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.

scholarly journals Infection of Macrophage-Like THP-1 Cells withMycobacterium avium Results in a Decrease in Their Ability to Phosphorylate Nucleolin

2000 ◽  
Vol 68 (6) ◽  
pp. 3121-3128 ◽  
Author(s):  
Rodolfo C. Garcia ◽  
Elena Banfi ◽  
Maria G. Pittis
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Host Response ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequencing ◽  
Intact Cells ◽  
Infected Cells

ABSTRACT This study of the phosphorylation ability of macrophage-like cells upon infection with Mycobacterium avium was undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [γ-32P]ATP, in the presence of Mg2+ and the absence of Ca2+, and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingestedEscherichia coli or killed M. avium. The 110-kDa phosphoprotein was present in the soluble fraction (230,000 ×g supernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of32P-labeled intact cells.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

1982 ◽  
Vol 152 (2) ◽  
pp. 757-761
Author(s):  
V L Sheladia ◽  
J P Chambers ◽  
J Guevara ◽  
D J Evans
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Amino Terminus ◽  
N Terminus ◽  
Type V

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.

scholarly journals Identification and Characterization of a Neospora caninum Microneme-Associated Protein (NcMIC4) That Exhibits Unique Lactose-Binding Properties

2004 ◽  
Vol 72 (8) ◽  
pp. 4791-4800 ◽  
Author(s):  
Nadine Keller ◽  
Michèle Riesen ◽  
Arunasalam Naguleswaran ◽  
Nathalie Vonlaufen ◽  
Rebecca Stettler ◽  
...  
Keyword(s):  
Host Cell ◽  
Gel Electrophoresis ◽  
Neospora Caninum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Vero Cells ◽  
Host Cells ◽  
Immunogold Electron Microscopy ◽  
Binding Properties ◽  
Microneme Protein

ABSTRACT Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M r but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

scholarly journals Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase

2007 ◽  
Vol 51 (12) ◽  
pp. 4512-4514 ◽  
Author(s):  
Fátima Fonseca ◽  
Ana Cristina Sarmento ◽  
Isabel Henriques ◽  
Bart Samyn ◽  
Jozef van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Molecular Mass ◽  
Clavulanic Acid ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class A

ABSTRACT The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

scholarly journals Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

1983 ◽  
Vol 213 (1) ◽  
pp. 187-191 ◽  
Author(s):  
A Lewendon ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Step ◽  
Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

scholarly journals The purification of shikimate dehydrogenase from Escherichia coli

1985 ◽  
Vol 226 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Chaudhuri ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Shikimate Dehydrogenase ◽  
E Coli ◽  
Gel Permeation

A procedure was developed for the purification of shikimate dehydrogenase from Escherichia coli. Homogeneous enzyme with specific activity 1100 units/mg of protein was obtained in 21% overall yield. The subunit Mr estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 32 000. The native Mr, estimated by gel-permeation chromatography on a TSK G2000SW column, was also 32 000. E. coli shikimate dehydrogenase is therefore a monomeric NADP-linked dehydrogenase.

scholarly journals The composition and the structure of bacterioferritin of Escherichia coli

1981 ◽  
Vol 197 (1) ◽  
pp. 171-175 ◽  
Author(s):  
J Yariv ◽  
A J Kalb ◽  
R Sperling ◽  
E R Bauminger ◽  
S G Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Electron Microscope ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iron Compound ◽  
Protein Subunit ◽  
Electron Dense Core

Bacterioferritin isolated from Escherichia coli is of two kinds: a protein containing a polynuclear iron compound, the bacterioferritin proper and a protein free of the polynuclear iron compound, the apo-bacterioferritin. Bacterioferritin of both kinds is characterized by absorption maxima at 417,530 and 560 nm, contributed by protohaem IX. Single crystals of bacterioferritin of the space group I432 suggest that the molecule is made up of 24 identical subunits related by a cubic point symmetry. The molecular weight of the protein subunit, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is 15000. In the electron microscope the bacterioferritin molecule appears to be a sphere of 9.5 nm (95 A) diameter composed of a negatively staining outer shell and an inner electron-dense core of 6 nm (60 A) diameter.

scholarly journals Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle

2007 ◽  
Vol 73 (7) ◽  
pp. 2037-2047 ◽  
Author(s):  
Ji Youn Lim ◽  
Haiqing Sheng ◽  
Keun Seok Seo ◽  
Yong Ho Park ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Differentially Expressed ◽  
Sequence Matching ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli ◽  
Cell Lysates

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ∼50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

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