Possible Cross-Reactivity of Human Anti-Mitochondrial Antibodies with Membrane Vesicles of Paracoccus denitrificans

1976 ◽  
Vol 4 (1) ◽  
pp. 138-139 ◽  
Author(s):  
THOMAS J. SAYERS ◽  
HAROLD BAUM
Keyword(s):  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Cross Reactivity

Competition between electron acceptors in membrane vesicles of Paracoccus denitrificans

1981 ◽  
Vol 9 (4) ◽  
pp. 324-326 ◽  
Author(s):  
ROBERT JONES ◽  
JULIAN O. D. COLEMAN ◽  
F. R. WHATLEY
Keyword(s):  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Electron Acceptors

scholarly journals Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

1999 ◽  
Vol 181 (15) ◽  
pp. 4605-4610 ◽  
Author(s):  
Delfina C. Dominguez ◽  
Hank Adams ◽  
James H. Hageman
Keyword(s):  
Bacillus Subtilis ◽  
Cellular Localization ◽  
Time Course ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Bovine Brain ◽  
Ruthenium Red ◽  
Thin Sections ◽  
Calcium Ion Transport

ABSTRACT To determine possible functions of the calmodulinlike protein ofBacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [45Ca]CaCl2, ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles ofB. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

scholarly journals New strategy to select cross-reactivity Meningococci strains: immunization with Outer membrane vesicles of serogroup C and cationic lipid as adjuvant

2021 ◽  
Author(s):  
Amanda Izeli Portilho ◽  
Gabriela Trzewikowski de Lima ◽  
Elizabeth De Gaspari
Keyword(s):  
Outer Membrane ◽  
Public Health Problem ◽  
Cationic Lipid ◽  
Humoral Response ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Outer Membrane Vesicles ◽  
High Avidity ◽  
Avidity Index ◽  
High Avidity Index

Background: Invasive meningococcal disease (IMD), caused by Neisseria meningitidis, is a public health problem, associated with high levels of morbidity and mortality, capable of causing outbreaks or epidemics, but preventable through vaccination. In Brazil, the main serogroups isolated are C and B. The last epidemic occurred in the 80s, in Sao Paulo, because of a B:4:P1.15 strain. Methods: Adult Swiss mice were immunized with outer membrane vesicles (OMV) of N. meningitidis strain C:4:P1.15, adjuvanted by the cationic lipid dioctadecyldimethylammonium bromide in bilayer fragments (DDA-BF), administered via prime-booster (intranasal/subcutaneous) scheme. The humoral response was accessed by Immunoblotting and ELISA, using homologous immunization strain and a different serogroup but equal serosubtype strain, N. meningitidis B:4:P1.15. Results: Immunoblotting revealed the recognition of antigens associated with the molecular weight of Porin A and Opacity proteins, which are immunogenic but highly heterogeneous, and Tbp and NspA, which are more homogeneous between meningococci strains. ELISA results showed antibody production that persisted after 190 days and recognized the C:4:P1.15 and the B:4:P1.15 strains, with high avidity index. The adjuvanted group recognized antigens following the IN prime and had a higher avidity index against the heterologous strain. Conclusions: DDA-BF improved the humoral response, but the OMV alone induced high avidity index antibodies as well. Even though these are preliminary results, we see it as a promising approach for affordable meningococcal immunization in developing countries, at outbreak or epidemic situations.

scholarly journals Cross-reactivity with Brazilian strains of Neisseria meningitidis B after immunization with outer membrane vesicles

2019 ◽  
Vol 7 ◽  
pp. 251513551989482
Author(s):  
Gabriela Trzewikoswki de Lima ◽  
Amanda Izeli Portilho ◽  
Elizabeth De Gaspari
Keyword(s):  
Public Health ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Immunosorbent Assay ◽  
Humoral Response ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Outer Membrane Vesicles ◽  
Antigen Recognition ◽  
Enzyme Linked Immunosorbent Assay

Background: Immunization against Neisseria meningitidis is important for public health. Vaccines composed of cross-reactivity antigens avoid strain-specific responses, ensuring more comprehensive protection. Methods: The cross-reactivity between three strains from the last outbreak of N. meningitidis in Brazil was assessed in our studies, using enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays. Results: Both assays verifed a similar humoral response between the strains evaluated. Patterns of antigen recognition differed with each dose evaluated. Conclusions: We observed that immunization with N. meningitidis B outer membrane vesicles (OMVs) led to the production of antibodies that recognized antigens of heterologous strains, indicating possible protection against these evaluated strains.

scholarly journals Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria.

1983 ◽  
Vol 96 (5) ◽  
pp. 1306-1315 ◽  
Author(s):  
D B Murphy ◽  
K T Wallis ◽  
R R Hiebsch
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Mitochondrial Atpase ◽  
Atpase Inhibitors ◽  
Native Gel ◽  
High Molecular Weight Proteins

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two-dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min-1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.

Respiration-driven accumulation of C4 dicarboxylic acids by isolated membrane vesicles of Paracoccus denitrificans

1979 ◽  
Vol 57 (5) ◽  
pp. 436-443 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Sole Carbon Source ◽  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Dicarboxylic Acids ◽  
Energy Coupling ◽  
Carbon Limitation ◽  
Dependent Manner ◽  
Dicarboxylate Transport ◽  
Osmotic Lysis ◽  
Isolated Membrane Vesicles

Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.

scholarly journals Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

Open Biology ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 170206 ◽  
Author(s):  
Febin Varghese ◽  
James N. Blaza ◽  
Andrew J. Y. Jones ◽  
Owen D. Jarman ◽  
Judy Hirst
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Inhibitor Protein ◽  
Wild Type ◽  
Efficient Energy ◽  
Inverted Membrane Vesicles ◽  
Atp Synthases

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans

1980 ◽  
Vol 58 (10) ◽  
pp. 996-1003 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Intact Cell ◽  
Respiratory Control ◽  
Membrane Vesicles ◽  
Bee Venom ◽  
Respiratory Enzymes ◽  
Protein Ratio ◽  
Membrane Topography ◽  
Considerable Controversy

There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria. It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans. The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD: ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of non-permeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control. Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.

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