bluuebunny/crossref_metadata_2025 huggingface dataset:
Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells.
Authors: Merisko E M , Farquhar M G , Palade G E
Published: May 2004

Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated STaphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent. The specificity of the immunoadsorption was monitored by SDS PAGE analysis and by competitive ELISA assays. SDS PAGE of the material immunoadsorbed from a fraction of porcine bran smooth microsomes showed a selective enrichment in a 180,000 mol wt protein. In an ELISA assay, this protein competed effectively--in binding anticlathrin--with clathrin extracted from a coated vesicle preparation. When the immunoadsorbed fraction was examined by electron microscopy, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles. Upon extensive dialysis (against MES buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin. This study demonstrates that anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles. In addition, the ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution.
Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin.
Authors: Wilcox D K , Kitson R P , Widnell C C
Published: May 2004

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase. The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated; the concentration of monensin required to inhibit pinocytosis (0.4 microM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion. Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by approximately 90% in monensin-treated cells. The effects of monensin were reversible: cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti-5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferase and 5'-nucleotidase increased from 1.128 to 1.148, and the median density of both N-acetyl-beta-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1.194 to 1.160. The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling. They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.
Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes.
Authors: Kreibich G , Ojakian G , Rodriguez-Boulan E , Sabatini D D
Published: May 2004

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.
Calcium movement and membrane potential changes in the early phase of neutrophil activation by phorbol myristate acetate: a study with ion-selective electrodes.
Authors: Mottola C , Romeo D
Published: May 2004

To quantitate calcium movements and membrane potential changes in stimulated neutrophils, we have measured net fluxes of Ca2+ and of the lipophilic cation tetraphenyl phosphonium by a very sensitive ion-selective electrode system. Activation of neutrophils by 3 X 10(-8) M phorbol 12-myristate, 13-acetate induces a release of approximately 20% of total cell calcium, with an initial lag period of less than 10 s. The Ca2+ outflux is markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurs by activation of the ATP-driven Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induces a transiently increased exchange of medium 45Ca with cell calcium, which is measurable a few seconds after cell exposure to the stimulant and peaks at approximately 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) results in a marked depolarization, with a lag period of approximately 60 s. The rate and extent of depolarization are reduced by 40 and 65%, respectively, in a low Na+ medium but are not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarizes neutrophils without either modifying their resting oxidative metabolism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibits no effect on the oxidative metabolism of neutrophils, does not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.
Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.
Authors: Marzella L , Ahlberg J , Glaumann H
Published: May 2004

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.
Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A.
Authors: Fisher K A
Published: May 2004

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely less than 2.5 X 10(-4) grains/microns 2/day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A (125I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane "halves." These data demonstrate that 125I-FITC-Con-A partitions exclusively with the extracellular "half" of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane "halves." This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se.
A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner.
Authors: Brown S S , Yamamoto K , Spudich J A
Published: May 2004

A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000-dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.
Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.
Authors: Roth J , Berger E G
Published: May 2004

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.
Monensin blocks the transport of diphtheria toxin to the cell cytoplasm.
Authors: Marnell M H , Stookey M , Draper R K
Published: May 2004

Lysosomotropic amines are believed to inhibit the transport of diphtheria toxin to the cell cytoplasm by raising the pH within intracellular vesicles. If so, then other drugs that dissipate intracellular proton gradients should have a similar effect on toxin transport. We found that monensin, a proton ionophore unrelated to lysosomotropic amines, is a potent inhibitor of the cytotoxic effect of diphtheria toxin. Monensin appears to block the escape of endocytosed toxin from a vesicle to the cytoplasm. Monensin fails to protect cells from the effects of diphtheria toxin that is bound to the cell surface and exposed to acidic medium, suggesting that the step normally blocked by the drug is circumvented under these conditions. The inhibition of toxin transport caused by monensin could not be relieved when monensin was replaced by ammonium chloride, nor when ammonium chloride was again replaced by monensin. This suggests that both drugs block the same step of toxin transport. The effect of monensin on the transport of diphtheria toxin to the cytoplasm is consistent with the proposal (Draper and Simon. 1980. J. Cell Biol. 87:849-854; Sandvig and Olsnes. 1980. J. Cell Biol. 87:828-832) that the toxin is endocytosed and then, in response to an acidic environment, penetrates through the membrane of an intracellular vesicle to reach the cytoplasm.
Quantification of ganglioside GM1 synthetase activity on intact chick neural retinal cells.
Authors: Pierce M
Published: May 2004

Neural retinal cells from 9-d-old chick embryos were assayed for uridine diphosphate (UDP)-galactose:ganglioside GM2 galactosyltransferase, or GM1 synthetase, activity using the oligosaccharide fragment of GM2, oligo-GM2, oligo-GM2, as the exogenous acceptor. The results demonstrated that this enzyme activity was present on the external surfaces of intact cells. Little difference between the specific activities of cell surface GM1 synthetase could be detected when cells derived from dorsal and ventral segments of the neural retina were compared. These results suggested that this cell-surface enzyme was not present in a concentration gradient along the dorsoventral axis of the neural retina.
Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac.
Authors: Clark C C , Crossland J , Kaplan G , Martinez-Hernandez A
Published: May 2004

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross-reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.
Analysis of DNA attached to the chromosome scaffold.
Authors: Kuo M T
Published: May 2004

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.
Participation of core histone "tails" in the stabilization of the chromatin solenoid.
Authors: Allan J , Harborne N , Rau D C , Gould H
Published: May 2004

We show here that the solenoid is maintained by the combination of linker histones and the nonglobular, highly basic "tails" of the core histones, which play only a minor part in the formation of the nucleosome core (Whitlock and Simpson, 1977. J. Biol. Chem. 252:6,516--6,520; Lilley and Tatchell, 1977. Nucleic Acids Res. 4:2,039--2,055; and Whitlock and Stein, 1978. J. Biol. Chem. 253:3,857--3,861). Polynucleosomes that contain core histones devoid of tails remain substantially unfolded under conditions otherwise favorable for the formation of solenoids. The tails can be replaced by extraneous basic polypeptides and in the presence of the linker histones the solenoid structure is then spontaneously recovered, as judged by a wide variety of structural criteria. The inference is that the core histone tail segments function by providing electrostatic shielding of the DNA charge and at the same time bridging adjacent nucleosomes in the solenoid. Our results carry the further implication that posttranscriptional modifications, such as acetylation of epsilon-amino groups, that reduce the positive charge of the core histone tails will tend to destabilize the higher-order structure and could thus render the DNA with which they are associated more readily available for transcription.
A model for the structure of chromatin in mammalian sperm.
Authors: Balhorn R
Published: May 2004

DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.
Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons
Authors: Chrispeels MJ , Higgins TJV , Spencer D
Published: May 2004

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm).

Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.
Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium.
Authors: Evinger-Hodges M J , Ewton D Z , Seifert S C , Florini J R
Published: May 2004

We have recently discovered that cells of Coon's Buffalo rat liver (BRL) line secrete a protein which is a potent inhibitor of skeletal myoblast differentiation in vitro. Using ion exchange and molecular exclusion chromatography, we have prepared this protein, which we designate "differentiation inhibitor" (DI), from the materials secreted by BRL cells maintained in serum-free medium. It is a relatively heat-stable protein which is inactivated by treatment with trypsin and mercaptoethanol and has an apparent molecular weight in the range 30,000--36,000. It exhibits no detectable mitogenic or lectin activity and differs from previously reported inhibitors of myoblast differentiation in several respects. It is active in all skeletal myoblast systems tested (Yaffe's L6 line as well as primary cultures of rat, chick, and Japanese quail myoblasts), and it blocks fusion, elevation of creatine kinase, and increased binding of alpha-bungarotoxin. Parallel fractionation of fetal bovine serum (FBS) and chick embryo extract (CEE) yields a peak of activity which similarly inhibits myoblast differentiation. We suggest that the differentiation inhibitor from BRL cells may correspond to the differentiation-inhibiting component(s) of FBS and CEE, and we call attention to the possibility that such a substance could play a role in embryonic growth of myoblasts and in satellite cell formation.
Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin
Authors: Nagata K , Sagara J , Ichikawa Y
Published: May 2004

A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography.

There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin.

The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline.

The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage.

By isoelectric focusing, the ratios of β- to γ-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.
Clogging of the glomerular basement membrane.
Authors: Kanwar Y S , Rosenzweig L J
Published: May 2004

The negative charges of the sulfated glycosaminoglycans (GAGs) of the glomerular basement membrane (GBM) were differentially neutralized by perfusin with high molarity buffers in order to determine whether or not these charges protect the GBM from being clogged by circulating plasma macromolecules. Progressive elimination of the negative charges resulted in clogging of the GBM by perfused native ferritin (NF) and bovine serum albumin as evidenced ultrastructurally by the increase in accumulation of NF in the GBM. In addition, the permeability of the GBM to 125I-insulin, a macromolecule which is normally freely permeable, and the glomerular filtration rate (as determined by [3H]inulin clearance) were markedly reduced after the GBM had been clogged with NF in the presence of high molarity buffer, thereby indicating that clogging severely reduces the ability of the GMB to act as a selective filter. These findings are consistent with the idea that the sulfated GAGs of the GBM serve as anticlogging agents.
Epidermal growth factor induces rapid centrosomal separation in HeLa and 3T3 cells.
Authors: Sherline P , Mascardo R N
Published: May 2004

Using indirect immunofluorescence, we have found that epidermal growth factor (EGF), at 100 ng/ml, induces centrosomal separation within 20 min in HeLa and 3T3 cells. The effect was evident both in unsynchronized cultures and in HeLa cells blocked in early S phase by hydroxyurea. EGF also induced centrosomal separation in quiescent 3T3 cells blocked in G0/G1 by serum deprivation, indicating that DNA replication is not necessary for this effect. The mechanism of this rapid centrosomal separation and its role in the mitogenic effects of EGF remains to be determined.
Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting.
Authors: Brunschwig J P , Brandt N , Caswell A H , Lukeman D S
Published: May 2004

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.
Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle.
Authors: Cadwell J J , Caswell A H
Published: May 2004

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38-42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.
Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.
Authors: Sarris A H , Palade G E
Published: May 2004

With the lithium diiodosalicylate (LIS1) extraction-phenol partition method, we have isolated a sialoglycoprotein fraction from DBA/2 mouse erythrocyte ghosts. We have demonstrated that the Laemmeli system for SDS PAGE can resolve this fraction into four monomers of which two (gp-2.1 and gp-3.1) appear to be authentic, whereas the other two (gp-2.2 and gp-3.2) are probably generated from gp-2.1 and gp-3.1, by limited proteolysis during the isolation procedure. All four components contain O-acetylated neuraminic acid residues, can be stained with Periodic acid-Schiff reagent (PAS) and with Coomassie Brilliant Blue (CB), and can be radioiodinated with the lactoperoxidase-glucose oxidase (LPO-GO) method. All monomers but especially gp-2.1 and gp-3.1 generate characteristic aggregates during solubilization in SDS. The aggregation is enhanced by boiling at high concentrations, and can be reversed by boiling at low concentrations. In addition, the fraction contains a diffuse component present also in ghosts which stains poorly with CB and with PAS and cannot be radioiodinated by the LPO-GO technique. SDS PAGE in the Steck and Yu gel system does not give an accurate separation of the sialoglycoprotein monomers.
A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction.
Authors: Ogihara S , Tonomura Y
Published: May 2004

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.
Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas.
Authors: Remillard S P , Witman G B
Published: May 2004

We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta-tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools.
Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin.
Authors: Tamir H , Theoharides T C , Gershon M D , Askenase P W
Published: May 2004

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.
Centrifugation shearing exposes filamentous networks in cortical regions of crane-fly spermatocytes.
Authors: Strauch A R , LaFountain J R
Published: May 2004

Spermatocytes of the crane-fly, Nephrotoma suturalis, were attached to electron microscope grids and then sheared by applying centrifugal force. Transmission electron microscopy of exposed regions of the cell cortex revealed networks containing arrays of filamentous structures. Networks were present in sheared spermatocytes at all stages of meiosis. The networks of dividing spermatocytes (meta- through telophase) were denser and appeared to contain more aggregated material then networks of prophase cells. The appearance of networks in spermatocytes resembled actin-containing networks of sheared and detergent-extracted human erythrocytes. Networks treated with myosin subfragment 1 under conditions in which muscle F-actin was clearly decorated were not distinguishable from those of untreated cells. Exposure to deoxyribonuclease-1 caused the disruption of networks in sheared spermatocytes as well as in erythrocytes. The results of deoxyribonuclease experiments are interpreted as an indication that actin is a component of the cell cortex in crane-fly spermatocytes.
Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor.
Authors: Fletcher W H , Byus C V
Published: May 2004

A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP-dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP-dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones.
Flagellar surface antigens in Euglena: immunological evidence for an external glycoprotein pool and its transfer to the regenerating flagellum.
Authors: Rogalski A A , Bouck G B
Published: May 2004

Antibodies raised against the Sarkosyl-insoluble, major flagellar glycoprotein fraction, mastigonemes, were used to determine the source of flagellar surface glycoproteins and to define the general properties of flagellar surface assembly in Euglena. After suitable absorption, mastigoneme antiserum reacts with several specific mastigoneme glycoproteins but does not bind either to the other major flagellar glycoprotein, xyloglycorien, or to other Sarkosyl-soluble flagellar components. When Fab' fragments of this mastigoneme-specific antiserum were used in combination with a biotin-avidin secondary label, antigen was localized not only on the flagellum as previously described but also in the contiguous reservoir region. If deflagellated cells are reservoir pulse-labeled with Fab' antibody, this antibody appears subsequently on the newly regenerated flagellum. This chased antibody is uniformly distributed throughout the length of the flagellum and shows no preferred growth zone after visualization with either fluorescein or ferritin-conjugated secondary label. From these and tunicamycin inhibition experiments it is concluded that (a) a surface pool of at least some flagellar surface antigens is present in the reservoir membrane adjacent to the flagellum and that (b) the reservoir antigen pool is transferred to the flagellar surface during regeneration.
Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels.
Authors: Gibson G J , Schor S L , Grant M E
Published: May 2004

Chick-embryo sternal chondrocytes have been cultured within three-dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.
Ultrastructure of clots during isometric contraction.
Authors: Cohen I , Gerrard J M , White J G
Published: May 2004

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension. The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction. Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i.e. fibrin). Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed. Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands. Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation. Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract. The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension. The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers.