bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescent morphological probe for hyaluronate.
Authors: Knudson C B , Toole B P
Published: May 2004

Hyaluronate levels change dramatically during morphogenesis of various tissues and organs. Morphological detection of the exact temporal and spatial distribution patterns of hyaluronate may help to elucidate its role in morphogenesis. Since no specific direct method for visualizing hyaluronate with the light or electron microscope is currently available, we have developed a morphological probe by exploiting the high-affinity interaction of cartilage proteoglycan with hyaluronate. The core protein of this proteoglycan consists of a region that binds specifically to hyaluronate with a high association constant, and a region to which the majority of sulfated polysaccharide chains are covalently attached. The polysaccharide chains were removed by treatment with chondroitinase ABC, and the core protein, labeled with rhodamine, was used as the probe. This fluorescent probe binds reversibly and specifically to [3H]hyaluronate in a binding assay using ammonium sulfate precipitation of the core protein. The probe has been used to visualize the cell surface hyaluronate of rat fibrosarcoma cells, 3T3 cells, and SV-40 transformed 3T3 cells, three cell types with significantly different amounts of cell surface-associated hyaluronate.
Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum.
Authors: Mierendorf R C , Cardelli J A , Dimond R L
Published: May 2004

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.
Slow posttranslational modification of a neurofilament protein.
Authors: Bennett G S , DiLullo C
Published: May 2004

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.
Discoidin I, an endogenous lectin, is externalized from Dictyostelium discoideum in multilamellar bodies.
Authors: Barondes S H , Haywood-Reid P L , Cooper D N
Published: May 2004

Discoidin I, a soluble lectin synthesized by aggregating Dictyostelium discoideum and implicated in their adhesion to the substratum, is localized in multilamellar bodies both intracellularly and upon externalization. These structures also contain a glycoconjugate that binds discoidin I. The multilamellar bodies apparently serve to package the lectin for externalization, and may then gradually release it to function extracellularly.
The microsporidian spore invasion tube. IV. Discharge activation begins with pH-triggered Ca2+ influx.
Authors: Pleshinger J , Weidner E
Published: May 2004

The microsporidian spore extrusion apparatus activates with a calcium influx from Spraguea lophii spore wall/plasma membrane; this influx requires preconditioning with an extrasporular shift in medium pH to the alkaline in the presence of the polyanions mucin or polyglutamate. Undischarged S. lophii spores display calcium bound to the wall/plasma membrane with a characteristic calcium-chlorotetracycline fluorescence; this fluorescence declines significantly during spore discharge. S. lophii spores do not discharge when spore wall/plasma membrane calcium is removed with EGTA. Extrasporular mucin or polyglutamate and a pH shift to the alkaline appear to be necessary preconditions for the triggering of the influx of spore wall/plasma membrane-bound 45Ca2+. Ionophore A-23187 also effectively activates spore discharge without other extrasporular polyanions. Micromolar concentrations of the calcium antagonists lanthanum or verapamil prevent spore discharge, and micromolar concentrations of calmodulin inhibitors chlorpromazine and trifluroperazine prevent spore discharge. Calmodulin, visualized with a calmodulin antibody and a peroxidase conjugate, is localized particularly on the plasma membrane and the polaroplast membranes of the extrusion apparatus.
Glycoproteins of the lysosomal membrane.
Authors: Lewis V , Green S A , Marsh M , Vihko P , Helenius A , Mellman I
Published: May 2004

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and/or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100- and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and endo-beta-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple N-linked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pI of the 120-kD protein from less than 4 to greater than 8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins lgp120, lgp100, lgp80.
Effect of taxol on secretory cells: functional, morphological, and electrophysiological correlates.
Authors: Thuret-Carnahan J , Bossu J L , Feltz A , Langley K , Aunis D
Published: May 2004

The effect of 0.5-1.0 microM taxol, a potent promoter of microtubule polymerization in vitro, was studied on the secretory activity of chromaffin cells of the adrenal medulla. Taxol was found to have a dual effect: the long-term effect (after a 1-h incubation) of taxol was to induce almost complete inhibition of catecholamine release, whereas after a short incubation (10 min) a massive, nicotine-independent release of catecholamine was produced. From results obtained using the patch-clamp technique to study the Ca++-dependent K+ channels (Ic channels), it was possible to conclude that taxol probably provokes an augmentation of free [Ca++]i in the cytoplasm, values increasing from 10(-8) M at rest to several 10(-7) M. The increased spontaneous release of stored neurohormones and the increased frequency of opening of Ic channels occur simultaneously and could both originate from a rise of [Ca++]i upon taxol addition. Immunofluorescence and ultrastructural studies showed that 13-h taxol treatment of chromaffin cells led to a different distribution of secretory organelles, and also to microtubule reorganization. In treated cells, microtubules were found to form bundles beneath the cell membrane and, at the ultrastructural level, to be packed along the cell axis. It is concluded that in addition to its action on microtubules, the antitumor drug taxol has side effects on the cell secretory activity, one of them being to modify free [Ca++]i.
Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.
Authors: Siegel V , Walter P
Published: May 2004

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.
Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.
Authors: McEwen B F , Arion W J
Published: May 2004

Pathogenic staphylococci secrete a number of exotoxins, including alpha-toxin. alpha-Toxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used alpha-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage less than or equal to 22%. Treatment conditions included incubation for 5-10 min at 37 degrees C and pH 7.0 with an alpha-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml alpha-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, alpha-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.
Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components.
Authors: Skinner M K , Tung P S , Fritz I B
Published: May 2004

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.
Characterization of a 36,000-dalton protein from the surface of Madin-Darby canine kidney cells involved in cell attachment and spreading.
Authors: Sabanero M , Gonzalez-Robles A , Meza I
Published: May 2004

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.
Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella.
Authors: Goodenough U W , Heuser J E
Published: May 2004

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.
Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.
Authors: Takano-Ohmuro H , Obinata T , Kawashima M , Masaki T , Tanaka T
Published: May 2004

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.
Dictyostelium amebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP.
Authors: Varnum B , Edwards K B , Soll D R
Published: May 2004

Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.
Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers.
Authors: Wagner D D , Mayadas T , Urban-Pickering M , Lewis B H , Marder V J
Published: May 2004

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.
Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method.
Authors: Valberg P A , Albertini D F
Published: May 2004

The motions of magnetic particles contained within organelles of living cells were followed by measuring magnetic fields generated by the particles. The alignment of particles was sensed magnetometrically and was manipulated by external fields, allowing non-invasive detection of particle motion as well as examination of cytoplasmic viscoelasticity. Motility and rheology data are presented for pulmonary macrophages isolated from lungs of hamsters 1 d after the animals had breathed airborne gamma-Fe2O3 particles. The magnetic directions of particles within phagosomes and secondary lysosomes were aligned, and the weak magnetic field produced by the particles was recorded. For dead cells, this remanent field was constant, but for viable macrophages, the remanent field decreased rapidly so that only 42% of its initial magnitude remained 5 min after alignment. A twisting field was applied perpendicular to the direction of alignment and the rate at which particles reoriented to this new direction was followed. The same twisting was repeated for particles suspended in a series of viscosity standards. Based on this approach, the low-shear apparent intracellular viscosity was estimated to be 1.2-2.7 X 10(3) Pa.s (1.2-2.7 X 10(4) poise). Time-lapse video microscopy confirmed the alignment of ingested particles upon magnetization and showed persistent cellular motility during randomization of alignment. Cytochalasin D and low temperature both reduced cytoplasmic activity and remanent-field decay, but affected rheology differently. Magnetic particles were observed in association with the microtubule organizing center by immunofluorescence microscopy; magnetization did not affect microtubule distribution. However, both vimentin intermediate filaments and f-actin reorganized after magnetization. These data demonstrate that magnetometry of isolated phagocytic cells can probe organelle movements, rheology, and physical properties of the cytoskeleton in living cells.
An efficient method for introducing macromolecules into living cells.
Authors: Doxsey S J , Sambrook J , Helenius A , White J
Published: May 2004

The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.
SERGE, the subcellular site of initial hepatic glycogen deposition in the rat: a radioautographic and cytochemical study.
Authors: Cardell R R , Michaels J E , Hung J T , Cardell E L
Published: May 2004

Hormonal control of hepatic glycogen and blood glucose levels is one of the major homeostatic mechanisms in mammals: glycogen is synthesized when portal glucose concentration is sufficiently elevated and degraded when glucose levels are low. We have studied initial events of hepatic glycogen synthesis by injecting the synthetic glucocorticoid dexamethasone (DEX) into adrenalectomized rats fasted overnight. Hepatic glycogen levels are very low in adrenalectomized rats, and DEX causes rapid deposition of the complex carbohydrate. Investigation of the process of glycogen deposition was performed by light and electron microscopic (EM) radioautography using [3H]galactose as a glycogen precursor. Rats injected with DEX for 2-3 h and [3H]galactose one hour before being killed displayed an increasing number of intensely labeled hepatocytes. EM radioautography revealed silver grains over small (+/- 1 micron) ovoid or round areas of the cytosome that were rich in smooth endoplasmic reticulum (SER) and contained a high concentration of small dense particles. These distinct areas or foci of SER and presumptive glycogen (SERGE) were most numerous during initial periods of glycogen synthesis. After longer exposure to DEX (4-5 h) more typical deposits of cytoplasmic glycogen were evident in the SERGE regions. Several criteria indicated that the SERGE foci contained glycogen or presumptive glycogen: resemblance of the largest dense particles to beta-glycogen particles in EM; association of 3H-carbohydrate with the foci; removal of particles and label with alpha-amylase; and positive reaction with periodic acid-chromic acid-silver methenamine. The concentration of SER in the small foci and the association of newly formed glycogen particles with elements of SER suggest a role for this organelle in the initial synthesis of glycogen.
Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin.
Authors: Chantler P D
Published: May 2004

A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell.
Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development.
Authors: Gibson G J , Flint M H
Published: May 2004

Our morphological studies have demonstrated that the appearance of localized, paired zones of primary calcification on either side of the midline of the 19-d embryonic chick sternum is heralded by the development of paired, translucent zones 2 d previously. Histological studies demonstrated that the majority of chondrocytes within these translucent zones are hypertrophic, and that the zones are surrounded by a margin of flattened nonhypertrophic cells. The discrete localization of these paired areas of hypertrophic chondrocytes and subsequent endochondral bone development allows for the direct correlation of the histological and biochemical characteristics of the zones sequentially during development and makes it possible to precisely match the synthetic activity to the cellular morphology, thereby eliminating possible minor but critical variations in developmental staging that could otherwise arise. Our studies have demonstrated that there is a direct spatial and temporal correlation between the degree of cellular maturation and the synthesis of type X collagen, and that the sudden and profound initiation of type X collagen synthesis on days 16-17 of development occurs concurrently with the attainment of hypertrophic characteristics by the majority of cells within the translucent zone. Before acquisition of these hypertrophic characteristics, the cells of this precalcification zone synthesize only type II and the minor cartilage collagens. Chondrocytes isolated from these regions in more immature sternae (i.e., 11+ d embryos) were found to synthesize high levels of type X collagen within 4 d of culture within collagen gels even though hypertrophic development and type X collagen synthesis by cells within this region would not normally have been apparent in ovo for several more days. These data indicate that there is a direct correlation between the development of hypertrophic characteristics and the synthesis of type X collagen, and that the maturation of chondrocytes in precalcification zones may be regulated by matrix components and/or stimulated by culture within collagen gels.
Individual neural cell types express immunologically distinct N-CAM forms.
Authors: Williams R K , Goridis C , Akeson R
Published: May 2004

The neural cell adhesion molecules, or N-CAMs, are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000, 140,000, and 120,000. In this article we identify, using monoclonal antibody (Mab) 3G6.41, an immunologically distinct adult rat N-CAM form and show that this form is selectively expressed by some clonal neural cell lines. Consecutive immunoprecipitation experiments indicate that rabbit anti-N-CAM can remove from solubilized cerebellar neuron primary cultures all 180,000- and 140,000-mol-wt N-CAM molecules that react with Mab 3G6.41. However Mab 3G6.41 cannot remove all N-CAM molecules that react with rabbit anti-N-CAM. Rabbit anti-N-CAM binds to and immunoprecipitates N-CAM forms from the rat neuronal cell lines B35, B65, and B104, the glial lines B12 and C6, and L6 myoblasts. Mab 3G6.41 does not bind to or immunoprecipitate N-CAM from the B12 and B65 lines but does react with the other four lines by both criteria. Many cells in primary cultures of postnatal rat that express glial fibrillary acidic protein also bind Mab 3G6.41. Thus a unique form of rat N-CAM recognized by Mab 3G6.41 is found on some but not all neuronal, glial, and muscle cells.
Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate.
Authors: Zanetti M , Ratcliffe A , Watt F M
Published: May 2004

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.
Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus.
Authors: Ono M , Mifune K , Yoshimura A , Ohnishi S , Kuwano M
Published: May 2004

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.
Identification of two lysosomal membrane glycoproteins.
Authors: Chen J W , Murphy T L , Willingham M C , Pastan I , August J T
Published: May 2004

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.
Evidence for an involvement of actin in the positioning and motility of centrosomes.
Authors: Euteneuer U , Schliwa M
Published: May 2004

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.
Effects of swainsonine on rat liver and kidney: biochemical and morphological studies.
Authors: Novikoff P M , Touster O , Novikoff A B , Tulsiani D P
Published: May 2004

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.
Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta.
Authors: Maruyama I , Bell C E , Majerus P W
Published: May 2004

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.
Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.
Authors: Bartnik E , Osborn M , Weber K
Published: May 2004

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.
Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells.
Authors: Pfeiffer S , Fuller S D , Simons K
Published: May 2004

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.
Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.
Authors: Vantard M , Lambert A M , De Mey J , Picquot P , Van Eldik L J
Published: May 2004

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.