bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.
Authors: Adoutte A , Ramanathan R , Lewis R M , Dute R R , Ling K Y , Kung C , Nelson D L
Published: May 2004

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.
A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells.
Authors: Bravo R , Celis J E
Published: May 2004

The polypeptides synthesized during the cell cycle of HeLa cells were analyzed by means of two-dimensional gel electrophoresis followed by fluorography under conditions in which the position of 700 polypeptides (acidic and basic) could be reproducibly assessed. Mitotic cells obtained by mechanical detachment and synchronized cells in other stages of the cell cycle were labeled with [35S]methionine for 30-min pulses or for long terms starting at the beginning of each phase. Visual comparison of the polypeptide maps obtained in the different stages of the cell cycle showed that these were strikingly similar, and there was no indication that the synthesis of any of the detected polypeptides was confined to only one of the cell cycle phases. Quantitation of 99 abundant polypeptides (acidic and basic) in pulse-labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypeptides, including total actin, alpha-actinin, 6 abundant basic nonhistone proteins, and 13 major acidic proteins present in Triton cytoskeletons, remains constant throughout the cell cycle. Among the few variable polypeptides (markers), we have identified alpha- and beta-tubulin (increase in M), the subunit of the 100-A filament protein "fibroblast type" (decreases in M), and a 36,000 mol wt acidic cytoarchitectural protein that increases in S. A few other unidentified polypeptides have also been found to vary in M and in M and G2, but no marker was found in G1.
Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture
Authors: Klagsbrun M
Published: May 2004

Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco's modified eagle's medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.
Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.
Authors: Rivin C J , Fangman W L
Published: May 2004

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.
Nonlinear current-voltage relationships in cultured macrophages.
Authors: Gallin E K , Livengood D R
Published: May 2004

Intracellular recordings of cultured mouse thioglycolate-induced peritoneal exudate macrophages reveal that these cells can exhibit two different types of electrophysiological properties characterized by differences in their current-voltage relationships and their resting membrane potentials. The majority of cells had low resting membrane potentials (-20 to -40 mV) and displayed current-voltage relationships that were linear for inward-going current pulses and rectifying for outward-going pulses. Small depolarizing transients, occurring either spontaneously or induced by current pulses, were seen in some cells with low resting membrane potentials. A second smaller group of cells exhibited more hyperpolarized resting membrane potentials (-60 to -90 mV) and S-shaped current-voltage relationships associated with a high-resistance transitional region. Cells with S-shaped current-voltage relationships sometimes exhibited two stable states of membrane potential on either side of the high-resistance transitional region. These data indicate that macrophages exhibit complex electrophysiological properties often associated with excitable cells.
Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study.
Authors: Molano J , Bowers B , Cabib E
Published: May 2004

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.
Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis.
Authors: McGraw C F , Somlyo A V , Blaustein M P
Published: May 2004

Ultrastructural techniques and electron probe microanalysis were used to determine whether or not the smooth endoplasmic reticulum (SER) within presynaptic nerve terminals is a Ca-sequestering site. The three-dimensional structure of the SER was determined from serial sections of synaptosomes. The SER consists of flattened cisterns that may branch and are frequently juxtaposed to mitochondria. To investigate intraterminal Ca sequestration, synaptosomes were treated with saponin to disrupt the plasmalemmal permeability barrier. When these synaptosomes were incubated in solutions containing Ca, ATP, and oxalate, electrondense Ca oxalate deposits were found in intraterminal mitochondria, SER cisterns, and large vesicular profiles. Saponin-treated synaptosomes that were incubated in the presence of mitochondrial poisons contained electron-dense deposits within SER cisterns and large vesicular profiles, but very rarely in mitochondria. Similar deposits were observed within saponin-treated synaptosomes that were not post-fixed with OSO4, and within saponin-treated synaptosomes that were prepared for analysis by freeze-substitution. Electron-probe microanalyses of these deposits confirmed the presence of large concentrations of Ca. When oxalate was omitted from the incubation solutions, no electron-dense deposits were present in saponin-treated synaptosomes. In other control experiments, either the Ca ionophore A23187 or the Ca chelator EGTA was added to the incubation media; electron-dense deposits were very rarely observed within the intraterminal organelles of these saponin-treated synaptosomes. The data indicate that presynaptic nerve terminal SER is indeed a Ca-sequestering organelle.
Chick embryo fibroblasts produce two forms of hyaluronidase
Authors: Orkin RW , Toole BP
Published: May 2004

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.
Changes in contractile proteins during differentiation of myeloid leukemia cells. I. Polymerization of actin.
Authors: Nagata K , Sagara J , Ichikawa Y
Published: May 2004

Quantitative and qualitative changes in cellular actin were followed during differentiation of a myeloid leukemia cell line, namely Ml, which was inducible with conditioned medium (CM). During 3 d of incubation with CM, when the Ml cells differentiated to macrophages and lost their mitotic activity, the actin content, F-actin ratio in total actin, and the actin synthesis showed an increase. A greater difference before and after differentiation was found in the ability of G-actin to polymerize. Actin harvested from CM-treated cells showed a greater ability to polymerize, depending on the increased concentration of MgCl2 and/or KCl and proteins, as compared with the actin from untreated Ml cells. Actin harvested from the Mml cell line, a macrophage line, had a particularly high polymerizability with or without CM treatment. In contrast, the actin from the D- subline, which is insensitive to CM, showed almost no polymerization.
Effects of cholesterol on lipid organization in human erythrocyte membrane.
Authors: Hui S W , Stewart C M , Carpenter M P , Stewart T P
Published: May 2004

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.
Early events in the cellular formation of proparathyroid hormone
Authors: Habener JF , Maunus R , Dee PC , Potts JT
Published: May 2004

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.
[3H]ouabain autoradiography of frog retina.
Authors: Stirling C E , Lee A
Published: May 2004

The kinetics and distribution of ouabain binding in retinas of Rana pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a bimolecular reaction. Estimated equilibrium binding levels gave a Michaelis-Menton relationship with a Km = 8.3 x 10(-8) M and a maximum binding level (Bmax) = 4.4 x 10(-8) mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform, and optic nerve fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging approximately 4% of that in the proximal receptor layers (Bmax = 4.6 x 10(-6) M). The origin of the outer segment activity is uncertain at light microscope resolution, as it may be a result of inner segment calyceal processes. Binding within the proximal receptor layers was also nonuniform. Several peaks were observed, with those at the inner segment and synaptic layers being especially noticeable. Assuming an absence of glial cell binding in the proximal receptor layers, we calculated there to be 13 x 10(6) ouabain or Na+,K+ pump sites per rod receptor. Limited measurements suggest a Bmax of approximately 8 x 10(-6) M for the inner plexiform layer.
Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. III. A morphometric analysis of the number and diameter of intramembrane particles.
Authors: Fesce R , Grohovaz F , Hurlbut W P , Ceccarelli B
Published: May 2004

The intramembrane particles on the presynaptic membrane and on the membrane of synaptic vesicles were studied at freeze-fractured neuromuscular junctions of the frog. The particles on the P face of the presynaptic membrane belong to two major classes: small particles with diameters less than 9 nm and large particles with diameters between 9 and 13 nm. In addition, there were a few extralarge particles with diameters greater than 13 nm. Indirect stimulation of the muscle, or the application of black widow spider venom, decreased the concentration of small particles on the presynaptic membrane but did not change the concentration of large particles. Three similar classes of particles were found on the P face of the membrane of the synaptic vesicles. The concentrations of large and extralarge particles on the vesicle membrane were comparable to the concentrations of these particles on the presynaptic membrane, whereas the concentration of small particles on the vesicle membrane was less than than the concentration of small particles on the presynaptic membrane. These results are compatible with the idea that synaptic vesicles fuse with the presynaptic membrane when quanta of transmitter are released. However, neither the large nor the extralarge particles on the P face of the presynaptic membrane can be used to trace the movement of vesicle membrane that has been incorporated into the axolemma.
Use of immobilized lactoperoxidase to label L cell proteins involved in adhesion to polystyrene.
Authors: Chin N W , Lanks K W
Published: May 2004

Proteins involved in the attachment of murine L cells to polystyrene have been identified by a technique designed to iodinate only those macromolecules coming into closet apposition to the substratum. Whereas soluble lactoperoxidase (LPO) catalyzes the radioiodination of a broad spectrum of polypeptides, the same enzyme immobilized on polystyrene tissue culture flasks discriminately labels 55,000 and 42,000 mol wt polypeptides that adhere tightly to the substratum after the cells are removed. One-dimensional peptide mapping following limited proteolysis showed that the labeled 55,000 mol wt polypeptide is similar to a component of comparable molecular weight present in the detergent-extracted cytoskeleton. The functional association of two cytoskeletal structures, presumably 10-nm filaments and actin, is discussed, and alternative explanations for their susceptibility to iodination by immobilized LPO are presented.
Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.
Authors: Meldolesi J , Corte G , Pietrini G , Borgese N
Published: May 2004

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.
Stoichiometry of wheat germ agglutinin as a morphology controlling agent and as a morphology controlling agent and as a morphology protective agent for the human erythrocyte.
Authors: Lovrien R E , Anderson R A
Published: May 2004

The lectin wheat germ agglutinin (WGA) is an unusually effective agent in controlling both the forward and reverse reactions of the reversible morphology conversion discocyte in equilibrium with echinocyte for the human erythrocyte. Under conditions severe enough to drive the reactions to completion in either direction without the lectin, WGA is able to stabilize both these morphologies and to fully prevent conversion of either morphology. The lectin can quantitatively block both reactions. The ability of WGA to carry out these functions has no obvious rate limitation. Its effectiveness depends mainly on its binding stoichiometry, particularly toward the transmembrane glycoprotein, glycophorin. The critical binding stoichiometries for both the lectin and the echinocytic agent were determined in relation to the binding isotherms using 125I-labeled WGA and 35S-labeled dodecyl sulfate. There appear to be two principal stoichiometries for WGA binding that are important in its control of erythrocyte morphology. The first stoichiometry marks the threshold of obvious protection of the discocyte against strong echinocytic agents such as detergents and, likely, is simply a 1:1 stoichiometry of WGA: glycophorin, assuming currently recognized values of 3--5 x 10(5) copies of glycophorin per cell. The second important stoichiometry, whereby the cell's morphology is protected against extremely severe stress, involves binding of approximately 4--5 WGA molecules per glycophorin. The controls that WGA exerts can be instantly abolished by added N-acetylglucosamine. However, N-acetylglucosamine ligands on the erythrocyte are of less importance than membrane neuraminic acid residues in enabling WGA to control the cell's morphology, as is shown by comparing intact cells with completely desialated cells. WGA can also be used to produce elliptocytes in vitro, but it does this at levels approaching monolayer coverage of the cell with WGA.
Cyanobacterial Phycobilisomes. Particles from synechocystis 6701 and two pigment mutants
Authors: Williams R , Gingrich J , Glazer A
Published: May 2004

The phycobilisomes of the unicellular cyanobacterium Synechocystis 6701, grown in white light, contain C-phycoerythrin, C-phycocyanin, and allophycocyanin in a molar ration of approximately 2:2:1, and in addition, polypeptides of 99, 46, 33.5, 31.5, 30.5, and 27 x 10(3) Daltons, as well as a trace of a approximately 9 x 10(3)-dalton component. Two nitrosoguanidine-induced mutants of this organism produce aberrant phycobilisomes. Crude cell extracts of these mutants, 6701-NTG25 and NTG31, contain phycoerythrin, phycocyanin, and allophycocyanin in a molar ration of 1:5:1:1 and 0.55:0.3:1.0, respectively. The phycobilisomes from both mutants lack the 33.5 x 10(3)-dalton polypeptide.

Wile-type phycobilisomes consist of a core composed of an equilateral array of three cylindrical elements surrounded by six rods in a fanlike arrangement. The rods are made up of stacked disks, 11 nm in diameter and 6 nm thick. In phycobilisomes of mutant 6701-NTG25, numerous particles with fewer than six rods are seen. Mutant 6701-NTG31 produces incomplete structures that extend from triangular core particles, through cores with one or two attached rods, to cores with as many as five rods. The structure of the core appears unaltered throughout. The amount of phycocyanin (relative to allophycocyanin) appears to determine the number of rods per core.

A common assembly form seen in 6701-NTG31 is the core with two rods attached at opposite sides. From observations of this form, it is concluded that the core elements are cylindrical, with a height of 14 nm and a diameter of 11 nm. No consistently recognizable structural details are evident within the core elements.
Association of an axonally transported polypeptide (H) with 100-A filaments. Use of immunoaffinity electron microscope grids.
Authors: Willard M , Simon C , Baitinger C , Levine J , Skene P
Published: May 2004

Polypeptide H (mol wt 195,000) is axonally transported in rabbit retinal ganglion cells at a velocity of 0.7--1.1 mm/d, i.e., in the most slowly moving of the five transport groups described in these neurons. To identify the organelle with which H is associated, we purified H, prepared antibodies directed against it, and adsorbed the antibodies onto Formvar-coated electron microscope grids. When the resulting "immuno-affinity grids" were incubated with extracts of spinal cord and then examined in the electron microscope, they contained as many as 100 times more 100-A filaments than did grids coated similarly with nonimmune IgG. The ability of the anti-H IgG to specifically adsorb filaments to grids was completely blocked by incubating the IgG with polypeptide H. The 100-A filaments adsorbed to anti-H immunoaffinity grids could be specifically decorated by incubating them with anti-H IgG. These observations demonstrate that H antigens (and most likely H itself) are associated with 100-A neurofilaments. In addition, they suggest that the use of immunoaffinity grids may be a useful approach for determining the organelle associations of polypeptides.
Purified lectin from skeletal muscle inhibits myotube formation in vitro.
Authors: MacBride R G , Przybylski R J
Published: May 2004

A lactose-extractable lectin obtained from 14--16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture. When applied to muscle cultures at 0.09 micrograms/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation. Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4 degrees C. Incubation in L-15 solution at 37 degrees C with or without 2 mM dithiothreitol resulted in inactivation in 2--3 h. To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca+2] medium to inhibit fusion, and then [Ca+2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93%. Continued incubation at 37 degrees C. without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.
Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine.
Authors: Specian R D , Neutra M R
Published: May 2004

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.
Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat.
Authors: Warshawsky H , Goltzman D , Rouleau M F , Bergeron J J
Published: May 2004

An in vivo binding assay using radioautography was employed to visualize calcitonin receptors in rat tissues. At 2 min after intravenous injection of biologically active 125I-salmon calcitonin, free hormone was separated from bound hormone by intracardiac perfusion with lactated Ringer's followed by fixation with 2.5% glutaraldehyde. Various tissues were removed and processed for light and electron microscope radioautography. These were compared to tissues removed from animals that received identical amounts of labeled hormone with a large excess of unlabeled calcitonin. Among the tissues investigated, kidney and bone demonstrated labeling. In kidney, most silver grains were located over vesicles below the brush border of cells of theproximal convoluted tubules. These grains were still present after simultaneous injection of excess unlabeled hormone and most likely represented binding to sites involved with ingestion and degradation of hormone from the urinary filtrate. In contrast, grains localized to the basal surfaces of distal convoluted tubule cells were significantly reduced in number in control animals and represented sites of saturable, specific hormone binding. In bone, specific binding sites were found only at the periphery of osteoclasts. These labeled cells were located at resorption sites examined in tibia, humerus, and alveolar bone. This demonstration of the localization of 124I-calcitonin in situ provides a new approach for study the interaction of calcium-regulating hormones with their target cells.
Chemotactic peptide receptor modulation in polymorphonuclear leukocytes.
Authors: Sullivan S J , Zigmond S H
Published: May 2004

The binding of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) to its receptor on rabbit polymorphonuclear leukocytes (PMNs) modulates the number of available peptide receptors. Incubation with FNLLP decreases subsequent binding capacity, a phenomenon that has been termed receptor down regulation. Down regulation of the chemotactic peptide receptor is concentration dependent in both the rate and extent of receptor loss. The dose response parallels that of FNLLP binding to the recptor. The time-course is rapid; even at concentrations of FNLLP as low as 3 x 10(-9) M, the new equilibrium concentration of receptors is reached within 15 min. Down regulation is temperature dependent, but does occur even at 4 degrees C. Concomitant with down regulation, some of the peptide becomes irreversibly cell associated. At 4 degrees C, there is a small accumulation of nondissociable peptide that rapidly reaches a plateau. At higher temperatures, accumulation of nondissociable peptide continues after the rceptor number has reached equilibrium, and the amount accumulated can exceed the initial number of receptors by as much as 300%. The dose response of peptide uptake at 37 degrees C reflects that of binding, suggesting that it is receptor mediated. This uptake may occur via a pinocytosis mechanism. Although PMNs have not been considered to be pinocytic, the addition of FNLLP causes a fourfold stimulation of the rate of pinocytosis as measured by the uptake of [3H]sucrose.
Inhibition and relaxation of sea urchin sperm flagella by vanadate.
Authors: Okuno M
Published: May 2004

Direct measurements of the stiffness (elastic bending resistance) of demembranated sera urchin sperm flagella were made in the presence of MgATP2- and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of approximately 0.9 x 10(-21) N m2, which is approximately 5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP- dose not substitute for MgATP2- in producing relaxed state. A progressive inhibition of movement is observed after addition of MgATP2- to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP2- and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP2- that is not enough to induce spontaneous beating. Vanadate enhances competitive inhibition of beat frequency by MgADP- but not by ADP3-, ATP4-, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dynein-nucleotide complexes induced by MgATP2- and MgADP-. The state accessible by MgATP2- binding must be a state in which the cross-bridges are detached and the flagellum is relaxed. The state accessible by MgADP- binding must be a cross-bridged state. Bound vanadate prevents the transition between these two states. Inhibition and relaxation by banadate in the presence of MgATP2- results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the deteched state.
Induction of the acrosome reaction in guinea pig spermatozoa by cGMP analogues.
Authors: Santos-Sacchi J , Gordon M
Published: May 2004

The effect of cyclic nucleotide analgoues upon the immediate induction of the guinea pig acrosome reaction (AR) was studied. Dibutyryl (dB) CGMP and 8-bromo-cGMP, when added to sperm suspensions after varying periods of preincubation in glucose-free BWW medium (NaCl 94.59 mM, KCl 4.7 mM, CaCl2 1.71 mM, KH2PO4 1.19 mM, MgSO4 1.19 mM, NaHCO3 25.07 mM, pyruvate 0.25 mM, lactate 21.58 mM, and bovine seru albumen 1 g/liter), induced the AR in a large proportion of spermatozoa relative to controls. The proportion of ARs induced upon the addition of dB cGMP or 8-bromo-cGMP (10mM) at 1 h was equivalent to that obtained after a 5-h incubation in glucose-free BWW alone. The effect of cGMP analogues was concentration dependent over the tested range of 2-12 mM (less than 1-20%). The simultaneous addition if imidazole (10 mM), a cAMP phosphodiesterase stimulator, potentiated the effect (imidazole + 12 mM 8-bromo-cGMP: 73%). cAMP analogues were without effect. The presence of extracellular Ca++ was required, and it is suggested that a rise in the cGMP/CAMP ratio triggers Ca++ influx and the AR.
Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.
Authors: McCarthy K D , de Vellis J
Published: May 2004

A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.
Myosin types in cultured muscle cells.
Authors: Cantini M , Sartore S , Schiaffino S
Published: May 2004

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.
Brush-border calmodulin. A major component of the isolated microvillus core.
Authors: Howe C L , Mooseker M S , Graves T A
Published: May 2004

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons.The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.
Prefracture and cold-fracture images of yeast plasma membranes.
Authors: Steere R L , Erbe E F , Moseley J M
Published: May 2004

Fracture-temperature related differences in the ultrastructure of plasmalemma P faces of freeze-fractured baker's yeast (Saccharomyces cerevisiae) have been observed in high-resolution replicas prepared in freeze-etch systems pumped to 2 X 10(-7) torr in which the specimens were protected from contamination by use of liquid nitrogen-cooled shrouds. Two major P-face images were observed regardless of the source of the yeast, the age of the culture, the growth temperature, the physiological condition, or the suspending medium used: (a) a "cold-fracture image" with many strands closely associuated with tubelike particles (essentially the same image as those previously published for yeast freeze-fractured at 77 degrees K), and (b) a "prefracture image" characterized by the presence of more distinct tubelike particles with few or no associated strands (for aging cultures, the image recently referred to as "paracrystalline arrays" of "craterlike particles"). Both types of P-face image can be found in separate areas of single replicas and occasionally even within a single plasma membrane. Whereas portions of replicas known to be fractured at any temperature colder than 218 degrees K reveal only the cold-fracture image, prefracture images are found in cells intentionally fractured at 243 degrees K and in cracks or fissures which develop during the freezing of other specimens. These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens at some temperature between 173 degrees and 77 degrees K, and not from the use of "starved" yeast cells.
Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor.
Authors: Griffin C G , Letourneau P C
Published: May 2004

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell-substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.
Isolation and characterization of rabbit endocervical cells
Authors: Chilton B , Nicosia S , Sowinski J , Wolf D
Published: May 2004

Three cytologically distinct cell populations were identified, in addition to ciliated cells, when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit endocervical cells. Two of these cell populations contained histochemically distinguishable (periodic acid- Schiff [PAS]) mucoproteins and were designated vacuolated and granular PAS-positive cells. The third, designated as vacuolated PAS-negative, did not contain secretory granules. Cell integrity was confirmed by trypan blue dye exclusion, [(3)H]leucine incorporation, and ultrastructural analysis. To demonstrate hormonal modulation of endocervical cell morphology, cell distribution profiles were compared from animals in different hormonal states. In the absence of estrogen dominance, PAS- positive cells from 5-d pseudopregnant rabbits were reduced 50 percent, while vacuolated PAS-negative cells increased fourfold as compared with estrous cell populations. The PAS-positive cells sedimented toward the top of the gradient where the bovine serum albumin concentrations were lower, consistent with a reduction in the number of secretory granules. In the sustained absence of ovarian steroid hormones, the number of PAS-positive mucous cells from ovariectomized rabbits was reduced to only 4 percent of the total endocervical cell population.

The biosynthetic capacity of isolated endocervical cells was determined by incubating the three nonciliated cell populations from estrous and 5-d pseudopregnant rabbits for 36 h with the mucin precursor, [(14)C]N-acetyl- D-glucosamine. Only PAS-positive cells incorporated significant amounts of labeled precursor. This study indicates that steroid hormones influence cervical secretions by modulating the type of endocervical cells.