bluuebunny/crossref_metadata_2025 huggingface dataset:
Synemin and vimentin are components of intermediate filaments in avian erythrocytes
Authors: Granger BL , Repasky EA , Lazarides E
Published: May 2004

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.
Ultrastructural localization of type V collagen in rat kidney.
Authors: Martinez-Hernandez A , Gay S , Miller E J
Published: May 2004

Antibodies specific for the alpha 1 (V) chain and native collagen molecules containing the alpha 1 (V) chain have been used in electron immunohistochemical studies of rat kidney to determine the ultrastructural distribution of this class of collagen molecules. In addition, antibodies against type I collagen and whole basement membrane were used as markers for interstitial collagen and authentic basement membranes. Our results indicate that type V collagen is present in the renal interstitium in different forms: in close apposition to interstitial collagen fibers; in the stromal aspect of vascular basement membranes; and as particulate material not bound to other structures. On the basis of these findings, we postulate a binding or connecting function for this collagen type.
Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells.
Authors: Munson R , Caldwell K L , Glaser L
Published: May 2004

The regulation of the synthesis of muscle-specific proteins has been examined in BC3H1 cells, a smooth muscle-like cell line isolated by Schubert et al. (J. Cell Biol., 1974, 61: 398-413.). The synthesis of both creatine kinase and the acetylcholine receptor appear to be under dual control, a positive control due to cell-cell contact which increases the rate of synthesis of this protein, and a negative signal, elicited by serum components, that decreases the rate of synthesis of these proteins. Induction of muscle-specific proteins in BC3H1 cells is a reversible process and can be arrested after partial induction has taken place by the addition of serum or high-molecular-weight protein fraction from serum to these cells. The high-molecular-weight protein fraction from serum is not by itself mitogenic for Bc3H1 cells and cannot be replaced by a variety of known hormones (mitogenic factors).
Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.
Authors: Goodenough U W , Detmers P A , Hwang C
Published: May 2004

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.
Abnormal Epidermal Keratinization in the repeated epilation mutant mouse
Authors: Holbrook KA , Dale BA , Brown KS
Published: May 2004

Repeated epilation (Er) is a radiation-induced, autosomal, incomplete dominant mutation in mice which is expressed in heterozygotes but is lethal in the homozygous condition. Many effects of the mutation occur in skin: the epidermis in Er/Er mice is adhesive (oral and nasal orifices fuse, limbs adhere to the body wall), hyperplastic, and fails to undergo terminal differentiation. Skin from fetal +/+, Er/+ and Er/Er mice at ages pre- and postkeratinization examined by light, scanning, and transmission electron microscopy showed marked abnormalities in tissue architecture, differentiation, and cell structure; light and dark basal epidermal cells were separated by wide intercellular spaces, joined by few desmosomes, and contained phagolysomes. The numbers of spinous, granular, and superficial layers were highly variable within any given region and among various regions of the body. In some areas, 2-8 layers of granular cells, containing large or diminutive keratohyalin granules, extended to the epidermal surface; in others, the granular layers were covered by several layers of partially keratinized or nonkeratinized cells. In rare instances, a single or small group of cornified cells was present among the granular layers but was not associated with the epidermal surface. Both the granular and nonkeratinized/partially keratinized upper epidermal layers Er/Er skin gave positive immunofluorescence with antiserum to the histidine-rich, basic protein, filaggrin. Proteins in epidermal extracts from +/+, Er/+ and Er/Er mice were separated and identified by radio- and immunolabeling techniques. The Er/Er extract was missing a 26.5- kdalton protein and had an altered ratio of bands in the keratin region. The 26.5-kdalton band was histidine-rich and cross-reacted with the antiserum to rat filaggrin. Several high molecular weight bands present in both Er/Er and +/+ extracts also reacted with the antiserum. These are presumed to be the precursors of filaggrin and to account for the immunofluorescence om Er/Er epidermis even though the product protein is absent. The morphologic and biochemical data indicated that the genetic defect has a general and profound influence on epidermal differentiation, including alteration of two proteins (filaggrin and keratin) important in normal terminal differentiation, tissue architecture, and cytology. Identification of epidermal abnormalities at early stages of development (prekeratinization) and defective structure of other tissues and gross anatomy suggest that the mutation is responsible for a defect in same regulatory step important in many processes of differentiation and development.
Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling.
Authors: Tietze C , Schlesinger P , Stahl P
Published: May 2004

Receptor-mediated endocytosis of rat preputial beta-glucuronidase and the glycoconjugate mannose-BSA by rat alveolar macrophages is inhibited by chloroquine and ammonium chloride. We have previously reported that these drugs cause a loss of cell surface binding activity and that they do not inhibit internalization of receptor ligand complexes when incubated with cells at 37 degrees C. In this report we more clearly delineate the intracellular site of weak base inhibition of receptor recycling and the mechanism of that inhibition. From our analysis of the kinetics of ligand transport we conclude that there are two functionally distinct intracellular pools of receptor. One of these, the cycling pool, is not sensitive to the presence of weak bases, and receptor-ligand complexes return from this pool to the cell surface intact. The second pool is responsible for the time-dependent intracellular delivery of ligand to acid vesicles, which is inhibited by weak bases. Chloroquine and ammonium chloride appear to inhibit the dissociation of receptor-ligand complexed in this second pool and thereby the production of free receptors for the continuation of receptor-mediated endocytosis. We examine the internalization and binding of ligand in normal and paraformaldehyde-treated cells and find that these are strongly affected by pH. In particular, the dissociation rate of receptor ligand complexes is enhanced greater than 7.5 fold by lowering the medium pH from 7 to 6. From these results we propose that weak bases raise the pH of acid intracellular compartments, slowing the rate of receptor-ligand dissociation and thereby reducing the cellular pool of free receptors available for further uptake of ligand. In addition, we demonstrate that receptor-ligand complexes cannot return to the cell surface from the amine-sensitive (acid) intracellular pool that led us to call this the nonreleasable pool. This final observation indicates that receptor movements through these two pools are functionally distinct processes.
Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.
Authors: McDonald J A , Kelley D G , Broekelmann T J
Published: May 2004

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.
Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting.
Authors: Sealock R
Published: May 2004

In this study, acetylcholine receptor-rich postsynaptic membranes from electric tissues of the electric rays Narcine brasiliensis and Torpedo californica are negatively contrasted for thin-section electron microscopy through the use of tannic acid. Both outer (extracellular) and inner (cytoplasmic) membrane surfaces are negatively contrasted, and can be studied together in transverse sections. The hydrophobic portion of the membrane appears as a thin (approximately 2 nm), strongly contrasted band. This band is the only image given by membrane regions which are devoid of acetylcholine receptor. In regions of high receptor density, however, both surfaces of the membrane are seen to bear or be associated with material which extends approximately 6.5 nm beyond the center of the bilayer. The material on the outer surface can be identified with the well-known extracellular portion of the receptor molecule. A major portion of the inner surface image is eliminated by extraction of the membranes at pH 11 to remove peripheral membrane proteins, principally the 43,000 Mr (43K) protein. The images thus suggest a cytoplasmic localization of the 43K protein, with its distribution being coextensive with that of the receptor. They also suggest that the 43K protein extends farther from the cytoplasmic surface than does the receptor.
Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton.
Authors: Painter R G , Ginsberg M
Published: May 2004

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A-Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.
Platelet-derived growth factor in chemotactic for fibroblasts.
Authors: Seppä H , Grotendorst G , Seppä S , Schiffmann E , Martin G R
Published: May 2004

Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet-derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.
Specific localization of scallop gill epithelial calmodulin in cilia
Authors: Stommel EW , Stephens RE , Masure HR , Head JF
Published: May 2004

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.
Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella.
Authors: Brokaw C J , Luck D J , Huang B
Published: May 2004

The mutation uni-1 gives rise to uniflagellate Chlamydomonas cells which rotate around a fixed point in the microscope field, so that the flagellar bending pattern can be photographed easily. This has allowed us to make a detailed analysis of the wild-type flagellar bending pattern and the bending patterns of flagella on several mutant strains. Cells containing uni-1, and recombinants of uni-1 with the suppressor mutations, suppf-1 and suppf-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beta frequency, apparently as the result of defective function of the outer dynein arms. The pf-17 mutation has been shown to produce nonmotile flagella in which radial spoke heads and five characteristic axonemal polypeptides are missing. Recombinants containing pf-17 and either suppf-2 or suppf-3 have motile flagella, but still lack radial-spoke heads and the associated polypeptides. The flagellar bending pattern of these recombinants lacking radial-spoke heads is a nearly symmetric, large amplitude pattern which is quite unlike the wild-type pattern. However, the presence of an intact radial-spoke system is not required to convert active sliding into bending and is not required for bend initiation and bend propagation, since all of these processes are active in suppfpf-17 recombinants. The function of the radial-spoke system appears to be to convert the symmetric bending pattern displayed by these recombinants into the asymmetric bending pattern required for efficient swimming, by inhibiting the development of reverse bends during the recovery phase of the bending cycle.
Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.
Authors: Penningroth S M , Cheung A , Olehnik K , Koslosky R
Published: May 2004

The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin (Lytechinus pictus) spermatozoa into ATP-free buffer. Relaxation in response to nucleotide was quantitated by measuring theta, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation. It was found that (a) the rate of flagellar relaxation induced by 4 X 10(-6) M ATP was inhibited 80% by vanadate concentrations of 3 X 10(-6) M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and epsilon-ATP) were also capable of effecting relaxation; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP relaxation depends on ATP hydrolysis; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon. A general model of relaxation is proposed based on active microtubule sliding.
Chemical modification of matrix porin from Escherichia coli: probing the pore topology of a transmembrane protein.
Authors: Schindler M , Rosenbusch J P
Published: May 2004

Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta-octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine-specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.
High voltage electron microscopy studies of axoplasmic transport in neurons: a possible regulatory role for divalent cations.
Authors: Stearns M E
Published: May 2004

Light and high voltage electron microscopy (HVEM) procedures have been employed to examine the processes regulating saltatory motion in neurons. Light microscope studies demonstrate that organelle transport occurs by rapid bidirectional saltations along linear pathways in cultured neuroblastoma cells. HVEM stereo images of axons reveal that microtubules (Mts) and organelles are suspended in a continuous latticework of fine microtrabecular filaments and that the Mts and lattice constitute a basic cytoskeletal structure mediating the motion of particles along axons. We propose that particle transport depends on dynamic properties of nonstatic microtrabecular lattice components. EXperiments were initiated to determine the effects of changes in divalent cation concentrations (Ca2+ and Mg2+) on: (a)the continuation of transport and (b) the corresponding structural properties of the microtrabecular lattice. We discovered that transport continues or is stimulated to a limited extent in cells exposed to small amounts of exogenously supplied Ca2+ and Mg2+ ions (less than 0.1 mM). Exposure of neurons to increased dosages of Ca2+ and Mg2+ (0.2-1.0 mM) stimulates transport for 2-4 min at 37 degrees C, but after a 5- to 20-min exposure the saltatory movements of organelles are observed gradually to become shorter in duration and rate particle motion ceases to occur. HVEM observations demonstrated that Ca2+ - and with the cessation of motion. Ca2+-containing solutions produced contractions of the microtrabecular filaments, whereas Mg2+-containing solutions had the opposing effect of stimulating an elongation and assembly (expansion) of microtrabeculae. On the basis of these observations we hypothesize that cycles of Ca2+/Mg2+-coupled contractions and expansions of the microtrabecular lattice probably regulate organelle motion in nerve cells.
Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin.
Authors: Trapp B D , Quarles R H
Published: May 2004

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.
Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture.
Authors: Barnes D W
Published: May 2004

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.
Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes.
Authors: Fujiki Y , Fowler S , Shio H , Hubbard A L , Lazarow P B
Published: May 2004

Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol. 93:97-102). The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar. The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides. The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER). Conversely, most ER proteins are absent from peroxisomes. By electron microscopy, purified peroxisomal membranes are approximately 6.8 nm thick and have a typical trilaminar appearance. The phospholipid/protein ratio of peroxisomal membranes is approximately 200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin. All the membranes investigated contain a polypeptide band with a molecular mass of approximately 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed.
Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution
Authors: Rosenfeld MG , Kreibich G , Popov D , Kato K , Sabatini DD
Published: May 2004

By in vitro translation of mRNA's isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA's for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes.

In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself.

Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.
Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins.
Authors: Suprenant K A , Dentler W L
Published: May 2004

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.
On the structural organization of isolated bovine lens fiber junctions.
Authors: Zampighi G , Simon S A , Robertson J D , McIntosh T J , Costello M J
Published: May 2004

Junctions between fiber cells of bovine lenses have been isolated in milligram quantities, without using detergents or proteases. The structure of the isolated junctions has been studied by thin-section, negative-stain, and freeze-fracture electron microscopy and by x-ray diffraction. The junctions are large and most often have an undulating surface topology as determined by thin sectioning and freeze-fracture. These undulations resemble the tongue-and-groove interdigitations between lens fiber cells previously seen by others (D. H. Dickson and G. W. Crock, 1972, Invest. Ophthalmol. 11:809-815). In sections, the isolated junctions display a pentalamellar structure approximately 13-14 nm in overall thickness, which is significantly thinner than liver gap junctions. Each junctional membrane contains in the plane of the lipid bilayers distinct units arranged in a square lattice with a center-to-center spacing of 6.6 nm. Freeze-fracture replicas of the junctions fractured transversely show that the repeating units extend across the entire thickness of each membrane. Each unit is probably constructed from four identical subunits, with each subunit containing a protein of an apparent molecular weight of 27,000. We conclude that the lens junctions are structurally and chemically, different from gap junctions and could represent a new kind of intercellular contact, not simply another crystalline state of the gap junction protein.
Localization of the casein gene family to a single mouse chromosome.
Authors: Gupta P , Rosen J M , D'Eustachio P , Ruddle F H
Published: May 2004

A series of mouse-hamster somatic cell hybrids containing a variable number of mouse chromosomes and a constant set of hamster chromosomes have been used to determine the chromosomal location of a family of hormone-inducible genes, the murine caseins. Recombinant mouse cDNA clones encoding the alpha-, beta-, and gamma-caseins were constructed and used in DNA restriction mapping experiments. All three casein cDNAs hybridized to the same set of somatic cell hybrid DNAs isolated from cells containing mouse chromosome 5, while negative hybridization was observed to ten other hybrid DNAs isolated from cells lacking chromosome 5. A fourth cDNA clone, designated pCM delta 40, which hybridized to an abundant 790 nucleotide poly(A)RNA isolated from 6-d lactating mouse mammary tissue, was also mapped to chromosome 5. The chromosomal assignment of the casein gene family was confirmed using a mouse albumin clone. The albumin gene had been previously localized to mouse chromosome 5 by both breeding studies and analogous molecular hybridization experiments. An additional control experiment demonstrated that another hormone-inducible gene, specifying a 620 nucleotide abundant mammary gland mRNA, hybridized to DNA isolated from a different somatic cell hybrid line. These studies represent the first localization of a peptide and steroid hormone-responsive gene family to a single mouse chromosome.
Release of clathrin from coated vesicles dependent upon a nucleoside triphosphate and a cytosol fraction.
Authors: Patzer E J , Schlossman D M , Rothman J E
Published: May 2004

Calf-brain coated vesicles were incubated with ATP and a cytosol fraction. As much as 90% of the clathrin was selectively released within 10 min at 37 degrees C without detectable proteolysis. This uncoating process required the presence of both ATP and cytosol. Empty cages of clathrin could also be dissociated in a similar manner. A nonhydrolyzable analogue, 5'-adenylylimidodiphosphate (AMP-PNP), would not substitute for ATP. Clathrin was dissociated from coats in a form unable to reassemble into cages under standard conditions. These reactions may reflect a segment of a clathrin-coated vesicle cycle in which coats are removed from vesicles after budding.
Expression of a mannosyl-fucosyl receptor for endocytosis on cultured primary macrophages and their hybrids.
Authors: Stahl P , Gordon S
Published: May 2004

The presence of a pinocytosis receptor, specific for mannose-fucose terminated glycoproteins, has been established on murine resident peritoneal macrophages, thioglycollate-elicited peritoneal macrophages, and macrophages derived from bone-marrow in culture. Macrophagelike cell lines (J-774 and P338.D1), a myelomonocytic cell line (427E), lymphocytes, polymorphonuclear leukocytes, and fibroblasts were negative. Binding and uptake of 125I-mannose-BSA and 125I-beta-glucuronidase, respectively, into thioglycollate-induced peritoneal macrophages is saturable (Kd 4 degrees C = 5.4 X 10(-9) M; Kuptake 37 degrees C = 7 X 10(-7) M) and sugar specific. Macrophage-macrophage (rat X mouse) hybrids prepared by fusing rat alveolar macrophages with J-774-B10 (HAT-sensitive macrophagelike cell line) expresses the mannose-fucose receptor. Karyotypes of the hybrids confirmed a 1:1 fusion of rat and mouse cells. The rat/mouse hybrids express a variety of rat and mouse antigens including Fc receptors. Fibroblast-macrophage hybrids and melanoma-macrophage hybrids were negative for mannose-fucose receptor activity. The expression of the mannose-fucose receptor by macrophages appears to be regulated independently of other macrophage markers.
Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.
Authors: Fehlmann M , Carpentier J L , Le Cam A , Thamm P , Saunders D , Brandenburg D , Orci L , Freychet P
Published: May 2004

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.
The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule.
Authors: Herzlinger D A , Easton T G , Ojakian G K
Published: May 2004

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.
Bone formation and calcification by isolated osteoblastlike cells.
Authors: Nijweide P J , van Iperen-van Gent A S , Kawilarang-de Haas E W , van der Plas A , Wassenaar A M
Published: May 2004

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.