bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.
Authors: Singer I I , Kawka D W , Kazazis D M , Clark R A
Published: May 2004

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.
Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila.
Authors: Wenkert D , Allis C D
Published: May 2004

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Earlier studies ( Allis , C. D., C. V. C. Glover , J. K. Bowen, and M. A. Gorovsky , 1980, Cell, 20:609-617; and Allis , C. D., Y. S. Ziegler , M. A. Gorovsky , and J. B. Olmsted, 1982, Cell, 31:131-136) demonstrated the existence of a macronuclear-specific histone variant, hv1 , which is enriched in small punctate regions in nucleoli of several mammalian cell lines. These observations suggest that this histone variant is highly conserved in evolution and may be associated with actively transcribed sequences. Despite large differences in structure and function during vegetative growth, macro- and micronuclei are related. During conjugation, the sexual phase of the life cycle in Tetrahymena, postzygotic division products of micronuclei give rise to new micro- and macronuclei, while the old macronucleus moves to the posterior of each cell and is eliminated. In this study using antiserum specific for hv1 , we determined by indirect immunofluorescence the time during conjugation at which hv1 first appears in the developing new macronuclei. In growing, starved, and young mating cells (2-5 h after mixing opposite mating types), only macronuclei are detected with affinity-purified antibodies against hv1 . Newly formed macronuclei are either not stained or only weakly stained in cells in which the old macronucleus is located in the center of the cell. However, new macronuclei are clearly observed in cells in which the old macronucleus has moved to the posterior of the cell (approximately 8 h). During later stages of conjugation (10-16 h), the intensity of hv1 staining in new macronuclei increases with time corresponding to the increasing DNA content of these nuclei. Disappearance of detectable hv1 from old macronuclei begins nearly 1 h after these nuclei reach the posterior cytoplasm (approximately 9-10 h) and is sometimes complete before these nuclei are eliminated from the cells. Autoradiography of cells labeled for brief periods with [3H]uridine shows that new macronuclei begin to synthesize RNA very soon after the second postzygotic division (approximately 8 h). During stages when hv1 is clearly detected in new macronuclei, anlagen are active in RNA synthesis. RNA synthesis in old macronuclei ceases very close to the time when RNA synthesis begins in new macronuclei. Thus, the addition of hv1 coincides closely with the transformation of a transcriptionally inactive germinal nucleus into that of a transcriptionally active somatic nucleus. We suspect that addition of hv1 plays a fundamental role in
Properties of the 120,000- and 95,000-dalton actin-binding proteins from Dictyostelium discoideum and their possible functions in assembling the cytoplasmic matrix.
Authors: Condeelis J , Vahey M , Carboni J M , DeMey J , Ogihara S
Published: May 2004

The cell cortex of Dictyostelium amebae contains an actin-rich cytoplasmic matrix. Changes in geometry of this matrix are believed to regulate protrusive activity and motility of the cell cortex. Two actin-binding proteins (120,000 and 95,000 daltons [120K and 95K]) are present in the cell cortex, and their properties, many of which are described here for the first time, suggest that they regulate growth and organization of cortical microfilaments. The 120K protein is a flexible dimer 35 nm in length with a native molecular mass of 241,000. It nucleates the polymerization of actin and crosslinks the filaments to form branched networks like those seen in situ in the cell cortex. The production of a branched network of short crosslinked filaments results in a lattice that would theoretically generate the maximum rigidity with minimum amount of polymer. This sort of lattice would be very useful as a space-filling cytoskeleton capable of resisting deformation. The 120K protein inhibits the actin-stimulated Mg ATPase of myosin. Competition for actin binding between 120K and myosin, the impenetrability of the 120K-actin network to myosin, and the rigidity of actin filaments that are crosslinked by 120K could all contribute to the decrease in the actin-stimulated Mg ATPase of myosin. The properties of 120K are consistent with a role for this protein in regulating the site of actin filament growth and gelation in the cell but not the assembly of actin-containing structures that would participate in force generation by a sliding-filament mechanism involving myosin. The 95K protein is a rigid dimer 40 nm in length with a native molecular mass of between 190,000 and 210,000. Its physical and antigenic properties lead us to conclude that the 95K protein is Dictyostelium alpha-actinin. Unlike 120K, it crosslinks actin filaments into lateral arrays and increases the actin-stimulated Mg ATPase of myosin. Both activities are regulated by Ca2+. The properties of 95K are consistent with a role in organizing actin filaments in the cell into lateral arrays that are capable of efficient interaction with myosin to produce force for cell motility.
Intracellular water and the cytomatrix: some methods of study and current views.
Authors: Clegg J S
Published: May 2004

The extent to which the properties of water in cells are like those of water in dilute aqueous solutions is a question of broad significance to cell biology. A detailed answer is not available at present, although evidence is accumulating that the properties of at least a large fraction of intracellular water are altered by interactions with cell ultrastructure, notably the cytomatrix. That and related evidence also suggests that the properties, composition, and activities of the "aqueous cytoplasm" of intact cells bear little resemblance to those of the "cytosol" obtained by cell fractionation. This paper will consider some of the evidence for these possibilities and some of their potential consequences with regard to cellular structure and function.
Diffusion in the aqueous compartment.
Authors: Mastro A M , Keith A D
Published: May 2004

Measurements of diffusion of molecules in cells can provide information about cytoplasmic viscosity and structure. In a series of studies electron-spin resonance was used to measure the diffusion of a small spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity of the cytoplasm from both rotational and translational motion would distinguish between the effects of viscosity and structure on diffusion. The diffusion constant measured in several cell lines averaged 3.3 X 10(-6) cm2/s. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion constant or the rotational correlation time was 2.0-3.0 centipoise, about two to three times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiologic conditions. However, when cells were subjected to hypertonic conditions, the translational motion of the spin label decreased threefold, whereas the rotational motion changed by less than 20%. These data suggest that the decrease in cell volume under hypertonic conditions is accompanied by an increase in cytoplasmic barriers and a decrease in the space between existing cytoplasmic components without a significant increase in viscosity in the aqueous phase. In addition, a comparison of reported diffusion values of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.
Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes.
Authors: Zentgraf H , Franke W W
Published: May 2004

Fractions of homogeneously-sized supranucleosomal particles can be obtained in high yield and purity from various types of cells by brief micrococcal nuclease digestion (10 or 20 s) of condensed chromatin in 100 mM NaCl followed by sucrose gradient centrifugation and agarose gel electrophoresis. These chromatin particles, which contain only DNA and histones, differed according to cell type. Sea urchin spermatozoa (Paracentrotus lividus) gave rise to heavy particles (ca. 260 S) with a mean diameter (48 nm). These resembled the unit chromatin fibrils fixed in situ, which contain an average of 48 nucleosomes, as determined both by electron microscopy after unraveling in low salt buffer and gel electrophoresis. In contrast, higher order particles from chicken erythrocyte chromatin were smaller (105 S; 36-nm diam) and contained approximately 20 nucleosomes. The smallest type of supranucleosomal particle was obtained from chicken and rat liver (39 S; 32-nm diam; eight nucleosomes). Oligomeric chains of such granular particles could be recognized in regions of higher sucrose density, indicating that distinct supranucleosomal particles of globular shape are not an artifact of exposure to low salt concentrations but can be obtained at near-physiological ionic strength. The demonstration of different particle sizes in chromatin from different types of nuclei is contrary to the view that such granular particles are produced by artificial breakdown into "detached turns" from a uniform and general solenoid structure of approximately six nucleosomes per turn. Our observations indicate that the higher order packing of the nucleosomal chain can differ greatly in different types of nuclei and the supranucleosomal organization of chromatin differs between cell types and is related to the specific state of cell differentiation.
Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat.
Authors: Steer C J , Bisher M , Blumenthal R , Steven A C
Published: May 2004

We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. Sci. USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions. The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response. Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver. Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes.
Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine.
Authors: Brown W J , Constantinescu E , Farquhar M G
Published: May 2004

The distribution of Man-6-P receptors was determined by immunoperoxidase cytochemistry in Clone 9 hepatocytes cultured in the presence or absence of tunicamycin and chloroquine, agents that perturb lysosomal enzyme sorting and lead to their secretion. In control (untreated) cells, receptors were localized in cis Golgi cisternae, coated vesicles, and in endosomes or lysosomes. After tunicamycin treatment, receptors were found in coated vesicles lined up along the cis cisternae but were not detected in endosomes or lysosomes. After chloroquine treatment, receptors were localized in large vacuolated endosomes or lysosomes but were not usually detected in Golgi cisternae or in coated vesicles. These results demonstrate a redistribution of receptors along the normal Man-6-P-dependent sorting pathway after these treatments. In ligand-deficient (tunicamycin-treated) cells, immunoreactive receptors accumulate at the presumptive sorting site in the cis Golgi and are depleted from endosomes and lysosomes. When the intralysosomal pH is increased (by chloroquine treatment) preventing ligand-receptor dissociation, receptors accumulate at the presumptive delivery site (lysosomes and endosomes) and are depleted from the cis Golgi region. The findings also suggest that (a) ligand binding triggers movement of the receptor to endosomes or lysosomes, and (b) ligand dissociation triggers their movement back to the cis Golgi region.
Comparison of the protein content of three different bovine secretory granule membrane types: a search for exocytosis-specific shared proteins.
Authors: Rubin R W , Lyubkin A K , Pressman B C
Published: May 2004

A two-dimensional polyacrylamide gel analysis of three types of bovine exocytotic granule membranes has been undertaken. Great care was taken to purify the membranes of biochemical homogeneity with minimal contamination from other membrane sources. The goal was to identify proteins that were present in all three membrane types. Although a number of minor components were observed that co-migrated for two membrane types, no proteins were detected that were present in all three granule membranes. We therefore conclude that such exocytosis-specific proteins do not exist or that they represent less than 0.1% of the total membrane protein present in a given isolated membrane preparation.
The higher-order structure of chromatin: evidence for a helical ribbon arrangement.
Authors: Woodcock C L , Frado L L , Rattner J B
Published: May 2004

Both intact and nuclease-isolated chromatin fibers have been examined at different degrees of salt-induced compaction, using a variety of preparation techniques. The results suggest that the initial folding step in nucleosome packing involves the formation of a zig-zag ribbon as has been proposed by others (Thoma F., T. Koller, and A. Klug, 1979, J. Cell Biol., 83:403-427; Worcel A., S. Strogartz, and D. Riley, 1981, Proc. Natl. Acad. Sci. USA, 78:1461-1465), and that subsequent compaction occurs by coiling of the ribbon to form a double helical structure. This type of folding generates a fiber in which the nucleosome-nucleosome contacts established in the zig-zag ribbon are maintained and in which the histone H1 molecules occupy equivalent sites. The diameter of the fiber is not dependent upon the nucleosome repeat length. Direct mass values for individual isolated fibers obtained from electron scattering measurements showed that the mass per length was dependent on ionic strength, and ranged from 6.0 X 10(4) daltons/nm at 10 mM NaCl to 27 X 10(4) daltons/nm at 150 mM salt. These values are equivalent to 2.5 nucleosomes/11 nm at 10 mM NaCl and to 11.6 nucleosomes/11 nm at 150 mM salt and are consistent with the range of packing ratios for the proposed helical ribbon.
A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.
Authors: de Harven E , Leung R , Christensen H
Published: May 2004

A method is described for the use of scanning electron microscopy on the surface of gold-labeled cells. It includes the use of 45- or 20-nm colloidal gold marker conjugated with Staphylococcal protein A. The marker is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging mode of the scanning electron microscope. When the backscattered electron signal is mixed with the secondary electron signal, an optimum correlation between the distribution of the labeled sites and the cell surface structures is demonstrated. The method is illustrated by its application to the identification of human circulating granulocytes. Its good resolution, high contrast, and good labeling efficiency offers a promising approach to the specific localization of cell surface antigenic sites labeled with particles of colloidal gold.
A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments.
Authors: Goodloe-Holland C M , Luna E J
Published: May 2004

The binding between sonicated Dictyostelium discoideum plasma membrane fragments and F-actin on Sephacryl S-1000 beads was found to be competitively inhibited by myosin subfragment-1. This inhibition is MgATP-sensitive, exhibits a Ki of approximately 5 X 10(-8) M, and is reciprocal, since membranes inhibit the binding of 125I-heavy meromyosin to F-actin on beads. These experiments demonstrate that membrane binding and S-1 binding to F-actin on beads are mutually exclusive and, therefore, that the membrane fragments bind predominantly to the sides, rather than to the ends, of the actin filaments. This conclusion is supported by electron micrographs that show many lateral associations between membrane fragments and bead-associated actin filaments. Such lateral associations could play an important role in the organization and lateral movement of membrane proteins by the cytomusculature.
Actin-membrane interaction in fibroblasts: what proteins are involved in this association?
Authors: Mangeat P , Burridge K
Published: May 2004

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.
Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.
Authors: Scherson T , Kreis T E , Schlessinger J , Littauer U Z , Borisy G G , Geiger B
Published: May 2004

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.
Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve.
Authors: Aquino D A , Margolis R U , Margolis R K
Published: May 2004

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Müller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.
Identification and extraction of proteins that compose the triad junction of skeletal muscle.
Authors: Caswell A H , Brunschwig J P
Published: May 2004

Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCl or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCl. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000 and 140,000 and migrates in the gel as a protein of Mr = 1.2 X 10(6) indicating a polymeric structure.
Growth-inhibitory activity of lymphoid cell plasma membranes. II. Partial characterization of the inhibitor.
Authors: Stallcup K C , Burakoff S J , Mescher M F
Published: May 2004

We have shown that plasma membranes from lymphoid cells have inhibitory activity for the growth of normal lymphocytes and lymphoid tumor cells (Stallcup, K. C., A. Dawson, and M. F. Mescher, J. Cell Biol. 99:1221-1226). This growth-inhibitory activity has been found to co-purify with major histocompatibility complex class I antigens (H-2K and D) when these cell surface glycoproteins are isolated from detergent lysates of cells by affinity chromatography on monoclonal antibody columns. When incorporated into liposomes, the affinity-purified H-2 antigens inhibited the growth of both normal lymphocytes and tumor cells at concentrations of 1-3 micrograms/ml. Inhibition was readily reversed upon removal of the liposomes from the cell cultures, even after several days of exposure of cells to the inhibitor. Inhibitory activity was insensitive to protease digestion or heat treatment, indicating that it was not due to the H-2 glycoproteins. This was confirmed by the demonstration that inhibitory activity could be separated from the H-2 protein by gel filtration in the presence of deoxycholate and could be extracted from membranes or H-2 antigen preparations with organic solvents. The results demonstrate that the growth-inhibitory component(s) of the plasma membrane is a minor lipid or lipid-like molecule which retains activity in the absence of other membrane components. The findings reported here and in the preceding article suggest that this novel membrane component may have a role in control of lymphoid cell growth, possibly mediated by cell contacts.
A selective defect in arachidonic acid release from macrophage membranes in high potassium media.
Authors: Aderem A A , Scott W A , Cohn Z A
Published: May 2004

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.
A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions.
Authors: Robbins A R , Oliver C , Bateman J L , Krag S S , Galloway C J , Mellman I
Published: May 2004

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.
Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland.
Authors: Spearman T N , Hurley K P , Olivas R , Ulrich R G , Butcher F R
Published: May 2004

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.
Alternate patterns of doublet microtubule sliding in ATP-disintegrated macrocilia of the ctenophore Beroë.
Authors: Tamm S L , Tamm S
Published: May 2004

We have used the unique properties of macrocilia from the lips of the ctenophore Beroë to test whether the ciliary beat cycle is caused by sequential activation of doublet sliding on opposite sides of the axoneme (Satir, P., 1982, Soc. Exp. Biol. Symp., 35: 179-201; Sugino, K., and Y. Naitoh, 1982, Nature (Lond.), 295: 609-611; Wais-Steider, J., and P. Satir, 1979, J. Supramol. Struct., 11:339-347). Macrocilia contain several hundred axonemes linked into rows by lamellae between doublets 3 and 8. These connections provide morphological markers for numbering the doublet microtubules in thin sections. Demembranated, detached macrocilia undergo ATP-induced sliding disintegration by extrusion of thick fragments and finer fibers from the proximal end. Disintegration can easily be followed with low-magnification brightfield or phase-contrast optics. Sliding occurs with or without added elastase, and is reversibly inhibited by vanadate. Thin sections through 16 ATP-disintegrated macrocilia showed two mutually exclusive patterns of doublet extrusion with equal frequency. Doublets 9, 1, and 2 or doublets 5, 6, and 7 were usually extruded, but not both groups. We conclude that both subsets of doublets slide by their own active arms, and that the two extrusion patterns represent alternate activation and inactivation of doublet sliding on opposite halves of the axoneme. These findings provide the first direct experimental support for a switching mechanism regulating microtubule sliding in cilia.
Adhesive multiplicity in the interaction of embryonic fibroblasts and myoblasts with extracellular matrices.
Authors: Decker C , Greggs R , Duggan K , Stubbs J , Horwitz A
Published: May 2004

Neff et al. (1982, J. Cell Biol., 95:654-666) have described a monoclonal antibody, CSAT, directed against a cell surface antigen that participates in the adhesion of skeletal muscle to extracellular matrices. We used the same antibody to compare and parse the determinants of adhesion and morphology on myogenic and fibrogenic cells. We report here that the antigen is present on skeletal and cardiac muscle and on tendon, skeletal, dermal, and cardiac fibroblasts; however, its contribution to their morphology and adhesion is different. The antibody produces large alterations in the morphology and adhesion of skeletal myoblasts and tendon fibroblasts; in contrast, its effects on the cardiac fibroblasts are not readily detected. The effects of CSAT on the other cell types, i.e., dermal and skeletal fibroblasts, cardiac muscle, 5-bromodeoxyuridine-treated skeletal muscle, lie between these extremes. The effects of CSAT on the skeletal myoblasts depends on the calcium concentration in the growth medium and on the culture age. We interpret these differential responses to CSAT as revealing differences in the adhesion of the various cells to extracellular matrices. This interpretation is supported by parallel studies using quantitative assays of cell-matrix adhesion. The likely origin of these adhesive differences is the progressive display of different kinds of adhesion-related molecules and their organizational complexes on increasingly adhesive cells. The antigen to which CSAT is directed is present on all of the above cells and thus appears to be a lowest common denominator of their adhesion to extracellular matrices.
ATP-induced gelation--contraction of microtubules assembled in vitro.
Authors: Weisenberg R C , Cianci C
Published: May 2004

We report here an ATP-dependent formation and contraction, or syneresis, of a microtubule gel using microtubule proteins prepared from calf brains. Gel contraction is typically observable 15-30 min after ATP addition to microtubules assembled to steady state, and is complete after approximately 60 min, at which time the gel volume is reduced by as much as 75%. In contracted gels, microtubule bundles and aster-like structures are observable. Gelation-contraction requires only microtubule proteins present after purification by three cycles of assembly and disassembly.
Phosphomannosyl receptors may participate in the adhesive interaction between lymphocytes and high endothelial venules.
Authors: Stoolman L M , Tenforde T S , Rosen S D
Published: May 2004

Normal and malignant lymphocytes can migrate from the bloodstream into lymph nodes and Peyer's patches. This process helps distribute normal lymphocytes throughout the lymphoid system and may provide a portal of entry for circulating malignant cells. An adhesive interaction between lymphocytes and the endothelium of postcapillary venules is the first step in the migratory process. We have recently shown that the simple sugars L-fucose and D-mannose, and an L-fucose-rich polysaccharide (fucoidin), can inhibit this adhesive interaction in vitro. We now report that mannose-6-phosphate, the structurally related sugar fructose-1-phosphate, and a phosphomannan, core polysaccharide from the yeast Hansenula holstii (PPME) are also potent inhibitors. Inhibitory activity was assessed by incubating freshly prepared suspensions of lymphocytes, containing the various additives, over air-dried, frozen sections of syngeneic lymph nodes at 7-10 degrees C. Sections were then evaluated in the light microscope for the binding of lymphocytes to postcapillary venules. Mannose-6-phosphate and fructose-1-phosphate were potent inhibitors of lymphocyte attachment (one-half maximal inhibition at 2-3 mM). Mannose-1-phosphate and fructose-6-phosphate had slight inhibitory activity, while glucose-1-phosphate, glucose-6-phosphate, galactose-1-phosphate, and galactose-6-phosphate had no significant activity (at 10 mM). In addition, the phosphomannan core polysaccharide was a potent inhibitor (one-half maximal inhibition at 10-20 micrograms/ml); dephosphorylation with alkaline phosphatase resulted in loss of its inhibitory activity. Preincubation of the lymphocytes, but not the lymph node frozen sections, with PPME resulted in persistent inhibition of binding. Neither the monosaccharides nor the polysaccharide suppressed protein synthesis nor decreased the viability of the lymphocytes. Furthermore, inhibitory activity did not correlate with an increase in negative charge on the lymphocyte surface (as measured by cellular electrophoresis). These data suggest that a carbohydrate-binding molecule on the lymphocyte surface, with specificity for mannose-phosphates and structurally related carbohydrates, may be involved in the adhesive interaction mediating lymphocyte recirculation.
Chromatin conformation of yeast centromeres.
Authors: Bloom K S , Amaya E , Carbon J , Clarke L , Hill A , Yeh E
Published: May 2004

The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and XI. A 289-base pair centromere (CEN3) sequence can stabilize yeast chromosome III through mitosis and meiosis. The orientation of the centromeric fragments within chromosome III has no effect on the normal mitotic or meiotic behavior of the chromosome. The structural integrity of the centromere region in these genomic substitution strains was examined by mapping nucleolytic cleavage sites within the chromatin DNA. A nuclease-protected centromere core of 220-250 base pairs was evident in all of the genomic substitution strains. The position of the protected region is determined strictly by the centromere DNA sequence. These results indicate that the functional centromere core is contained within 220-250 base pairs of the chromatin DNA that is structurally distinct from the flanking nucleosomal chromatin.
Sequence of a highly divergent beta tubulin gene reveals regional heterogeneity in the beta tubulin polypeptide.
Authors: Sullivan K F , Cleveland D W
Published: May 2004

The nucleotide sequence of a chicken genomic DNA segment containing the chicken beta 4 tubulin gene has been determined. The predicted amino acid sequence of beta 4 is surprisingly divergent from that of the chicken beta 2 gene that encodes the dominant neural beta tubulin. beta 4 differs from beta 2 at 36 residue positions and encodes a polypeptide that is four amino acids longer, yielding a divergence of 8.9% between the two beta tubulin isotypes. While many of the amino acid substitutions are conservative, several involve significant alteration in the physiochemical properties of the residue. Furthermore, the amino acid substitution positions are not randomly located within the primary sequence but are distinctly clustered: major divergence occurs in the carboxy-terminal region beyond residue 430 and within the second protein coding exon segments of the genes. In addition, large regions of absolute sequence conservation are also present. Certain sequences within the heterogeneous regions are conserved in other species, indicating that these regions are under positive evolutionary selection pressure and are therefore probably essential for some aspect of beta-tubulin function. These findings strongly suggest that regional amino acid sequence heterogeneity may play an important role in the establishment of functionally differentiated beta tubulin polypeptides.
Expression of unusually large keratins during terminal differentiation: balance of type I and type II keratins is not disrupted.
Authors: Kim K H , Marchuk D , Fuchs E
Published: May 2004

When a basal epidermal cell undergoes a commitment to terminally differentiate, it ceases to divide and begins to migrate outward towards the surface of the skin. Dramatic changes in its cytoskeletal architecture take place, accompanied by numerous changes in the expression of keratins, a family of related polypeptides that form 8-nm filaments in these cells. We show here that a shift to the synthesis of unusually large keratins occurs that does not seem to disrupt the ratio of two distinct subfamilies of keratins. Preliminary studies indicate that this differentiation-specific shift may be at the level of transcriptional rather than post-transcriptional regulation. The striking similarities between these large keratins and the type I and type II keratins of basal epidermal cells suggests the important role that both classes of large keratin sequences must play in the assembly of the intermediate filaments within the differentiating keratinocyte.
Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.
Authors: Choi Y D , Dreyfuss G
Published: May 2004

The C proteins (C1 and C2) are major constituents of the 40S subparticle of heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) (Beyer, A.L., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon, 1977, Cell, 11:127-138) and are two of the most prominent proteins that become cross-linked by ultraviolet light to heterogeneous nuclear RNA (hnRNA) in vivo. Studies are described here on the characterization of the C proteins in vertebrate cells using monoclonal and polyclonal antibodies. Monoclonal antibodies to genuine RNP proteins, including the C proteins, were obtained by immunizing mice with purified complexes of poly(A)+ hnRNA and poly(A)+ mRNA with their contacting proteins in vivo obtained by ultraviolet cross-linking the complexes in intact cells (Dreyfuss, G., Y.D. Choi, and S.A. Adam, 1984, Mol. Cell. Biol., 4:1104-1114). One of the monoclonal antibodies identified the C proteins in widely divergent species ranging from human to lizard. In all species examined, there were two C proteins in the molecular weight range of from 39,000 to 42,000 for C1, and from 40,000 to 45,000 for C2. The two C proteins were found to be highly related to each other; they were recognized by the same monoclonal antibodies and antibodies raised against purified C1 reacted also with C2. In avian, rodent, and human cells the C proteins were phosphorylated and were in contact with hnRNA in vivo. Immunofluorescence microscopy demonstrated that the C proteins are segregated to the nucleus. Within the nucleus the C proteins were not found in nucleoli and were not associated with chromatin as seen in cells in prophase. These findings demonstrate that C proteins with similar characteristics to those in humans are ubiquitous components of hnRNPs in vertebrates.