bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles.
Authors: Hirano S , Nose A , Hatta K , Kawakami A , Takeichi M
Published: May 2004

Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin-based cytoskeletal elements.
The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum.
Authors: Siu C H , Cho A , Choi A H
Published: May 2004

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80-Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.-H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell-cell binding at the aggregation stage of development via homophilic interaction.
Association of a plasminogen activator inhibitor (PAI-1) with the growth substratum and membrane of human endothelial cells.
Authors: Levin E G , Santell L
Published: May 2004

We have studied the distribution of the plasminogen activator inhibitor type 1 (PAI-1) in cultures of confluent human umbilical vein endothelial cells. Plasminogen activator inhibitor activity measured by the 125I-fibrin plate assay was detected in the cytosol (2.85 +/- 0.16 U), 100,000 g particulate fraction (1.26 +/- 0.30 U), and in the growth substratum (9.82 +/- 1.80 U). Characterization of the protein responsible for this activity by reverse fibrin autography, immunoprecipitation, and immunoblotting demonstrated that it had an Mr of 46,000 and was antigenically related to PAI-1. Only the active form of the inhibitor was found in all three fractions. Inhibitor in the cytosol and particulate fraction converted to the latent form during 37 degrees C incubation while the substratum inhibitor remained fully active. Extracellular PAI-1 was detected in the growth substratum before its appearance in conditioned medium and represented the major protein deposited beneath the cells. The inhibitor was only transiently localized in the substratum, disappearing within 6 h and concomitantly appearing in the culture medium. Incubation of isolated metabolically labeled substratum with tissue plasminogen activator (tPA) resulted in the appearance and release of an immunologically related inactive 44,000 Mr form as well as the tPA-PAI-1 complex (110,000 Mr). PAI-1 was also converted into its 44,000-Mr form and released by treatment of the substratum with human leukocyte elastase. The rapid deposition and predominance of PAI-1 in the underlying compartment of endothelial cells may explain how the basement membrane is protected from proteolytic degradation by plasmin-generating enzymes.
Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.
Authors: Frost G H , Thompson W C , Carney D H
Published: May 2004

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.
Transcytosis of albumin in capillary endothelium.
Authors: Milici A J , Watrous N E , Stukenbrok H , Palade G E
Published: May 2004

We have used a variety of immunocytochemical procedures to localize albumin in transit through the capillary endothelium of the murine myocardium and thereby identify endothelial cell structures involved in albumin efflux. The most informative results were obtained with a protocol that included (a) removal of endogenous albumin by perfusion of the heart with PBS supplemented with 14 mM glucose, (b) perfusion of the heart vasculature with exogenous (bovine) albumin for various short time periods, (c) fixation of the vessels by formaldehyde-glutaraldehyde mixtures, (d) processing of fixed myocardium specimens through L. R. White embedding followed by sectioning, or direct thin frozen sectioning, and (e) reacting the surface of such specimens with antialbumin antibodies followed by gold-labeled secondary antibodies. The results indicate that (a) monomeric albumin binds (with low affinity) to the luminal surface of the capillary endothelium, (b) it is restricted in transit through the endothelium to plasmalemmal vesicles, and (c) it appears in the pericapillary spaces less than 15 s after the beginning of its perfusion. No albumin concentration gradients, centered with their maxima on the exits from intercellular spaces, were detected at any time points, including the shortest ones (15 and 30 s) investigated. Additional information comparing monomeric vs. polymeric albumin transcytosis was obtained using albumin-gold complexes. The results are discussed in terms of vesicular transport of albumin across the endothelium and the relations of this type of transport to the postulated pore systems of the physiological literature.
Tyrosine sulfation is a trans-Golgi-specific protein modification.
Authors: Baeuerle P A , Huttner W B
Published: May 2004

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.
The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins.
Authors: Dorner A J , Bole D G , Kaufman R J
Published: May 2004

The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.
Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.
Authors: Steinberg T H , Newman A S , Swanson J A , Silverstein S C
Published: May 2004

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.
Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.
Authors: Yamashiro D J , Maxfield F R
Published: May 2004

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.
Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.
Authors: Yamashiro D J , Maxfield F R
Published: May 2004

In the preceding paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2713-2721), we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wild-type Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5-1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308), our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para-Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled alpha 2-macroglobulin or fluorescein-isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH greater than 6.5 in the CHO mutants when compared with wild-type cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.
Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line.
Authors: Urban J , Parczyk K , Leutz A , Kayne M , Kondor-Koch C
Published: May 2004

The biosynthesis, processing, and apical secretion of a group of polypeptides (Kondor-Koch, C., R. Bravo, S. D. Fuller, D. Cutler, and H. Garoff. 1985. Cell. 43:297-306) are studied in MDCK cells using a specific polyclonal antiserum. These polypeptides are synthesized as a precursor protein which has an apparent Mr of 65,000 in its high mannose form. This precursor is converted into a protein with an apparent Mr of 80,000 containing complex carbohydrates and sulfate. After intracellular cleavage of the 80-kD protein, the 35-45-kD subunits are secreted as an 80-kD glycoprotein complex (gp 80) linked together by disulfide bonds. Secretion of the protein complex occurs by a constitutive pathway at the apical surface of the epithelial monolayer. Since the immediate post-translational precursor, the 65-kD protein, is hydrophilic in nature as shown by its partitioning behavior in a phase-separated Triton X-114 solution, gp 80 is segregated into the apical exocytotic pathway as a soluble molecule. The proteolytic maturation of gp 80 is blocked in the presence of chloroquine and its secretion is retarded. The 80-kD precursor is released at the apical cell surface, demonstrating that proteolytic processing is not necessary for the apical secretion of this protein. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, indicating a role of the carbohydrate moieties in the vectorial transport of this protein.
Immunochemical localization of myosin heavy chain isoforms and paramyosin in developmentally and structurally diverse muscle cell types of the nematode Caenorhabditis elegans.
Authors: Ardizzi J P , Epstein H F
Published: May 2004

The nematode Caenorhabditis elegans contains two major groups of muscle cells that exhibit organized sarcomeres: the body wall and pharyngeal muscles. Several additional groups of muscle cells of more limited mass and spatial distribution include the vulval muscles of hermaphrodites, the male sex muscles, the anal-intestinal muscles, and the gonadal sheath of the hermaphrodite. These muscle groups do not exhibit sarcomeres and therefore may be considered smooth. Each muscle cell has been shown to have a specific origin in embryonic cell lineages and differentiation, either embryonically or postembryonically (Sulston, J. E., and H. R. Horvitz. 1977. Dev. Biol. 56:110-156; Sulston, J. E., E. Schierenberg, J. White, and J. N. Thomson. 1983. Dev. Biol. 100:64-119). Each muscle type exhibits a unique combination of lineage and onset of differentiation at the cellular level. Biochemically characterized monoclonal antibodies to myosin heavy chains A, B, C, and D and to paramyosin have been used in immunochemical localization experiments. Paramyosin is detected by immunofluorescence in all muscle cells. Myosin heavy chains C and D are limited to the pharyngeal muscle cells, whereas myosin heavy chains A and B are localized not only within the sarcomeres of body wall muscle cells, as reported previously, but to the smooth muscle cells of the minor groups as well. Myosin heavy chains A and B and paramyosin proteins appear to be compatible with functionally and structurally distinct muscle cell types that arise by multiple developmental pathways.
Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages.
Authors: Tokuyasu K T , Maher P A
Published: May 2004

Our initial attempts to immunolabel intact myocardial walls of 4-12 somite stage chick embryos were hindered by the presence of the cardiac jelly that covers the inner myocardial wall surface and prevents the access of antibodies to that surface. We overcame this difficulty by treating the specimens with hyaluronidase, which made the cardiac jelly permeable to the antibodies. An additional nonionic detergent treatment made the two or more cell layers of the myocardial wall accessible to the antibodies from both surfaces of the wall. Specimens treated in this manner were fluorescently labeled with antibodies to titin, myosin, or actin or with NBD-phallacidin for F-actin and examined as whole mount preparations or cut into semithin sections after resin embedding. These preparations and sections revealed that titin, a putative scaffolding protein of sarcomeres, is present in a punctate state and also in a diffuse form throughout the cytoplasm of cardiac myocytes in the premyofibril stages (4-7 somite stages) as well as in the early stages of myofibril formation. We interpreted the punctate and diffuse states to represent an aggregated state of several titin molecules and a dispersed state of individual titin molecules, respectively. In the 4-7 somite cardiac primodia, myosin and actin show only a uniform labeling throughout the cytoplasm of the myocytes. These observations are in contrast to a previous report that titin and myosin are tightly linked during in vitro skeletal myofibrillogenesis (Hill, C. S., S. Duran, Z. Ling, K. Weber, and H. Holtzer, 1986, J. Cell Biol., 103:2185-2196). In the 8-11 somite stage hearts, the number of individual titin spots rapidly reduces, while the number of myofibrils with periodically aligned titin spots increases, which strongly suggests that the titin spots are incorporated into the newly arising myofibrils. Titin spots were seen as doublets only after titin spots were incorporated into the first myofibrils. However, the fact that the distance between the components of the narrowest doublet was close to the resolution limit of the light microscope left open the possibility that undiscernible doublets of submicroscopic separations might exist in the premyofibril stages. The myosin labeling revealed the sarcomeric periodicity in an earlier stage of myofibril development than the F-actin labeling. In addition, we made two morphogenic observations.(ABSTRACT TRUNCATED AT 400 WORDS)
Distribution and cellular localization of actin depolymerizing factor.
Authors: Bamburg J R , Bray D
Published: May 2004

Actin depolymerizing factor (ADF) is a low molecular mass (19 kD) protein that forms a tightly bound dimeric complex with actin. We have raised a rabbit antiserum to chick brain ADF and used it to analyze the distribution and cellular localization of ADF. We find that ADF is a major constituent of all chick embryonic and most adult tissues examined, accounting for 0.1-0.4% of the total protein. Some tissues have as much as 0.6 mol ADF per mole actin. Adult heart and skeletal muscle are unusual in having very low levels of ADF: less than 0.02% of the soluble protein. During the development of skeletal muscle, ADF levels are maximal up to approximately 11 d in ovo and then decline to reach their adult levels by 14 d posthatching. Brain tissue and cultured cell lines from several other vertebrates, including mammals, all possess proteins of identical size to ADF that are recognized by the ADF antiserum. No proteins are specifically recognized by the ADF antiserum in extracts from Acanthamoeba castellanii or from nerve tissue of several invertebrates. Indirect immunofluorescence shows that ADF is present throughout the cytosol of most cells and at the leading edge of ruffled membranes and in the neuronal growth cone. Its abundance and widespread distribution together with its ability to sequester actin molecules, even those in an already polymerized state, suggest that ADF is a major factor in the regulation of actin filaments in many vertebrate cells.
Processing of rotavirus glycoprotein VP7: implications for the retention of the protein in the endoplasmic reticulum.
Authors: Stirzaker S C , Whitfeld P L , Christie D L , Bellamy A R , Both G W
Published: May 2004

Rotaviruses are icosahedral particles that assemble in the lumen of the endoplasmic reticulum (ER). The viral glycoprotein, VP7, is also directed into this compartment and is retained for assembly onto the surface of viral cores. VP7 is therefore a resident ER glycoprotein with a luminal orientation. The VP7 gene possesses two potential in-frame initiation codons, each preceding a hydrophobic domain. Mature VP7 is derived from a precursor by cleavage but the site of cleavage has not been determined because viral VP7 has a blocked amino terminus. Using site-directed mutagenesis of the gene and in vitro transcription and translation systems, we have investigated the synthesis and processing of the primary products synthesized from each initiation codon. Proteins translated from either codon were processed in vitro to yield products indistinguishable in size. The primary translation products therefore appeared to be cleaved at the same site. The site was located empirically between Ala50 and Gln51 and mutation of the gene to convert Ala50----Val prevented processing. Amino-terminal sequence analyses of proteins synthesized in vitro, and characterization of an amino-terminal fragment of VP7 purified from virus unequivocally established Gln51 as the amino-terminal residue. Pyroglutamic acid was tentatively identified as the blocking group. Processing of VP7 therefore removes both amino-terminal hydrophobic domains from the protein. Some other mechanism not requiring the presence of these hydrophobic sequences must account for the retention of this novel glycoprotein in the ER.
Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.
Authors: Bird P , Gething M J , Sambrook J
Published: May 2004

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.
Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential.
Authors: Imanaka T , Small G M , Lazarow P B
Published: May 2004

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S-Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.
Rat basophilic leukemia cells stiffen when they secrete.
Authors: Liu Z Y , Young J I , Elson E L
Published: May 2004

RBL cells provide a useful model of the IgE and antigen-dependent stimulus-secretion coupling of mast cells and basophils. We have measured cellular deformability to investigate the participation of cytoskeletal mechanical changes. Cross-linking cell-surface IgE-receptor complexes with multivalent ligands not only triggered secretion but also caused the cells to stiffen, i.e., to become more resistant to deformation. This mechanical response required receptor cross-linking, had a time course similar to that of secretion, and was reversed by DNP-L-lysine, a competitive inhibitor of antigen binding. Hence the same stimulus seems to elicit both stiffening and secretion. Cytochalasin D, which inhibits actin filament assembly, prevented or reversed stiffening, thereby implicating the cytoskeleton in the mechanical response. Increasing intracellular calcium ion concentration with the ionophore A23187 stiffened cells and stimulated secretion. Activation of protein kinase C with a phorbol ester also stiffened cells and enhanced both the stiffening and secretion caused by the ionophore. Yet cytochalasin D enhances secretion whereas activation of protein kinase c alone is insufficient for secretion. Therefore stiffening is neither necessary nor sufficient for secretion. These results characterize a cytoskeletal mechanical response triggered by the same receptor-dependent stimulus that elicits secretion and by second messengers that are thought to mediate between the receptor signal and secretion. The function of the mechanical response, however, remains to be determined.
Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state.
Authors: Koenig C S , Dabiké M , Bronfman M
Published: May 2004

Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl-secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine-stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by dense membrane profiles, analogous to the luminal processes of stimulated oxyntic cells. SDS-PAGE showed that, in purified membrane fractions of stimulated glands, the concentration of 28-, 43-, and 200-kD polypeptides increased while that of 95- and 250-kD polypeptides decreased. The present results define the tubular system of oxyntic cells as an organelle with properties different from those of endoplasmic reticulum, mitochondria, and plasma membrane. The biochemical and physico-chemical properties of this membraneous system changed when the organization of the membranes and the cytoskeletal matrix of the apical pole was modified by the onset of HCl secretion.
Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment.
Authors: De Lozanne A , Berlot C H , Leinwand L A , Spudich J A
Published: May 2004

The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.
Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping.
Authors: Dechesne C A , Bouvagnet P , Walzthöny D , Léger J J
Published: May 2004

Two mAbs, one specific for cardiac alpha-myosin heavy chains (MHC) and the other specific for cardiac beta-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their alpha-or beta-MHC content. alpha alpha-MHC and beta beta-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the alpha-MHC-specific mAb and the beta-MHC-specific mAb, alpha beta-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of alpha alpha-and beta beta-MHC homodimers and alpha beta-MHC heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of alpha- and beta-MHC in alpha alpha-MHC homodimers, beta beta-MHC homodimers, and alpha beta-MHC heterodimers, as initially proposed by Hoh, J. F. Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).
Regulation of human mesothelial cell differentiation: opposing roles of retinoids and epidermal growth factor in the expression of intermediate filament proteins.
Authors: Kim K H , Stellmach V , Javors J , Fuchs E
Published: May 2004

We report here the discovery that retinoids are potent regulators of epithelial morphology and keratin expression in cultured human mesothelial cells. When LP-9 cells are cultured in medium supplemented with vitamin A-depleted serum, they grow with an extreme spindle-shaped morphology and synthesize abundant levels of vimentin, but very little keratin. When retinoic acid is added to the medium at 1 X 10(-8) to 1 X 10(-6) M, keratin synthesis is increased, vimentin synthesis is decreased, and the cells assume an epithelioid morphology. Keratin synthesis, but not epithelioid morphology, seems to be dependent on cell density as well: even when vitamin A is present, sparse cultures cannot fully maintain keratin synthesis. In contrast, epidermal growth factor (EGF) acts in an antagonistic fashion to suppress both keratin synthesis and epithelial morphology. The effects of vitamin A, EGF, and cell shape on intermediate filament (IF) expression seem to occur in a growth-independent manner, and they appear to be at the level of transcription or mRNA stability. Even so, their effects on IF expression do not appear to be rapid ones, and hence it is unlikely that these agents interact directly at the gene level to cause changes in IF gene expression.
Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation.
Authors: King I A , Tabiowo A , Fryer P R
Published: May 2004

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity-purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin-sensitive components of desmosomes. Western blotting showed that the 78-kD glycopolypeptide was immunologically related to several other Con A-binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane-bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105-kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.
Fibronectin receptors of human keratinocytes and their expression during cell culture.
Authors: Toda K , Tuan T L , Brown P J , Grinnell F
Published: May 2004

Keratinocyte attachment to fibronectin (FN) substrata was inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by the variant peptide Gly-Arg-Gly-Glu-Ser-Pro. The RGDS-containing peptide did not inhibit keratinocyte adhesion to collagen. Keratinocyte adhesion to FN substrata also was inhibited by polyclonal anti-FN receptor antibodies originally prepared against the 140-kD FN receptors of Chinese hamster ovary (CHO) cells. Anti-CHO FN receptor antibodies did not, however, inhibit keratinocyte adhesion to collagen substrata. A monoclonal antibody designated VM-1 that was prepared against human basal keratinocytes inhibited keratinocyte adhesion to collagen but not to FN. Based on these results, we conclude that keratinocytes have distinct FN and collagen receptors. Experiments were performed to compare the expression of FN receptors on keratinocytes freshly isolated from skin and keratinocytes harvested from cell cultures. Cells harvested from keratinocyte cultures were able to neutralize the inhibitory activity of anti-CHO FN receptor antibodies and were able to attach and spread on anti-CHO FN receptor-coated substrata. Cells freshly harvested from skin, however, did not neutralize the antibodies, nor did they attach and spread on antibody-coated substrata. To learn more about the biochemical nature of the keratinocyte FN receptors, we performed immunoaffinity chromatography and immunoprecipitation experiments using the anti-CHO FN receptor antibodies. Extracts from metabolically radiolabeled, 10-d cultured keratinocytes contained FN receptors that had a 135-kD component under reducing conditions and 115- and 155-kD components under nonreducing conditions. Similar components were observed in extracts from surface-radiolabeled cells indicating that the FN receptors were expressed on keratinocyte cell surfaces. On the other hand, extracts from metabolically radiolabeled, 1-d cultured keratinocytes lacked intact FN receptors but contained a component that migrated at 48 kD under reducing conditions and 50 kD under nonreducing conditions. Because this fragment was not detected in surface-radiolabeled keratinocytes that were freshly isolated from skin, it seems likely that the fragment was located inside the cells rather than on the cell surface. A 50-kD FN receptor fragment also was observed in extracts from 10-d cultured keratinocytes if leupeptin and pepstatin were omitted from the extraction buffer. The results suggested that human keratinocytes cultured for 10 d express the 140-kD class of FN receptors, but that these receptors are not expressed on the surfaces of keratinocytes freshly isolated from skin.(ABSTRACT TRUNCATED AT 400 WORDS)
Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape.
Authors: Jiang L W , Schindler M
Published: May 2004

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.
The polarity and stability of microtubule capture by the kinetochore.
Authors: Huitorel P , Kirschner M W
Published: May 2004

We have studied the capture of microtubules by isolated metaphase chromosomes, using microtubules stabilized with taxol and marked with biotin tubulin to distinguish their plus and minus ends. The capture reaction is reversible at both the plus and minus ends. The on rate of capture is the same for both polarities but the dissociation rate from the kinetochore is seven times slower with microtubules captured at their plus ends than those captured at their minus ends. At steady state this disparity in off rates leads to the gradual replacement of microtubules captured at their minus ends with those captured at their plus ends. These results suggest that the kinetochore makes a lateral attachment near the end of the microtubule in the initial capture reaction and shows a structural specificity that may be important in proper bipolar attachment of the chromosome to the spindle.