bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts.
Authors: Moutsatsos I K , Davis J M , Wang J L
Published: May 2004

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.
Secretion of a cytoplasmic lectin from Xenopus laevis skin.
Authors: Bols N C , Roberson M M , Haywood-Reid P L , Cerra R F , Barondes S H
Published: May 2004

The skin of Xenopus laevis contains a soluble beta-galactoside-binding lectin with a approximately 16,000-mol-wt subunit. It resembles similar lectins purified from a variety of tissues from other vertebrates, and differs from two other soluble X. laevis lectins from oocytes and serum that bind alpha-galactosides. The skin lectin is concentrated in the cytoplasm of granular gland and mucous gland cells, as demonstrated by immunohistochemistry with the electron microscope. Upon injection with epinephrine, there is massive secretion of the cytoplasmic lectin from the granular gland cells.
Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling.
Authors: Patzak A , Winkler H
Published: May 2004

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.
Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization.
Authors: Menon A K , Holowka D , Webb W W , Baird B
Published: May 2004

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.
Effect of membrane splitting on transmembrane polypeptides.
Authors: Fisher K A , Yanagimoto K C
Published: May 2004

We investigated the effect of membrane splitting on the primary structure of human erythrocyte membrane polypeptides. Monolayers of intact, chemically unmodified cells were freeze-fractured and examined by one-dimensional SDS PAGE. Silver-stained gels revealed all major polypeptides that stain with Coomassie Blue as well as all bands that stain with periodic acid Schiff's reagent. Both nonglycosylated and glycosylated membrane polypeptides could be detected at concentrations of only a few nanograms per band. Membrane splitting had no effect on the position or number of bands. Monolayers of intact erythrocytes that had been enzymatically radioiodinated with lactoperoxidase were examined by electrophoresis, fluorography, and liquid scintillation counting. Radioactivity was quantified before and after monolayer formation and splitting, and at several stages of gel staining, drying, and fluorography. Although overexposed fluorographs revealed several minor radioiodinated bands in addition to band 3 and the glycophorins, no new bands were detected in split membrane samples derived from intact cells. These observations support the conclusion that neither the band 3 anion channel nor the glycophorin sialoglycoproteins are fragmented during freeze-fracturing. Although both band 3 and glycophorin partition to the cytoplasmic side of the membrane, preliminary quantitative observations suggest an enrichment of glycophorin in the split extracellular "half" membrane. We conclude that the process of membrane splitting by planar monolayer freeze-fracture does not cleave the covalent polypeptide backbone of any erythrocyte membrane protein, peripheral or integral.
Major loss of junctional coupling during mitosis in early mouse embryos.
Authors: Goodall H , Maro B
Published: May 2004

Junctional coupling was assessed during the transition from the fourth to the fifth cell cycle of mouse embryogenesis by injection of the dye carboxyfluorescein and by measurement of electrical continuity between cells. Junctional coupling, which arises de novo in early 8-cell mouse embryos, subsequently becomes reduced towards the end of the cell cycle as the blastomeres enter into mitosis. Arrest of the cell cycle in metaphase by nocodazole, an inhibitor of tubulin polymerization, reveals that cell coupling becomes undetectable at mitosis. Junctional coupling then is resumed during interphase of the 16-cell stage. Nocodazole itself has no effect on junctional coupling in interphase cells, regardless of the extent of intercellular flattening, whereas taxol, a microtubule-stabilizing agent, does reduce the extent of coupling in interphase cells.
Resting myosin cross-bridge configuration in frog muscle thick filaments.
Authors: Cantino M , Squire J
Published: May 2004

Clear images of myosin filaments have been seen in shadowed freeze-fracture replicas of single fibers of relaxed frog semitendinosus muscles rapidly frozen using a dual propane jet freezing device. These images have been analyzed by optical diffraction and computer averaging and have been modelled to reveal details of the myosin head configuration on the right-handed, three-stranded helix of cross-bridges. Both the characteristic 430-A and 140-150-A repeats of the myosin cross-bridge array could be seen. The measured filament backbone diameter was 140-160 A, and the outer diameter of the cross-bridge array was 300 A. Evidence is presented that suggests that the observed images are consistent with a model in which both of the heads of one myosin molecule tilt in the same direction at an angle of approximately 50-70 degrees to the normal to the filament long axis and are slewed so that they lie alongside each other and their radially projected density lies along the three right-handed helical tracks. Any perturbation of the myosin heads away from their ideal lattice sites needed to account for x-ray reflections not predicted for a perfect helix must be essentially along the three helical tracks of cross-bridges. Little trace of the presence of non-myosin proteins could be seen.
End-to-end annealing of microtubules in vitro.
Authors: Rothwell S W , Grasser W A , Murphy D B
Published: May 2004

Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte beta-tubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.
Immunofluorescence examination of beta tubulin expression and marginal band formation in developing chicken erythroblasts.
Authors: Murphy D B , Grasser W A , Wallis K T
Published: May 2004

Chicken erythrocyte beta tubulin, a tubulin variant with unique biochemical and assembly properties, is found to be specifically contained in two chicken blood cell types--erythrocytes and thrombocytes. The beta tubulin variant is absent or present in low amounts in a variety of white blood cell types and other body tissues, as determined by immunofluorescence microscopy and a semi-quantitative immunoblotting procedure. During differentiation in the marrow the beta tubulin variant appears suddenly in mid-stage erythroblasts at the onset of hemoglobin synthesis, and forming marginal bands are seen in all subsequent polychromatophilic erythroblast stages. The developmental sequence of events in marginal band formation entails microtubule nucleation at the centrosome, followed by microtubule elongation, consolidation of loose parallel microtubules into a compact bundle, and microtubule association with the cell membrane.
Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching.
Authors: Wadsworth P , Salmon E D
Published: May 2004

One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 micron wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 micron diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assembly-disassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33 degrees C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the half-spindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.
Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium.
Authors: Gilson C A , Ackland N , Burnside B
Published: May 2004

Teleost retinal cones elongate in the dark and contract in the light. In isolated retinas of the green sunfish Lepomis cyanellus, cone myoids undergo microtubule-dependent elongation from 5 to 45 micron. We have previously shown that cone contraction can be reactivated in motile models of cones lysed with Brij-58. Reactivated contraction is both actin and ATP dependent, activated by calcium, and inhibited by cAMP. We report here that we have obtained reactivated cone elongation in lysed models prepared by the same procedures. Reactivated elongation is ATP dependent, activated by cAMP, and inhibited by calcium. The rate of reactivated elongation is proportional to the cAMP concentration between 10 microM and 0.5 mM, but is constant between 10 microM and 1.0 mM Mg-ATP. No elongation occurs if cAMP or Mg-ATP concentration is less than or equal to 5 microM. Mg-ATP is required for both cAMP-dependent and cAMP-independent processes, suggesting that Mg-ATP is required both for a regulatory process entailing cAMP-dependent phosphorylation and for a force-producing process. Free calcium concentrations greater than or equal to 10(-7) reduce the elongation rate by 78% or more, completely inhibiting elongation at 10(-5) M. This inhibition is not due to competition from calcium-activated contraction. Cytochalasin D blocks reactivated contraction, but does not abolish calcium inhibition of reactivated elongation. Thus calcium directly affects the elongation mechanism. Calcium inhibition is calmodulin dependent. The calmodulin inhibitor trifluoperazine abolishes calcium inhibition of elongation. Furthermore, calcium blocks elongation only if present during the lysis step; subsequent calcium addition has no effect. However, if calcium plus exogenous calmodulin are subsequently added, elongation is again inhibited. Thus calcium inhibition appears to require a soluble calmodulin which is lost shortly after lysis.
Identification of a 34-kD polypeptide as a light chain of microtubule-associated protein-1 (MAP-1) and its association with a MAP-1 peptide that binds to microtubules.
Authors: Kuznetsov S A , Rodionov V I , Nadezhdina E S , Murphy D B , Gelfand V I
Published: May 2004

We examined the association of a 34-kD light chain component to the heavy chains of MAP-1 using a monoclonal antibody that specifically binds the 34-kD component and labels neuronal microtubules in a specific and saturable manner. Immunoprecipitation of MAP-1 heavy chains together with the 34-kD component by the antibody indicates that the 34-kD polypeptide forms a complex with MAP-1 heavy chains. Both major isoforms of MAP-1 heavy chains (MAP-1A and MAP-1B) were found in the immunoprecipitate. Digestion of MAP-1 with alpha-chymotrypsin and analysis of the chymotryptic peptides reveals a 120-kD fragment of the MAP-1 heavy chain that binds to microtubules and is precipitable with the 34-kD light chain antibody, suggesting that the 34-kD light chain also binds to this domain of the molecule. Since microtubules that contain the 120-kD fragment lack the long lateral projections characteristic of microtubules with intact MAP-1, the 34-kD light chains may be localized at or near the microtubule surface.
Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers.
Authors: Amato P A , Taylor D L
Published: May 2004

The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no detectable cellular polarity in the association of AF-actin with pre-existing stress fibers relative to perinuclear or peripheral regions. The rate of incorporation was quantified by image analysis of images generated with a two-dimensional photon counting microchannel plate camera. After equilibration of up to 2 h post-injection, FRAP demonstrated that actin subunits exchanged rapidly between filaments in stress fibers and the surrounding cytoplasm. When co-injected with rhodamine-labeled bovine serum albumin as a control, only actin was detected in the phase-dense stress fibers. The control protein was excluded from fibers and any linear fluorescence of the control was demonstrated as a pathlength artifact. The incorporation of AF-actin into stress fibers was studied by immunoelectron microscopy using anti-fluorescein as the primary antibody and goat anti-rabbit IgG coupled to peroxidase as the secondary antibody. At 5 min post-injection, reaction product was localized periodically in some fibers with a periodicity of approximately 0.75 microns. In large diameter fibers at 5 min post-injection, the analog was seen first on the surface of fibers, with individual filaments resolvable within the core. In the same cell, thinner diameter fibers were labeled uniformly throughout the diameter. By 20 min post-injection, most fibers were uniformly labeled. We conclude that the rate of actin subunit exchange in vivo is extremely rapid with molecular incorporation into actin filaments of stress fibers occurring as early as a few minutes post-injection. Exchange appears to first occur in filaments along the surface of stress fibers and then into more central regions in a periodic manner. We suggest that the periodic localization of actin at very early time points is due to a local microheterogeneity in which microdomains of fast vs. slower incorporation result from the periodic localization of actin-binding protein, such as alpha-actinin, along the length of the fiber.
A re-examination of the interaction of vinculin with actin.
Authors: Wilkins J A , Lin S
Published: May 2004

Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined.
Monoclonal antibodies distinguish titins from heart and skeletal muscle.
Authors: Hill C , Weber K
Published: May 2004

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.
Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration.
Authors: Kinsella M G , Wight T N
Published: May 2004

The rates of 35S-sulfate incorporation into proteoglycan were compared in multi-scratch wounded and confluent cultures of bovine aortic endothelial cells to determine whether proteoglycan synthesis is altered as cells are stimulated to migrate and proliferate. Incorporation was found to be stimulated in a time-dependent manner, reaching maximal levels 44-50 h after wounding, as cells migrated into wounded areas of the culture dish. Quantitative autoradiography of 35S-sulfate-labeled single-scratch wounded cultures demonstrated a 2-4-fold increase in the number of silver grains over migrating cells near the wound edge when compared to cells remote from the wound edge. Furthermore, when cell proliferation was blocked by inhibition of DNA synthesis, the increase in 35S-sulfate incorporation into proteoglycan after wounding was unaffected. These data indicate that cell division is not required for the modulation of proteoglycan synthesis to occur after wounding. Characterization of the newly synthesized proteoglycan by ion-exchange and molecular sieve chromatography demonstrated that heparan sulfate proteoglycan constitutes approximately 80% of the labeled proteoglycan in postconfluent cultures, while after wounding, chondroitin sulfate proteoglycan and/or dermatan sulfate proteoglycan (CS/DSPG) increases to as much as 60% of the total labeled proteoglycan. These results suggest that CS/DSPG synthesis is stimulated concomitant with the stimulation of endothelial cell migration after wounding.
Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins.
Authors: Cheresh D A , Pierschbacher M D , Herzig M A , Mujoo K
Published: May 2004

Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.
Commitment to expression of the metalloendopeptidases, collagenase and stromelysin: relationship of inducing events to changes in cytoskeletal architecture.
Authors: Werb Z , Hembry R M , Murphy G , Aggeler J
Published: May 2004

Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-O-tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 micrograms/ml CB for 3-24 h, or with various concentrations of CB (0-2 micrograms/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 micrograms/ml CB for only 3 h, greater than 85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immunofluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (less than 0.5 micrograms/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for collagenase and stromelysin gene expression.
Molecular assembly, secretion, and matrix deposition of type VI collagen.
Authors: Engvall E , Hessle H , Klier G
Published: May 2004

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.
Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.
Authors: Kordeli E , Cartaud J , Nghiêm H O , Pradel L A , Dubreuil C , Paulin D , Changeux J P
Published: May 2004

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiêm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.
Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.
Authors: Nicolet M , Pinçon-Raymond M , Rieger F
Published: May 2004

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.
Localization of Aplysia neurosecretory peptides to multiple populations of dense core vesicles.
Authors: Kreiner T , Sossin W , Scheller R H
Published: May 2004

Many neurons in the mollusc Aplysia are identifiable and provide a useful model system for investigating the cellular mechanisms used by the neuroendocrine system to mediate simple behaviors. In this study we determined the subcellular localization of eight Aplysia neuropeptides using immunogold labeling techniques, and analyzed the size distribution of dense core and granular vesicles in peptidergic neurons. Recent observations demonstrate that many neurons use multiple chemical messengers. Thus, an understanding of the functional significance of cotransmitters requires an analysis of their relative subcellular distributions. The peptides are expressed in a subset of neurons, or the exocrine atrial gland, and are primarily localized to dense core vesicles. Multiple regions of precursors which are cleaved into several components are co-localized. Each neuron has a distinct size distribution of peptide-containing dense core vesicles ranging in size from 65 to 600 nm. The atrial gland contains very large (up to 2 micron) peptide-containing granules. Single neurons have multiple populations of granules whose quantal sizes agree with predictions based on physical constraints. Some cells contain very large peptide-containing granules which are found in the cell soma and not in processes. Thus, the genetic determination of neuronal cell type includes not only transmitter choices but also multiple modes of packaging the intercellular messengers.
Selective effects of ascorbic acid on acetylcholine receptor number and distribution.
Authors: Knaack D , Shen I , Salpeter M M , Podleski T R
Published: May 2004

Ascorbic acid in soluble extracts of neural tissue can account for the increase in surface acetylcholine receptors (AChR's) seen on L5 myogenic cells treated with crude brain extract (Knaack, D., and T. R. Podleski, 1985, Proc. Natl. Acad. Sci. USA., 82:575-579). The present study further elucidates the nature of the response of L5 cells to ascorbic acid. Light autoradiography showed that ascorbic acid treatment affects both the number and distribution of surface AChR's. Ascorbic acid, like crude brain extracts, caused a three- to fourfold increase in average AChR site density. However, the number of AChR clusters induced by ascorbic acid was only one-fifth that observed with crude brain extract. The rate constant for degradation of AChR in ascorbic acid-treated cells of 0.037 +/- 0.006 h-1 (t1/2 = 19 h) was not significantly different from that in untreated controls of 0.050 +/- 0.001 h-1 (t1/2 = 14 h). The increase in AChR site density is primarily due to a 2.8-fold increase in the average rate of AChR incorporation. Ascorbic acid also stimulates thymidine incorporation and increases the total number of nuclei per culture. However, cellular proliferation is not responsible for the increase in AChR's since 10 microM cytosine arabinofuranoside blocks the mitogenic effect without affecting the AChR increase. The specificity of ascorbic acid on AChR expression was established by showing that (a) ascorbic acid produced only a slight increase in total protein, which can be accounted for by the mitogenic effect, and (b) the normal increase seen in creatine kinase activity during muscle differentiation was not altered by the addition of ascorbic acid. We conclude that the action of ascorbic acid on AChR number cannot be explained by changes in cell growth, survival, differentiation, or protein synthesis. Therefore, in addition to a minor stimulation of AChR clustering, ascorbic acid specifically affects some aspect of the AChR biosynthetic pathway.
Molecular specialization of astrocyte processes at nodes of Ranvier in rat optic nerve.
Authors: Ffrench-Constant C , Miller R H , Kruse J , Schachner M , Raff M C
Published: May 2004

The HNK-1 and L2 monoclonal antibodies are thought to recognize identical or closely associated carbohydrate epitopes on a family of neural plasma membrane glycoproteins, including myelin-associated glycoprotein, the neural cell adhesion molecule, and the L1 and J1 glycoproteins, all of which have been postulated to play a part in mediating cell-cell interactions in the nervous system. We have used these two antibodies in immunofluorescence and immunogold-electron microscopic studies of semithin and ultrathin frozen sections of adult rat optic nerve, respectively, and we show that they bind mainly to astrocyte processes around nodes of Ranvier. Most other elements of the nerve, including astrocyte cell bodies and large astrocytic processes, are not labeled by the antibodies. To our knowledge, this is the first demonstration that perinodal astrocyte processes are biochemically specialized. We provide evidence that one of the HNK-1+/L2+ molecules concentrated around perinodal astrocyte processes is the J1 glycoprotein; our findings, taken together with previously reported observations, suggest that the other known HNK-1+/L2+ molecules are not concentrated on these processes. Since anti-J1 antibodies previously have been shown to inhibit neuron to astrocyte adhesion in vitro, we hypothesize that J1 may play an important part in the axon-glial interactions that presumably are involved in the assembly and/or maintenance of nodes of Ranvier.
Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study.
Authors: Jiang L W , Schindler M
Published: May 2004

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.
Nerve-induced remodeling of muscle basal lamina during synaptogenesis.
Authors: Anderson M J
Published: May 2004

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.
Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells.
Authors: Presky D H , Schonbrunn A
Published: May 2004

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.
Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity.
Authors: Cutler D F , Garoff H
Published: May 2004

Three mutations of the membrane-binding region of the Semliki Forest virus (SFV) p62 polypeptide (the precursor for virion E3 and E2) have been made by oligonucleotide-directed mutagenesis of a cDNA clone encoding the SFV structural proteins. One of the mutations (A2) substitutes a Glu for an Ala in the middle of the hydrophobic stretch which spans the bilayer. A1 and A3 alter the two basic charged amino acids in the cytoplasmic domain next to the hydrophobic region. The wild-type charge cluster of Arg-Ser-Lys (+2) has been changed to Gly-Ser-Met (0;A3) or to Gly-Ser-Glu (-1;A1). The mutant p62 proteins have been analyzed both in the presence and the absence of E1, the other half of the heterodimer spike complex of SFV. The mutant proteins expressed in COS-7 cells are glycosylated and are of the expected sizes. When co-expressed with E1, all three mutants are cleaved to yield the E2 protein and transported to the surface of COS-7 cells. When expressed in the absence of E1, the mutant p62 proteins remain uncleaved but still reach the cell surface. Once at the cell surface, all three mutants, when co-expressed with E1, can promote low pH-triggered cell-cell fusion. These results show that the three mutant p62/E2 proteins are still membrane associated in a functionally unaltered way.