM MgSO4 have 87% of their outer dynein arms attached to an adjacent B subfiber. The distribution of attached arms is uniform with respect to doublet position in the cilium. When both 0.1 mM ATP and Mg++ are added to the activation and fixation solutions, the frequency of bridged arms is reduced to 48%. At the same time, the distribution of the attached arms appears to have been systematically modified with respect to doublet position and the active bend plane. Those doublet pairs positioned in the bend plane where interdoublet sliding is minimal retain a greater number of bridged arms than those doublet pairs positioned outside the bend plane where sliding is maximal. These observations imply a functional coupling of the Mg++-induced bridging of the dynein arms and the subsequent binding and hydrolysis of ATP that results in a force-generating cross-bridge cycle.
Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell
Authors: Osborn M , Webster RE , Weber K
Published: May 2004

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.
Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin
Authors: Farquhar MG
Published: May 2004

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.
Experimental induction of gene activity in the salivary gland chromosomes of Trichosia pubescens (Diptera: Sciaridae).
Authors: Amabis J M , Janczur C
Published: May 2004

During the course of experiments with larvae of Trichosia pubescens, we have unexpectedly found that diethyl ether or chloroform anesthesia induces a large puff in a specific band in the polytene chromosomes of the salivary glands. This puff develops a few minutes after the treatment, attaining its maximum size after 60-100 min, and regresses completely 200 min after its activation. Through autoradiography, an intense incorporation of RNA precursors into that puff was observed. A few other smaller puffs are also induced by the treatment. The treatment with diethyl ether or chloroform does not induce puffing in the polytene cells of malpighian tubules and of midgut.
Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization.
Authors: Scheele G A , Palade G E , Tartakoff A M
Published: May 2004

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.
Mechanism for the selection of nuclear polypeptides in Xenopus oocytes.
Authors: Feldherr C M , Pomerantz J
Published: May 2004

The function of the nuclear envelope in regulating the cellular distribution of proteins was studied by experimentally altering nuclear permeability and determing the effect of the procedure on the incorporation of exogenous and endogenous polypeptides into the nucleoplasm. Using fine glass needles, nuclear envelopes were disrupted by puncturing oocytes in that region of the animal pole occupied by the germinal vesicle. This resulted in a highly significant increase in the nuclear uptake of cytoplasmically injected [125I]-bovine serum albumin ([125I]BSA), deomonstrating that the envelopes had lost their capacity to act as effective barriers to the diffusion of macromolecules. Endogenous proteins were labeled by incubating oocytes in L-[3H]lecuine. After appropriate intervals, nuclei were isolated from punctured and control cells and analyzed for tritiated polypeptides. Both total precipitable counts and the proportion of label in different size classes of polypeptides were compared. The results showed that puncturing the oocytes had no apparent quantitative or qualitative effects on the uptake of endogenous polypeptides by the nuclei. It can be concluded that the accumulation of specific nuclear proteins is not controlled by the envelope but rather by selective binding within the nucleoplasm.
Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites.
Authors: Sanes J R , Marshall L M , McMahan U J
Published: May 2004

Axons regenerate to reinnervate denervated skeletal muscle fibers precisely at original synaptic sites, and they differentiate into nerve terminals where they contact muscle fibers. The aim of this study was to determine the location of factors that influence the growth and differentiation of the regenerating axons. We damaged and denervated frog muscles, causing myofibers and nerve terminals to degenerate, and then irradiated the animals to prevent regeneration of myofibers. The sheath of basal lamina (BL) that surrounds each myofiber survives these treatments, and original synaptic sites on BL can be recognized by several histological criteria after nerve terminals and muscle cells have been completely removed. Axons regenerate into the region of damage within 2 wk. They contact surviving BL almost exclusively at original synaptic sites; thus, factors that guide the axon's growth are present at synaptic sites and stably maintained outside of the myofiber. Portions of axons that contact the BL acquire active zones and accumulations of synaptic vesicles; thus by morphological criteria they differentiate into nerve terminals even though their postsynaptic targets, the myofibers, are absent. Within the terminals, the synaptic organelles line up opposite periodic specializations in the myofiber's BL, demonstrating that components associated with the BL play a role in organizing the differentiation of the nerve terminal.
Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.
Authors: Salpeter M M , Rogers A W , Kasprzak H , McHenry F A
Published: May 2004

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.
Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex.
Authors: Gilula N B , Epstein M L , Beers W H
Published: May 2004

Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.
Stereological analysis of hepatic fine structure in the Fischer 344 rat. Influence of sublobular location and animal age.
Authors: Schmucker D L , Mooney J S , Jones A L
Published: May 2004

Stereological analysis of hepatic fine structure in Fischer 344 male rats at 1, 6, 10, 16, 20, 25, and 30 mo of age revealed differences in the amounts and distributions of hepatocellular organelles as a function of sublobular location or animal age. Between 1 and 16 mo of age, both the centrolobular and periportal hepatocytes increased in volume by 65 and 35%, respectively. Subsequently, the cell volumes declined until the hepatocytes of 30-mo-old rats approached the size of those found in the youngest animals. Regardless of animal age, the centrolobular cells were consistently larger than the corresponding periportal hepatocytes. The cytoplasmic and ground substance compartments reflected similar changes in their volumes, although there was no significant alteration in the nuclear volume. The volumes of the mitochondrial and microbody compartments increased and decreased concomitant with the changes in average hepatocyte size. Both lobular zones in the 30-mo-old rats contained significantly smaller relative volumes of mitochondria than similar parenchyma in 16-mo-old animals. The volume density of the dense bodies (lysosomes) increased markedly in both lobular zones between 1 and 30 mo of age, confirming reports of an age-dependent increase in this organelle. The surface area of the endoplasmic reticulum in the centrolobular and periportal hepatocytes reached its maximum level in the 10-mo-old rats and subsequently declined to amounts which approximated those measured in the 1-mo-old animals. This age-related loss of intracellular membrane is attributable to a significant reduction in the surface area of the smooth-surfaced endoplasmic reticulum (SER) in animals beyond 16 mo of age. The amount of rough-surfaced endoplasmic reticulum (RER) in the periportal parenchymal cells was unaffected by aging, but the centrolobular hepatocytes of 30-mo-old animals contained 90% more RER than similar cells in the youngest rats. The centrolobular parenchyma contained more SER and the portal zones more RER throughout the age span studied. These quantitative data suggest that (a) certain hepatic fine structural parameters undergo marked changes as a function of animal age, (b) there exists a gradient in hepatocellular fine structure across the entire liver lobule, and (c) there are remarkable similarities in hepatocyte ultrastructure between very young and senescent animals, including cell size and the amount of SER.
Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.
Authors: Papermaster D S , Schneider B G , Zorn M A , Kraehenbuhl J P
Published: May 2004

Immunocytochemical techniques have localized a large protein which is an intrinsic membrane component of isolated frog rod outer segments (ROS). This large protein whose apparent mol wt is 290,000 daltons comprises about 1--3% of the ROS membrane mass. Its molar ratio to opsin is between 1:300 and 1:900. Adequate immune responses were obtained with less than 30 microgram (100 pmol) of antigen per rabbit. Antibodies to the large protein were used for its localization on thin sections of frog retina embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specifically bound antibodies were detected by an indirect sequence with ferritin-conjugated antibodies. This technique detected the protein which is represented by 1,000--3,000 molecules per disk. This indicates that the procedure is sufficiently sensitive for analysis of membrane components in low molar proportions. The large protein was specifically localized to the incisures of ROS disks which divide the disks into lobes and to the disk margin. Thus, opsin is mobile within the membrane of the disk while the large protein is apparently constrained to the disk edges. This finding raises the possibility that special functions are also localized ot his unusual region of high curvature, and that collisions of bleached opsin with these edges are physiologically important in couter segment function.
Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes.
Authors: Volsky D J , Loyter A
Published: May 2004

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).
Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes.
Authors: Matsuura S , Fujii-Kuriyama Y , Tashiro Y
Published: May 2004

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.
Loss and reappearance of gap junctions in regenerating liver.
Authors: Yee A G , Revel J P
Published: May 2004

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.
Neurofilament protein is phosphorylated in the squid giant axon.
Authors: Pant H C , Shecket G , Gainer H , Lasek R J
Published: May 2004

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.
Fluctuations in the production of specific cellular peptides during the growth of animal cells.
Authors: Lee G T , Engelhardt D L
Published: May 2004

Patterns of newly synthesized proteins of Vero cells in different growth states were obtained using two-dimensional gel electrophoresis. The 240 most prevalent peptide spots were then compared. Cells in exponential growth and in the stationary phase were found to have patterns of peptide spots characteristic of their state of growth. The transition between these patterns is progressive, and the cells acquire a pattern characteristic of quiescent cells by the late exponential phase. These observations suggest that a series of modulations in gene expression occurs during the transition of growth states in animal cells that leads to the specific appearance or disappearance of certain cellular peptides.