scholarly journals Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association

2004 ◽  
Vol 70 (3) ◽  
pp. 1434-1441 ◽  
Author(s):  
Jennifer R. Kimbell ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Vibrio Fischeri ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apical Cell ◽  
Sodium Dodecyl ◽  
Duct Cell ◽  
Host Cells ◽  
Animal Cells ◽  
Pass Through ◽  
Induced Changes ◽  
Host Tissues

ABSTRACT The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells.

scholarly journals Alterations in the Proteome of the Euprymna scolopes Light Organ in Response to Symbiotic Vibrio fischeri

2000 ◽  
Vol 66 (9) ◽  
pp. 4091-4097 ◽  
Author(s):  
Judith Doino Lemus ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Vibrio Fischeri ◽  
Polyacrylamide Gel Electrophoresis ◽  
Critical Time ◽  
Euprymna Scolopes ◽  
Age Related ◽  
Marine Luminous Bacterium ◽  
Cooperative Association ◽  
Induced Changes ◽  
Host Tissues ◽  
Age Related Changes

ABSTRACT During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically, V. fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V. fischeri cells accelerates certain developmental processes of the symbiotic organ. These data suggest that V. fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

scholarly journals Induction of a Gradual, Reversible Morphogenesis of Its Host’s Epithelial Brush Border by Vibrio fischeri

1998 ◽  
Vol 66 (2) ◽  
pp. 777-785 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Brush Border ◽  
Vibrio Fischeri ◽  
Animal Cell ◽  
Morphological Changes ◽  
Confocal Scanning Laser Microscopy ◽  
Bacterial Cells ◽  
Filamentous Actin ◽  
Animal Cells ◽  
Fourfold Increase ◽  
Induced Changes

ABSTRACT Bacteria exert a variety of influences on the morphology and physiology of animal cells whether they are pathogens or cooperative partners. The association between the luminous bacterium Vibrio fischeri and the sepiolid squid Euprymna scolopesprovides an experimental model for the study of the influence of extracellular bacteria on the development of host epithelia. In this study, we analyzed bacterium-induced changes in the brush borders of the light organ crypt epithelia during the initial hours following colonization of this tissue. Transmission electron microscopy of the brush border morphology in colonized and uncolonized hosts revealed that the bacteria effect a fourfold increase in microvillar density over the first 4 days of the association. Estimates of the proportions of bacterial cells in contact with host microvilli showed that the intimacy of the bacterial cells with animal cell surfaces increases significantly during this time. Antibiotic curing of the organ following colonization showed that sustained interaction with bacteria is essential for the retention of the induced morphological changes. Bacteria that are defective in either light production or colonization efficiency produced changes similar to those by the parent strain. Conventional fluorescence and confocal scanning laser microscopy revealed that the brush border is supported by abundant filamentous actin. However, in situ hybridization with β-actin probes did not show marked bacterium-induced increases in β-actin gene expression. These experiments demonstrate that the E. scolopes-V. fischeri system is a viable model for the experimental study of bacterium-induced changes in host brush border morphology.

End-linked homodimers in fibrinogen Osaka VI with a Bβ-chain extension lead to fragile clot structure

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3779-3785 ◽  
Author(s):  
Teruko Sugo ◽  
Chizuko Nakamikawa ◽  
Nobuhiko Yoshida ◽  
Kazuki Niwa ◽  
Masazumi Sameshima ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Massive Bleeding ◽  
Chain Extension ◽  
Severe Bleeding ◽  
Transmission Electron ◽  
Pass Through ◽  
Clot Structure

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bβ gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient.

scholarly journals Lectin-Like Glycoprotein PsNLEC-1 Is Not Correctly Glycosylated and Targeted in Boron-Deficient Pea Nodules

2001 ◽  
Vol 14 (5) ◽  
pp. 663-670 ◽  
Author(s):  
Luis Bolaños ◽  
Arancha Cebrián ◽  
Miguel Redondo-Nieto ◽  
Rafael Rivilla ◽  
Ildefonso Bonilla
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Root Nodules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Immunogold Localization ◽  
Electrophoretic Mobilities ◽  
Cytoplasmic Vesicles ◽  
Infected Cells

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle

1996 ◽  
Vol 270 (1) ◽  
pp. C107-C114 ◽  
Author(s):  
C. Mundina-Weilenmann ◽  
L. Vittone ◽  
H. E. Cingolani ◽  
C. H. Orchard
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Troponin I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Physiological Salt Solution ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Induced Changes

Acidosis inhibits Ca2+ transport by the sarcoplasmic reticulum of cardiac muscle and decreases the Ca2+ sensitivity of the contractile proteins, although the mechanisms underlying these changes are unclear. We have investigated the hypothesis that changes in the phosphorylation of the regulatory proteins phospholamban and troponin I might play a role in the acidosis-induced changes in the function of the sarcoplasmic reticulum and the myofilaments, respectively. Langendorff-perfused rat hearts were labeled with 32P and then perfused with either control (pH 7.4) or acid (pH 6.8) physiological salt solution, in both the absence and presence of isoproterenol. The incorporation of 32P into phospholamban and troponin I was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sarcoplasmic reticulum and myofibrillar proteins, followed by autoradiography and liquid scintillation counting. The data show that acidosis has no effect on the phosphorylation of phospholamban in the absence of isoproterenol but that, in the presence of isoproterenol, acidosis increased the phosphorylation of phospholamban. However, acidosis increased the phosphorylation of troponin I, in both the absence and the presence of isoproterenol. Acidosis did not alter the adenosine 3',5'-cyclic monophosphate content of the hearts but did inhibit type 1 phosphatase. These data show that acidosis can alter the phosphorylation of these two proteins and suggest that these changes underlie, in part the changes observed in cardiac muscle during acidosis.

scholarly journals The ORF45 Protein of Kaposi's Sarcoma-Associated Herpesvirus Is Associated with Purified Virions

2003 ◽  
Vol 77 (7) ◽  
pp. 4221-4230 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
Early Stage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kaposi’S Sarcoma ◽  
Viral Envelope ◽  
Host Cells ◽  
Early Gene ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) ORF45 is encoded by an immediate-early gene in the KSHV genome. This protein was recently shown to interact with interferon regulatory factor 7 and inhibit virus-mediated alpha/beta interferon induction (Zhu et al., Proc. Natl. Acad. Sci. USA 99:5573-5578, 2002). ORF45 was characterized as a phosphorylated protein, and it is localized in the cytoplasm of infected cells. In this report, we provide evidence that ORF45 is associated with KSHV virions. (i) ORF45 was detected in gradient-purified virions by Western blotting along with known structural proteins of KSHV including gB, K8.1, and major capsid protein. In contrast, ORF50/Rta, K8α, and ORF59/PF8 were not detected in the same virion preparation. (ii) ORF45 comigrates with KSHV virions in sucrose gradient ultracentrifugation. (iii) Virion-associated ORF45 was resistant to trypsin digestion but became sensitive after the virions were treated with detergent which destroys the viral envelope. (iv) ORF45 remained associated with tegument-nucleocapsid complex when virion-specific glycoproteins were removed after detergent treatment. (v) An ORF45 protein band was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified KSHV virions and identified by mass spectrometry. (vi) By immunoelectron microscopy, virus-like structures were specifically stained by anti-ORF45 antibody. Based on the evidence, we conclude that ORF45 is associated with purified KSHV virions and appears to be a tegument protein. The presence of ORF45 in KSHV virions raised the possibility that this protein may be delivered to host cells at the start of infection and therefore have the opportunity to act at the very early stage of the infection, suggesting an important role of ORF45 in KSHV primary infection.

scholarly journals α-Enolase Resides on the Cell Surface of Mycoplasma fermentans and Binds Plasminogen

2007 ◽  
Vol 75 (12) ◽  
pp. 5716-5719 ◽  
Author(s):  
Amichai Yavlovich ◽  
Hagai Rechnitzer ◽  
Shlomo Rottem
Keyword(s):  
Cell Surface ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aminocaproic Acid ◽  
Immunoblot Analysis ◽  
Glycolytic Enzyme ◽  
Host Cells ◽  
Mycoplasma Fermentans ◽  
Triton X

ABSTRACT Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with ε-aminocaproic acid. The eluted ∼50-kDa protein was identified by mass spectrometric techniques as α-enolase. The possibility that α-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-α-enolase antiserum, which showed that α-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-α-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface α-enolase is a major Plg binding protein of M. fermentans.

scholarly journals Production of Secretory Immunoglobulin A against Shiga Toxin-Binding Subunits in Mice by Mucosal Immunization

2004 ◽  
Vol 72 (2) ◽  
pp. 889-895 ◽  
Author(s):  
Yasuyuki Imai ◽  
Rio Nagai ◽  
Yousuke Ono ◽  
Tomoyuki Ishikawa ◽  
Hiroki Nakagami ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin A ◽  
Host Cells ◽  
Secretory Iga ◽  
Major Histocompatibility Complex Haplotype ◽  
Intranasal Immunization ◽  
B Subunits ◽  
The Cross

ABSTRACT The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.

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