bluuebunny/crossref_metadata_2025 huggingface dataset:
A conserved tripeptide sorts proteins to peroxisomes.
Authors: Gould S J , Keller G A , Hosken N , Wilkinson J , Subramani S
Published: May 2004

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.
Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.
Authors: Cabib E , Sburlati A , Bowers B , Silverman S J
Published: May 2004

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.
Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric viral proteins.
Authors: Weltzin R , Lucia-Jandris P , Michetti P , Fields B N , Kraehenbuhl J P , Neutra M R
Published: May 2004

M cells of intestinal epithelia overlying lymphoid follicles endocytose luminal macromolecules and microorganisms and deliver them to underlying lymphoid tissue. The effect of luminal secretory IgA antibodies on adherence and transepithelial transport of antigens and microorganisms by M cells is unknown. We have studied the interaction of monoclonal IgA antibodies directed against specific enteric viruses, or the hapten trinitrophenyl (TNP), with M cells. To produce monospecific IgA antibodies against mouse mammary tumor virus (MMTV) and reovirus type 1, Peyer's patch cells from mucosally immunized mice were fused with myeloma cells, generating hybridomas that secreted virus-specific IgA antibodies in monomeric and polymeric forms. One of two anti-MMTV IgA antibodies specifically bound the viral surface glycoprotein gp52, and 3 of 10 antireovirus IgA antibodies immunoprecipitated sigma 3 and mu lc surface proteins. 35S-labeled IgA antibodies injected intravenously into rats were recovered in bile as higher molecular weight species, suggesting that secretory component had been added on passage through the liver. Radiolabeled or colloidal gold-conjugated mouse IgA was injected into mouse, rat, and rabbit intestinal loops containing Peyer's patches. Light microscopic autoradiography and EM showed that all IgA antibodies (antivirus or anti-TNP) bound to M cell luminal membranes and were transported in vesicles across M cells. IgA-gold binding was inhibited by excess unlabeled IgA, indicating that binding was specific. IgG-gold also adhered to M cells and excess unlabeled IgG inhibited IgA-gold binding; thus binding was not isotype-specific. Immune complexes consisting of monoclonal anti-TNP IgA and TNP-ferritin adhered selectively to M cell membranes, while TNP-ferritin alone did not. These results suggest that selective adherence of luminal antibody to M cells may facilitate delivery of virus-antibody complexes to mucosal lymphoid tissue, enhancing subsequent secretory immune responses or facilitating viral invasion.
Drosophila spectrin: the membrane skeleton during embryogenesis.
Authors: Pesacreta T C , Byers T J , Dubreuil R , Kiehart D P , Branton D
Published: May 2004

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.
Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates.
Authors: Uyeda T Q , Furuya M
Published: May 2004

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.
Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.
Authors: Sanders M A , Salisbury J L
Published: May 2004

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279-285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium-binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.
Three-dimensional reconstruction of the Z disk of sectioned bee flight muscle.
Authors: Cheng N Q , Deatherage J F
Published: May 2004

The three-dimensional structure of the central region of the Z disk of honeybee flight muscle has been determined to a resolution of 70 A by three-dimensional reconstruction from electron micrographs of tilted thin sections. The reconstructions show a complex assembly in which actin filaments terminate and are cross-linked together; a number of structural domains of this network are resolved in quantitative three-dimensional detail. The central region of the Z disk contains two sets of overlapping actin filaments of opposite polarity, which originate in the sarcomeres adjacent to the Z disk, and connections between these filaments. The filaments are deflected by the attachment of cross-links; spacing between filaments change by greater than 100 A during their passage through the Z disk. Each actin filament is linked by connecting structures to four filaments of opposite polarity and two filaments are of the same polarity. Four types of connecting density domain are observed in association with pairs of filaments of opposite polarity: C1, C2, C3, and C5. Two of these, C3 and C5, are associated with the ends of actin filaments. Another connection, C4, is associated with three filaments of the same polarity; C4 is threefold symmetric.
Arrangement of filaments and cross-links in the bee flight muscle Z disk by image analysis of oblique sections.
Authors: Deatherage J F , Cheng N Q , Bullard B
Published: May 2004

Information from oblique thin sections and from three-dimensional reconstructions of tilted, transverse thin sections (Cheng, N., and J. F. Deatherage. 1989. J. Cell Biol. 108:1761-1774) has been combined to determine the three-dimensional structure of the honeybee flight muscle Z disk at 70-A resolution. The overall symmetry and structure of the Z disk and its relationship to the rest of the myofibril have been determined by tracing filaments and connecting elements on electron images of oblique sections which have been enhanced by a local crystallographic averaging technique. In the three-dimensional structure, the connecting density between actin filaments can be described as five compact, crystallographically nonequivalent domains. Features C1 and C2 are located on the transverse twofold rotation axes in the central plane of the Z disk. They are associated with the sides of actin filaments of opposite polarity. Features C3, C4, and C5 are present in two symmetry-related sets which are located on opposite sides of the central plane. C3 and C5 are each associated with two filaments of opposite polarity, interacting with the side of one filament and the end of the other filament. C3 and C5 may be involved in stabilizing actin filament ends inside the Z disk. The location of the threefold symmetric connection C4, relative to the thick filament of the adjacent sarcomere, is determined and its possible relationship to the C filament is considered.
The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation.
Authors: Taubman M B , Smith C W , Izumo S , Grant J W , Endo T , Andreadis A , Nadal-Ginard B
Published: May 2004

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.
Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.
Authors: Horovitz O , Knaack D , Podleski T R , Salpeter M M
Published: May 2004

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.
The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells.
Authors: Musil L S , Frail D E , Merlie J P
Published: May 2004

Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes (Frail, D.E., L.S. Musil, a. Bonanno, and J.P. Merlie, 1989. Neuron. 2:1077-1086) led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with [35S]methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.
Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers.
Authors: Benfenati F , Greengard P , Brunner J , Bähler M
Published: May 2004

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
Membrane anchoring of heparan sulfate proteoglycans by phosphatidylinositol and kinetics of synthesis of peripheral and detergent-solubilized proteoglycans in Schwann cells.
Authors: Carey D J , Evans D M
Published: May 2004

Previous studies have shown that Schwann cells synthesize both peripheral and integral hydrophobic cell surface heparan sulfate proteoglycans (HSPGs). The experiments reported here were undertaken to investigate the mode of attachment of these proteins to the cell surface and their potential interrelationship. The binding of the hydrophobic HSPGs to membranes appears to be via covalently linked phosphatidylinositol based on the observation that incubation of the detergent-solubilized protein with purified phosphatidylinositol-specific phospholipase C significantly reduces the ability of the HSPGs to associate with phospholipid vesicles in a reconstitution assay. The peripherally associated HSPGs were released from the cells by incubation in the presence of heparin (10 mg/ml), 10 mM phytic acid (inositol hexaphosphate), or 2 M NaCl. These treatments also solubilized basement membrane HSPGs synthesized by the Schwann cells. These data suggest that the peripheral HSPGs are bound to the surface by electrostatic interactions. The peripheral and hydrophobic HSPGs were identical in overall size, net charge, length of glycosaminoglycan chains, and patterns of N-sulfation. To determine whether the peripheral HSPGs were derived from the membrane-bound form by cleavage of the membrane anchor, we examined the kinetics of synthesis and degradation of the two forms of HSPGs. The results obtained indicated the existence of two pools of detergent-solubilized HSPG with fast (t1/2 = 6 h) and slow (t1/2 = 55 h) turnover kinetics. The data were consistent with a model in which the peripheral HSPGs were derived from the slowly turning over pool of detergent-solubilized HSPGs.
Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event.
Authors: Danilov Y N , Juliano R L
Published: May 2004

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.
RGD-dependent linkage between plant cell wall and plasma membrane: consequences for growth.
Authors: Schindler M , Meiners S , Cheresh D A
Published: May 2004

Soybean (Glycine max [L.] Merr. cv. Mandarin) root cells (SB-1 cell line) grown in suspension culture containing Glycyl-Arginyl-Glycyl-Aspartyl-Seryl-Proline (GRGDSP) (0.25 mg/ml), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins, demonstrated (a) significantly enhanced growth rate, and (b) aberrant cell wall/plasma membrane interactions and organization. Substitution of the Asp (D) by a Glu (E) amino acid in the hexapeptide, or inversion of the RGD sequence to GDR, abolished the morphological and growth effects observed for GRGDSP in plant cells. Immunoblots, which were prepared from beta-octylglucoside extracts of whole soybean cells and protoplasts, probed with polyclonal antibodies raised against human vitronectin receptor (hVNR) complex, demonstrated a single band with an apparent molecular mass of 70-72 kD. Chromatography of beta-octylglucoside extracts of SB-1 cells on a Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose affinity column demonstrated the retention of a single 70-72 kD polypeptide that reacted specifically with anti-hVNR antiserum. In contradistinction, no cross-reactivity was observed with antifibronectin receptor antiserum. Epifluorescence microscopy of whole soybean cells, after moderate treatment with pectinase, demonstrated punctate fluorescent patches at the cell membrane/wall boundary when probed with anti-hVNR and rhodamine-derivatized secondary antibodies. We propose that coordination and control of plant cell division and proper cell wall biosynthesis may be mediated by an RGD-dependent recognition system in which RGD binding protein(s) promote cell membrane-cell wall attachment.
Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants.
Authors: Roossien F F , de Rijk D , Bikker A , Roos E
Published: May 2004

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.
Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.
Authors: Stephens R W , Pöllänen J , Tapiovaara H , Leung K C , Sim P S , Salonen E M , Rønne E , Behrendt N , Danø K , Vaheri A
Published: May 2004

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.
Alteration by heat shock and immunological characterization of Drosophila small nuclear ribonucleoproteins.
Authors: Wright-Sandor L G , Reichlin M , Tobin S L
Published: May 2004

Sera from human patients with systemic lupus erythematosus (SLE) have been shown to react with snRNP particles of both mammals and Drosophila (Mount, S. M. and J. A. Steitz. 1981. Nucleic Acids Res. 9:6351-6368). We have utilized fully characterized monospecific sera and specifically purified antibodies to carry out indirect immunofluorescence experiments with frozen sections of Drosophila embryos. Embryos subjected to severe heat shock before sectioning showed reduced binding of anti-Sm sera. Anti-nRNP sera reacted identically with antigens of heat shocked and non-heat-shocked sections. The reduction in anti-Sm fluorescence was restored by a brief salt wash. These results imply a noncovalent alteration in the conformation of Sm antigens with the administration of heat shock that can revert with exposure to salt. Drosophila antigens have been compared to mammalian standards, showing partial identity with bovine spleen extract (BSE) antigens when reacted with anti-Sm sera. The antigenic relatedness between affinity-purified heat-shocked and non-heat-shocked Drosophila antigens and their mammalian homologues was examined by quantitative ELISA methodology. In all cases, the Drosophila antigens from heat-shocked and non-heat-shocked embryos were identical. We theorize that the heat shock-induced alteration of Sm antigen reverst during extraction. Because the snRNP antigens have been shown to be involved in splicing, and because splicing is inhibited during heat shock (Yost, H. J., and S. Lindquist. 1986. Cell. 45:185-193), our results provide information on the nature and stability of a change in these antigens which may be a central element in control of the heat shock response.
Multiple inducers of the Drosophila heat shock locus 93D (hsr omega): inducer-specific patterns of the three transcripts.
Authors: Bendena W G , Garbe J C , Traverse K L , Lakhotia S C , Pardue M L
Published: May 2004

The Drosophila hsr omega locus produces one of the largest and most active heat shock puffs, yet it does not encode a heat shock protein. Instead, this locus produces a distinctive set of three transcripts, all from the same start site. The largest transcript, omega 1, is limited to the nucleus and appears to have a role there. A second nuclear transcript, omega 2, is produced by alternative termination and contains the sequence found in the 5' 20-25% of omega 1 (depending on the Drosophila species). The cytoplasmic transcript, omega 3, is produced by removal of a 700-bp intron from omega 2. All three hsr omega RNAs are produced constitutively and production is enhanced by heat shock. In addition to being a member of the set of heat shock puffs, the hsr omega puff is induced by agents that do not affect other heat shock loci, suggesting that hsr omega is more sensitive to environmental changes than other loci. We report here that agents that induce puffing of hsr omega loci in polytene nuclei also lead to an increase in hsr omega transcripts in diploid cells. We also show that the relative levels of omega 1 and omega 3 can be modulated independently by several agents. All drugs that inhibit translation, either initiation or elongation, stabilize the omega 3 transcript, which normally turns over within minutes in control cells. Drugs (such as benzamide and colchicine) that induce puffing of hsr omega, but not other heat shock loci, lead to large increases in omega 1. Although the constitutive level of omega 1 is relatively stable, the drug-induced excess is lost rapidly when the drug is withdrawn. The relative levels of hsr omega transcripts may reflect different states in cellular metabolism.
Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin.
Authors: Vance B A , Kowalski C G , Brinckerhoff C E
Published: May 2004

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.
Unusual behavior of the cytoplasmic transcript of hsr omega: an abundant, stress-inducible RNA that is translated but yields no detectable protein product.
Authors: Fini M E , Bendena W G , Pardue M L
Published: May 2004

Although a major site of transcription in heat shock, the Drosophila hsr omega gene does not encode any known heat shock proteins. Instead, studies of the hsr omega transcripts suggest that the RNA molecules, rather than encoded proteins, are the active products of this gene. The cytoplasmic RNA, omega 3, is spliced and polyadenylated and yet has only very small open reading frames (ORFs), and these are poorly conserved in different Drosophila species. Surprisingly, the work reported here leads us to conclude that one of the tiny ORFs in this RNA is translated. This ORF, designated ORF-omega, is notable in being the only ORF that shows sequence conservation in the three Drosophila species examined. However, translation of this ORF does not lead to detectable accumulation of the protein product. We suggest that ORF-omega may be an example of an unusual type of translated ORF. The act of translation itself may be important rather than the generation of a functional protein product. This nonproductive translation may play a role in regulation of cellular activities.
Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.
Authors: Aris J P , Blobel G
Published: May 2004

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.
Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells.
Authors: Kumar R V , Wolfman A , Panniers R , Henshaw E C
Published: May 2004

Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium. This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers. To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77). BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM. This effect was reversed by calcium but not by other cations tested. The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes. Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3. Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.
Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP).
Authors: Hurtley S M , Bole D G , Hoover-Litty H , Helenius A , Copeland C S
Published: May 2004

We have characterized the association between the binding protein, BiP (also known as GRP 78), and misfolded forms of the influenza virus hemagglutinin precursor, HA0. BiP is a heat-shock-related protein that binds to unassembled immunoglobulin heavy chain and to a variety of misfolded proteins in the lumen of the ER. A small fraction (5-10%) of newly synthesized HA0 in CV-1 cells was found to be misfolded and retained in the ER. When glycosylation was blocked with tunicamycin, all of the HA0 produced was similarly misfolded. The misfolded HA0 was retained as relatively small (9-25-S) complexes associated with BiP. In these complexes the top domains of HA0 were correctly folded judging by their reactivity with monoclonal antibodies, but the polypeptides were cross-linked via anomalous interchain disulfides. The association with BiP was non-covalent and easily broken by warming to 37 degrees C or by adding ATP to the lysate. Pulse-chase experiments showed that HA0's self-association into complexes occurred immediately after synthesis and was followed rapidly by BiP association. The misfolded, BiP-associated HA0 was not transported to the plasma membrane but persisted as complexes in the ER for a long period of time before degradation (t1/2 = 6 h). The results suggested that BiP may be part of a quality control system in the ER and that one of its functions is to detect and retain misfolded proteins.
Concentration of transferrin receptor in human placental coated vesicles.
Authors: Turkewitz A P , Harrison S C
Published: May 2004

Coated vesicles were purified from human placenta by sedimentation, isopycnic centrifugation, and gel filtration. Quantitative Western blotting of the endogenous transferrin receptor (tfR) demonstrated the presence, on average, of roughly one receptor per vesicle. TfR appeared undersaturated with transferrin. After solubilizing vesicles in nonionic detergent, we looked for evidence of tfR interactions with other proteins. Solubilized tfR had an unexpectedly high mobility by gel filtration, apparently resulting from its self-association. This property was not seen in purified tfR or in tfR from a different cell fraction. The tfR complexes, though noncovalent, were largely resistant to conditions that disassemble coat proteins, and they did not appear to contain any other protein species.
Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.
Authors: Stoorvogel W , Geuze H J , Griffith J M , Schwartz A L , Strous G J
Published: May 2004

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.
Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors.
Authors: Messner D J , Griffiths G , Kornfeld S
Published: May 2004

We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.