Quantitative immunocytochemical localization of four immunodominant antigens of Toxoplasma gondii

1990 ◽  
Vol 48 (3) ◽  
pp. 946-947
Author(s):  
A. Bonhomme ◽  
F. Boulanger ◽  
S. Lebonvallet ◽  
N. Bonnet ◽  
L.M. Bharadwaj ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Surface Membrane ◽  
Surface Protein ◽  
Surface Proteins ◽  
Gold Complex ◽  
Image Analysis System ◽  
Thin Sections ◽  
Immunodominant Antigens ◽  
Analysis System ◽  
Major Surface

Four immunodominant antigens frequently observed in congenital, acquired and reactivated toxoplasmosis have been localized on Toxoplasma gondii ultrathin sections using four monoclonal antibodies (Mab GII9, IV47, IE10 II38) which detect membrane and cytoplasmic antigens. Thin sections as well as the whole surface of RH tachyzoïtes of Toxoplasma gondii are immunolabeled with the different Mab and Biotin-streptavidin-colloïdal gold complex. The quantitative analysis of immunogold labeling was performed with the help of a semi-automatic procedure developped by us, using a digital image analysis system (Bio 500-BIOCOM, les Ullis, FRANCE). The number of gold particles per unit surface are quantified inside three cellular compartments defined on the video screen : surface membrane, submembrane area and rhoptries.On the whole cell surface, proteins of 30 kDa (P30) detected with Mab GII9 were the major surface protein (Fig 1). By quantitative evaluation, they were 50 fold more abundant than P45-50kDa (recognized by Mab II38) proteins and 7 fold more abundant than P66-70kDa (recognized by Mab IE10) proteins. For the proteins of 28 kDa detected with Mab IV47 the labeling was very intensive in the submembrane area (Fig 3).

Enrichment and biochemical characterization of Toxoplasma gondii tachyzoite plasmalemma

Parasitology ◽  
1997 ◽  
Vol 114 (5) ◽  
pp. 421-426 ◽  
Author(s):  
A. RABJEAU ◽  
F. FOUSSARD ◽  
G. MAURAS ◽  
J. F. DUBREMETZ
Keyword(s):  
Toxoplasma Gondii ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Surface Membrane ◽  
Surface Protein ◽  
Protozoan Parasite ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Partial Dissociation ◽  
Major Surface

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0·7M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS–PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20–100 nm) which possess a low density (1·085–1·090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1·12–1·16 g/cm3).

The interendothelial junction in myocardial capillaries: evidence for the existence of regularly spaced, cleft-spanning structures

1992 ◽  
Vol 101 (3) ◽  
pp. 647-655
Author(s):  
C. Schulze ◽  
J.A. Firth
Keyword(s):  
Coronary Circulation ◽  
Image Analysis System ◽  
Thin Sections ◽  
Computerized Image Analysis ◽  
Zonula Occludens ◽  
Transmission Electron ◽  
Rat Hearts ◽  
Analysis System ◽  
Wide Zone ◽  
Hydrophilic Solutes

Water and hydrophilic solutes cross the endothelium of continuous capillaries via the paracellular cleft and possibly other routes. This pathway shows a selectivity to molecule size and charge. However, it is not yet known which systems confer this selectivity. Isolated rat hearts were perfusion-fixed through the coronary circulation, stained with lanthanum or tannic acid, and further processed for transmission electron microscopy. Thin sections viewed at × 160,000 magnification revealed regularly spaced, cleft-spanning structures in the wider zone of a small percentage of clefts in addition to at least one zonula occludens. Goniometric tilting of the specimen in steps of 5 degrees perpendicular to the plane of the wide zone showed that such “linkers” can be revealed in at least 40% of all clefts. They become visible at some tilt angles, although the same area of the cleft is featureless at other angles. Single linker spacing measurements were obtained using a computerized image analysis system, and compiled in a frequency distribution chart. On the basis of these data, two models of a regular linker distribution within the cleft are illustrated. Our results provide evidence for the presence of regularly spaced, cleft-spanning structures within the interendothelial cleft which may have implications for endothelial cell-cell adhesion and permeability.

scholarly journals Development of a Multivalent ISCOM Vaccine against Anaplasmosis

1993 ◽  
Author(s):  
Guy H. Palmer ◽  
Eugene Pipano ◽  
Terry F. McElwain ◽  
Varda Shkap ◽  
Donald P. Knowles, Jr.
Keyword(s):  
T Lymphocytes ◽  
Outer Membrane ◽  
Membrane Surface ◽  
Surface Protein ◽  
The United States ◽  
Surface Proteins ◽  
Efficient Production ◽  
Major Surface ◽  
Homologous Challenge

Anaplasmosis is an arthropod+borne disease of cattle caused by the rickettsia Anaplasma marginale and an impediment to efficient production of healthy livestock in both Israel and the United States. Our research focuses on development of a recombinant membrane surface protein (MSP) immunogen to replace current vaccines derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Briefly, we accomplished the following in our BARD supported research: i) characterization of the intramolecular and intermolecular relationships of the native Major Surface Proteins (MSP) in the outer membrane; ii) expression, purification, and epitope characterization of the recombinant MSP-2, MSP-3, MSP-4, and MSP-5 proteins required to construct the recombinant ISCOM; iii) demonstration that the outer membrane-Quil A induces CD4+ T lymphocytes specific for the outer membrane polypeptides; iv) identification of CD4+ T lymphocytes that recognize outer membrane polypeptide epitopes conserved among other wise antigenically distinct strains; v) determination that immunization with the outer membrane-Quil A construct does not affect the ability of ticks to acquire or transmit A. marginale; and vi) demonstration that the outer membrane-Quil A construct induces complete protection against rickettsemia upon homologous challenge and significant protection against challenge with antigenically distinct strains, including tick transmission. Importantly, the level of protection against homologous challenge in the MSP vaccinates was comparable to that induced by live blood-based vaccines and demonstrates that development of a new generation of vaccines is feasible.

scholarly journals Similarities between the primary structures of two distinct major surface proteins of Toxoplasma gondii.

1994 ◽  
Vol 269 (23) ◽  
pp. 16217-16222 ◽  
Author(s):  
M.F. Cesbron-Delauw ◽  
S. Tomavo ◽  
P. Beauchamps ◽  
M.P. Fourmaux ◽  
D. Camus ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Surface Proteins ◽  
Major Surface Proteins ◽  
Primary Structures ◽  
Major Surface

scholarly journals Conformational Dependence ofAnaplasma marginale Major Surface Protein 5 Surface-Exposed B-Cell Epitopes

1998 ◽  
Vol 66 (6) ◽  
pp. 2619-2624 ◽  
Author(s):  
Devere Munodzana ◽  
Terry F. McElwain ◽  
Donald P. Knowles ◽  
Guy H. Palmer
Keyword(s):  
Surface Protein ◽  
Covalent Modification ◽  
Surface Proteins ◽  
Immunodominant Epitope ◽  
Disulfide Bonding ◽  
Amino Terminal ◽  
Major Surface Proteins ◽  
Major Surface ◽  
Carboxy Terminal ◽  
Epitope Binding

ABSTRACT The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denaturedA. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

scholarly journals Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis

2019 ◽  
Vol 20 (3) ◽  
pp. 552 ◽  
Author(s):  
Noelia Dasilva-Freire ◽  
Andrea Mayado ◽  
Cristina Teodosio ◽  
María Jara-Acevedo ◽  
Iván Álvarez-Twose ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Systemic Mastocytosis ◽  
Expression Profiles ◽  
Surface Membrane ◽  
Surface Protein ◽  
Surface Proteins ◽  
Significant Fraction ◽  
Surface Protein Expression ◽  
Protein Expression Profiles

Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM.

scholarly journals Thermostability of Reovirus Disassembly Intermediates (ISVPs) Correlates with Genetic, Biochemical, and Thermodynamic Properties of Major Surface Protein μ1

2002 ◽  
Vol 76 (3) ◽  
pp. 1051-1061 ◽  
Author(s):  
Jason K. Middleton ◽  
Tonya F. Severson ◽  
Kartik Chandran ◽  
Anne Lynn Gillian ◽  
John Yin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Thermal Inactivation ◽  
Surface Protein ◽  
Surface Proteins ◽  
Major Surface Protein ◽  
First Order ◽  
Major Surface Proteins ◽  
The Difference ◽  
Major Surface ◽  
Type 3

ABSTRACT Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein μ1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound μ1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of μ1 as a determinant of thermostability. Intact virions, which contain ς3 bound to μ1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for ς3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.

scholarly journals A liver-specific nuclear factor interacts with the promoter region of the large surface protein gene of human hepatitis B virus.

1989 ◽  
Vol 9 (11) ◽  
pp. 5189-5197 ◽  
Author(s):  
H K Chang ◽  
B Y Wang ◽  
C H Yuh ◽  
C L Wei ◽  
L P Ting
Keyword(s):  
Hepatitis B Virus ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Surface Protein ◽  
Surface Proteins ◽  
Nuclear Factor ◽  
Hepatoma Cell Lines ◽  
B Virus ◽  
Major Surface Proteins ◽  
Major Surface

The outer envelope of the 42-nm virion of the human hepatitis B virus (HBV) is composed of the large, the middle, and the major surface proteins. Whereas the middle and the major surface proteins are transcribed from the SPII promoter of the pre-S/S gene, the large surface protein is transcribed from the SPI promoter located upstream of SPII. We have previously shown that transcription of SPI (comprising nucleotides [nt] -380 to +17) occurs preferentially in differentiated hepatoma cell lines (H.K. Chang and L.P. Ting, Virology 170:176-183, 1989). In this report, we further demonstrated that a sequence of 95 base pairs in the upstream region of SPI (nt -95 to +17) was necessary and sufficient for such preferential expression in differentiated hepatoma cells. By analysis of the expression of the chloramphenicol acetyltransferase gene in a series of mutants with deletions at the 5' end of SPI, we identified a positive transcriptional cis-acting element mapping at nt -95 to -72 which appears to play a key role in the regulation of the expression of the large surface protein. This region shared a high degree of sequence homology with regulatory sequences of several liver-specific genes from human, mouse, and rat, with a consensus sequence (G/A)GTTA(A/C)TNNT(C/T)NNC(A/C). We further identified a nuclear factor present in the nuclear extracts of differentiated human hepatoma cell lines which interacted specifically with this element of the SPI promoter. This nuclear factor was similar to the rat liver-specific factor HNF-1, since an oligonucleotide containing the recognition sequence of HNF-1 could efficiently compete for the human factor in a footprinting assay. The sequence at nt -93 to -68 which was bound by this factor in SPI was termed the HNF-1-binding element. Activation of the SPI promoter by human differentiated hepatocyte nuclear factor 1, described in this report, probably explains, first, the formation of the 42-nm virion specifically in liver but not in several other tissues despite the synthesis of the middle and the major surface proteins in those tissues, and second, why only differentiated hepatoma cell lines are able to produce 42-nm-like virion particles on transfection by HBV DNA.

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