bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells.
Authors: Torrisi M R , Bonatti S
Published: May 2004

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.
Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.
Authors: Behrens J , Birchmeier W , Goodman S L , Imhof B A
Published: May 2004

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.
alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.
Authors: Pachter J S , Liem R K
Published: May 2004

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.
beta-Internexin, a ubiquitous intermediate filament-associated protein.
Authors: Napolitano E W , Pachter J S , Chin S S , Liem R K
Published: May 2004

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).
Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers.
Authors: Hoffman P N , Thompson G W , Griffin J W , Price D L
Published: May 2004

The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons.
Association of the epidermal growth factor receptor kinase with the detergent-insoluble cytoskeleton of A431 cells.
Authors: Landreth G E , Williams L K , Rieser G D
Published: May 2004

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble. Cytoskeletons prepared from EGF-treated A431 cells, when incubated with gamma-32P-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.
Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous.
Authors: Zervos A S , Hope J , Evans W H
Published: May 2004

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.
The distribution of tau in the mammalian central nervous system.
Authors: Binder L I , Frankfurter A , Rebhun L I
Published: May 2004

We have determined the biochemical and immunocytochemical localization of the heterogeneous microtubule-associated protein tau using a monoclonal antibody that binds to all of the tau polypeptides in both bovine and rat brain. Using immunoblot assays and competitive enzyme-linked immunosorbent assays, we have shown tau to be more abundant in bovine white matter extracts and microtubules than in extracts and microtubules from an enriched gray matter region of the brain. On a per mole basis, twice-cycled microtubules from white matter contained three times more tau than did twice-cycled microtubules from gray matter. Immunohistochemical studies that compared the localization of tau with that of MAP2 and tubulin demonstrated that tau was restricted to axons, extending the results of the biochemical studies. Tau localization was not observed in glia, which indicated that, at least in brain, tau is neuron specific. These observations indicate that tau may help define a subpopulation of microtubules that is restricted to axons. Furthermore, the monoclonal antibody described in this report should prove very useful to investigators studying axonal sprouting and growth because it is an exclusive axonal marker.
Myomesin and M-protein: expression of two M-band proteins in pectoral muscle and heart during development.
Authors: Grove B K , Cerny L , Perriard J C , Eppenberger H M
Published: May 2004

The expression of the myofibrillar M-band proteins myomesin and M-protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M-protein by 1.5 d. Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.
Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis.
Authors: Arndt-Jovin D J , Robert-Nicoud M , Baurschmidt P , Jovin T M
Published: May 2004

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.
The complement of desmosomal plaque proteins in different cell types.
Authors: Cowin P , Kapprell H P , Franke W W
Published: May 2004

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.
Polarized compartmentalization of organelles in growth cones from developing optic tectum.
Authors: Cheng T P , Reese T S
Published: May 2004

We have used computer-assisted reconstructions of continuous serial sections to study the cytoplasmic organization of growth cones in vivo. Optic tecta from 6.25-6.5-d-old chicken embryos were quick-frozen and then freeze-substituted in acetone-osmium tetroxide or, for comparison, prepared by conventional fixation. Images of eight freeze-substituted and two conventionally fixed growth cones were reconstructed from aligned serial micrographs. After freeze-substitution, numerous lumenless membrane-bound sacs arrayed in multilamellar stacks appear to replace the abundant smooth endoplasmic reticulum found after chemical fixation. Microtubule fascicles progressively diverge from their typical fascicular organization in the initial segment of the growth cone and are absent in the varicosity and the more distal segment. Mitochondria, in contrast, are concentrated in the proximal segment of the varicosity; multilamellar stacks and endosome-like vacuoles are in the distal segment; and coated pits and vesicles are concentrated near the terminal filopodium, which is the most distal and organelle-poor domain of the growth cone. These observations suggest that dilation and fusion of the lumenless, membrane-bound sacs that occurs during chemical fixation give rise to the network of smooth endoplasmic reticulum. The three-dimensional reconstructions show that the cytoplasmic components of growth cones, including the membrane-bound sacs and multilamellar stacks revealed by freeze substitution, are polarized along the axis of these growth cones, which suggests that they have a role in recycling of membrane during elongation of the growth cone.
Packing volume of sedimented microtubules: regulation and potential relationship to an intracellular matrix.
Authors: Brown P A , Berlin R D
Published: May 2004

To determine the contribution of microtubules to a hypothetical intracellular matrix, we have analyzed the space occupied by microtubules in vitro. Taxol-stabilized microtubules assembled from purified (three-times-cycled) bovine brain microtubule protein were pelleted by centrifugation under standardized conditions. The specific volume of the pellet, defined as the microliter volume per milligram protein, was 22.4. As suggested by others, this volume was strongly dependent on microtubule-associated proteins (MAPs), as shown by quantitation of the effects of purified MAP supplementation on specific volume. The specific volumes of microtubule pellets stripped of MAPs by high salt or chymotryptic digestion approached the mathematically optimal (least occupied space) and increased 14-fold with the highest MAP concentrations employed. Packing was also dependent on pH. Specific volumes comparable to those of MAP-depleted microtubules were attainable at pH's from 5.5 to 6.0, and specific volumes more than doubled at pH 7.5. MAP content was unaffected by pH. We present a theoretical analysis that suggests that as microtubules are centrifuged the mixture behaves as a liquid crystal. With packing, the mixture undergoes an isotropic-nematic phase transition in which the microtubules become oriented principally as parallel rods, mimicking their orientation in vivo. From the known concentration of microtubules in vivo, it can be inferred from our measurements that in some cells a large fraction, perhaps 40-50% of the cytosolic volume, is occupied by microtubules that form a mechanically irreducible space. Further theoretical analysis employing Ogston's formulation of the penetrability of fibrous networks suggests that the space between microtubules (in contrast to the extracellular matrix) imposes little barrier to the diffusion of macromolecules. A microtubule array thus achieves mechanical stability without affecting transport by diffusion. The space can accommodate other fibrous networks that could then affect transport, and, as we show, the space itself may be regulated by MAP content and intracellular pH.
Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization.
Authors: Francis G R , Waterston R H
Published: May 2004

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.
The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique.
Authors: Goodenough U W , Heuser J E
Published: May 2004

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.
Isolation of intermediate filament assemblies from human hair follicles.
Authors: Jones L N , Pope F M
Published: May 2004

We used developing human hair follicle cells for the isolation of hard alpha-keratin structural components. Intracellular dispersions examined by electron microscopy contained both individual alpha-keratin filaments and the tactoid-like filament assemblies observed in situ organized along subfibrillar arms of macrofibrils. The assemblies of average width 47 nm were composed of closely packed alpha-keratin filaments and originated from the initial filament arrays observed in sections of developing mammalian hair follicles. We have distinguished two types of assemblies: the para-like or hexagonally packed and the ortho-like spiral or whorl type. Axial banding extended across the width of filament assemblies, which suggested that hard alpha-keratin filaments pack in lateral register and form a lattice that contains interfilamentous bridges. We observed axial banding patterns with periods ranging from 20 to 22 nm, consistent with the 22-nm periodic structure deduced from x-ray diffraction studies and present in models proposed for hard alpha-keratin and other intermediate filaments. Preliminary biochemical studies of filaments and filament assemblies indicate that they consist of the closely related group of proteins (low-sulfur proteins) ubiquitous among extracts of hard mammalian keratins. Isolated hard alpha-keratin filament assemblies provide a new and valuable structural entity for investigating the assembly mechanisms involved in the formation of the filament-matrix framework found in hard mammalian keratin appendages.
Biochemical studies on cell fusion. I. Lipid composition of fusion-resistant cells.
Authors: Roos D S , Choppin P W
Published: May 2004

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.
Trophic effects of skeletal muscle extracts on ventral spinal cord neurons in vitro: separation of a protein with morphologic activity from proteins with cholinergic activity.
Authors: Smith R G , McManaman J , Appel S H
Published: May 2004

Protein factors derived from skeletal muscle separately promote neurite elongation and acetylcholine synthesis in cultured rat ventral spinal neurons. Morphologic factor activity (neurite-inducing activity) is specifically found in rat skeletal muscle and cord neuron extracts, decreases with the postnatal age of the rats from which muscle extract is prepared, and increases in rat hindlimb muscle after 5 d of denervation. Cholinergic factor activity (acetylcholine synthesis-stimulating activity) is found in extracts of rat cerebral cortex and cardiac muscle in addition to spinal cord and skeletal muscle, increases with animal age, and decreases following 5 d of denervation. Biochemically, the factors responsible for these activities differ in their lability to denaturing conditions, apparent molecular weights, isoelectric points, and lectin-binding specificities. Under reducing conditions, morphologic activity is isolated in a single acidic glycoprotein with an Mr of 35,000, while acetylcholine synthesis-stimulating activity is found in multiple species of different molecular weights. Thus, acetylcholine synthesis-promoting activities and neurite growth-promoting activity appear to reside in different molecules. Significant purification of several of these factors has been achieved.
A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II.
Authors: Schreiber A B , Kenney J , Kowalski J , Thomas K A , Gimenez-Gallego G , Rios-Candelore M , Di Salvo J , Barritault D , Courty J , Courtois Y
Published: May 2004

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.
Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures.
Authors: Miller J B , Crow M T , Stockdale F E
Published: May 2004

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.
An unusual lysosome compartment involved in vitellogenin endocytosis by Xenopus oocytes.
Authors: Wall D A , Meleka I
Published: May 2004

We have investigated the lysosomal compartment of Xenopus oocytes to determine the possible role of this organelle in the endocytic pathway of the yolk protein precursor, vitellogenin. Oocytes have lysosome-like organelles of unusual enzymatic composition at all stages of their development, and the amount of hydrolase activity increases steadily throughout oogenesis. These unusual lysosomes appear to be located primarily in a peripheral zone of oocyte cytoplasm. At least two distinct populations of lysosomal organelles can be identified after sucrose density gradient fractionation of vitellogenic oocytes. Most enzyme activity resides in a compartment of large size and high density that appears to be a subpopulation of yolk platelets that are less dense than most platelets within the cell. The appearance of this high density peak of lysosomal enzyme activity coincides with the time of onset of vitellogenin endocytosis during oocyte development. The data suggest that endocytic vesicles that contain vitellogenin fuse with modified lysosomes shortly after their internalization by the oocyte. Pulse-chase experiments with radiolabeled vitellogenin suggest that the ligand passes through the low density platelet compartment en route to the heavy platelets. The accumulation of yolk proteins apparently results from a failure of these molecules to undergo complete digestion after their entry into an unusual lysosomal compartment. The yolk platelets that these proteins finally enter for prolonged storage appear to be a postlysosomal organelle.
Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes.
Authors: Daukas G , Zigmond S H
Published: May 2004

We have found that hypertonic medium inhibited the receptor-mediated uptake of the chemotactic peptide N-formylnorleucylleucylphenylalanine without affecting fluid-phase endocytosis by polymorphonuclear leukocytes (PMNs). Morphological and biochemical evidence demonstrated that cells in hypertonic medium did not accumulate peptide in a receptor-mediated manner. However, the cells continued to form endosomes containing fluid-phase markers. Furthermore, the content of these endosomes was processed normally, i.e., both digested and intact material were released into the medium. The inhibition of receptor-mediated uptake was a function of the tonicity. Partial inhibition occurred in 0.45 and 0.6 osmolar medium and maximal inhibition occurred in 0.75 osmolar medium. The inhibition was independent of the solute used to increase the tonicity: sodium chloride, sucrose, and lactose all inhibited uptake to similar extents. Hypertonic medium had little effect on saturable peptide binding. However, it did prevent the clustering of surface molecules as indicated by the inhibition of capping of fluorescent concanavalin A. In addition, hypertonic medium prevented the peptide-stimulated increase in cytosolic calcium levels as measured by quin 2 fluorescence. The tonicity dependence of the inhibition of quin 2 fluorescence paralleled the inhibition of receptor-mediated uptake.
Wheat germ agglutinin blocks the biological effects of nerve growth factor.
Authors: Landreth G E , Williams L K , McCutchen C
Published: May 2004

The binding of nerve growth factor (NGF) to specific cell surface receptors initiates a variety of effects that lead to the morphological and biochemical differentiation of clonal pheochromocytoma, PC12, cells. The lectin wheat germ agglutinin (WGA) alters the characteristics of NGF-receptor interaction. We have found that treatment of PC12 cells with WGA dramatically and reversibly inhibits the ability of NGF to elicit three distinct biological effects characteristic of NGF action. Two of these events, the rapid ruffling of cell-surface membranes and the stimulation of the phosphorylation of a 250-kD cytoskeletal protein in situ, occur rapidly and are an immediate consequence of receptor occupancy. Both of these effects are blocked by pretreatment of the cells with WGA. WGA was also found to inhibit the NGF-stimulated regeneration of neurites that occurs over 1-2 d. Both the WGA inhibition of neurite outgrowth and the phosphorylation of the 250-kD cytoskeletal protein were reversed upon addition of the specific sugar N-acetylglucosamine. These data demonstrate that the WGA-induced changes in the NGF-receptor interaction reflect important alterations in the ability of the receptor to transmit biological signals, resulting in the abrogation of the biological effects of NGF on these cells.
Gap junction structures after experimental alteration of junctional channel conductance.
Authors: Miller T M , Goodenough D A
Published: May 2004

Gap junctions are known to present a variety of different morphologies in electron micrographs and x-ray diffraction patterns. This variation in structure is not only seen between gap junctions in different tissues and organisms, but also within a given tissue. In an attempt to understand the physiological meaning of some aspects of this variability, gap junction structure was studied following experimental manipulation of junctional channel conductance. Both physiological and morphological experiments were performed on gap junctions joining stage 20-23 chick embryo lens epithelial cells. Channel conductance was experimentally altered by using five different experimental manipulations, and assayed for conductance changes by observing the intercellular diffusion of Lucifer Yellow CH. All structural measurements were made on electron micrographs of freeze-fracture replicas after quick-freezing of specimens from the living state; for comparison, aldehyde-fixed specimens were measured as well. Analysis of the data generated as a result of this study revealed no common statistically significant changes in the intrajunctional packing of connexons in the membrane plane as a result of experimental alteration of junctional channel conductance, although some of the experimental manipulations used to alter junctional conductance did produce significant structural changes. Aldehyde fixation caused a dramatic condensation of connexon packing, a result not observed with any of the five experimental uncoupling conditions over the 40-min time course of the experiments.
Analysis of the microtubule-binding domain of MAP-2.
Authors: Gottlieb R A , Murphy D B
Published: May 2004

We examined the microtubule-binding domain of the microtubule-associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule-binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule.
Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors.
Authors: Drubin D G , Feinstein S C , Shooter E M , Kirschner M W
Published: May 2004

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.
Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.
Authors: Roos E , Spiele H , Feltkamp C A , Huisman H , Wiegant F A , Traas J , Mesland D A
Published: May 2004

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.
Zeugmatin: a new high molecular weight protein associated with Z lines in adult and early embryonic striated muscle.
Authors: Maher P A , Cox G F , Singer S J
Published: May 2004

Monoclonal antibodies were generated to a purified preparation of the fascia adherens domains of the intercalated discs of chicken cardiac cell membranes. One of these antibodies, McAb 20, immunofluorescently labeled the Z lines of adult skeletal muscle, the Z lines and intercalated discs of adult cardiac muscle, and the dense bodies and dense plaques of adult gizzard smooth muscle. In addition, McAb 20 was found to label regenerating muscle cells in a cross-striated pattern much like that of Z lines in 24-h muscle cell cultures before the appearance of Z lines was detectable by phase or Nomarski optics and before the concentration of alpha-actinin occurred at the Z lines. Thus, McAb 20 appears to be directed against an antigen involved in early myofibrillar organization. Preliminary biochemical characterization of the antigen recognized by McAb 20 indicates that it is a high molecular weight doublet of over 5 X 10(5) kD that is highly susceptible to proteolysis. By virtue of its presence in Z lines, and its possible role in the end-on attachment of microfilaments to Z lines and membranes, we have named this protein zeugmatin (xi epsilon nu gamma mu alpha identical to yoking).
Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.
Authors: Sellers J R , Spudich J A , Sheetz M P
Published: May 2004

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.