Immunologic evidence that staphylococcal alpha toxin is oriented on membranes

1979 ◽  
Vol 25 (6) ◽  
pp. 686-692 ◽  
Author(s):  
C. Y. Lo ◽  
H. B. Fackrell
Keyword(s):  
Membrane Binding ◽  
The Other ◽  
Erythrocyte Membranes ◽  
Alpha Toxin ◽  
Binding Region ◽  
Indirect Hemagglutination

Antibodies to staphylococcal alpha toxin were separated into two distinct populations. One population prevented binding of alpha toxin onto erythrocyte membranes. The other population neutralized after the toxin was bound onto erythrocytes and thereby brought about an indirect hemagglutination reaction.The data suggest that alpha toxin has a membrane-binding region.

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312 ◽  
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

Abstract This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

scholarly journals Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.

1997 ◽  
Vol 8 (3) ◽  
pp. 421-430 ◽  
Author(s):  
S Sun ◽  
M Footer ◽  
P Matsudaira
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Solvent Accessibility ◽  
The Other ◽  
Actin Binding ◽  
Binding Region ◽  
Binding Studies ◽  
Chemically Modified ◽  
Cross Links ◽  
Actin Binding Site

In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837.

scholarly journals A19F-NMR study of the membrane-binding region of D-lactate dehydrogenase ofescherichia coli

1993 ◽  
Vol 2 (11) ◽  
pp. 1938-1947 ◽  
Author(s):  
Zhen-Yu Sun ◽  
Hoai-Thu N. Truong ◽  
E. A. Pratt ◽  
David C. Sutherland ◽  
Christine E. Kulig ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Membrane Binding ◽  
Ofescherichia Coli ◽  
Binding Region ◽  
Nmr Study

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Isolation of Vi Antigen from Escherichia coli by a New Method

1970 ◽  
Vol 1 (1) ◽  
pp. 69-73
Author(s):  
Vito M. Esposito ◽  
John C. Feeley
Keyword(s):  
Escherichia Coli ◽  
Trichloroacetic Acid ◽  
Sedimentation Coefficient ◽  
The Other ◽  
Distilled Water ◽  
Saline Extract ◽  
Indirect Hemagglutination ◽  
Indirect Hemagglutination Test ◽  
The Relationship ◽  
Vi Antigen

Vi antigen was isolated from a trichloroacetic acid-deproteinized saline extract of acetone-killed and -dried cells of Escherichia coli (5396/38) by ethyl alcohol precipitation and absorption on diethylaminoethyl-Sephadex. Stepwise elution with distilled water, 0.15 m NaCl, and 0.5 m NaCl resulted in recovery of Vi antigen and two additional antigenic fractions (designated antigens 1 and 2). Vi antigen was an acidic polymer which flocculated readily with albumin, contained 6.0% nitrogen, and had a sedimentation coefficient of 14.7 S . Vi was shown to be antigenic in the rabbit by the indirect hemagglutination test and in mice by tests for protection against challenge with Salmonella typhosa , wherein it was more protective by the intraperitoneal (ip) than by the subcutaneous (sc) route. Vi antigen prepared by this method was much more protective for mice than was the Vi antigen used in previous studies. The other antigens, not fully characterized, were also capable of protecting mice. Antigen 1 was more effective by the sc than the ip route, whereas antigen 2 was more effective by the ip route. The relationship between Vi antigen and antigens 1 and 2, as demonstrated by hemagglutination tests with rabbit antisera, remains unclear.

Alterations in the band3/Rh Protein Complex in Erythrocyte Membranes of Protein 4.2 (Chartres) deficient Cells Occur Late during Erythropoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3460-3460
Author(s):  
Emi le van den Akker ◽  
Timothy J Satchwell ◽  
Jo F Flatt ◽  
Stephanie Pellegrin ◽  
M. Maigre ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Band 3 ◽  
Red Cells ◽  
The Other ◽  
Erythrocyte Membranes ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Core Domain ◽  
Protein 4.2 ◽  
Exon 9

Abstract We report on a 40 year old patient with mild hereditary spherocytosis (RBC: 4.43×1012/dL; Reticulocyte count: 253×109/dL; Hb: 14g/dL), whose red blood cells completely lack protein 4.2. Genetic analysis showed that the patient was a double heterozygote for EPB42 deletions; one allele lacked exon 9 but the sequence remained in frame (protein 4.2 Chartres I) and the other allele contained a di-nucleotide deletion resulting in a premature stop signal (protein 4.2 Chartres II). Homology modelling showed that the hairpin region that forms the proposed band 3 binding site is still present in both mutants. However, the deletion of exon 9 removes a large portion of Domain 2 (core domain) of protein 4.2, potentially removing a band 3 binding groove, and the truncation mutant lacks a portion of the core domain and the whole domains 3 and 4. Therefore, these mutations are likely to destabilize protein 4.2 either directly, or indirectly by disturbing the interaction of protein 4.2 with band 3. Flow cytometry, SDS-PAGE and Western blotting of erythrocyte membranes showed a significant reduction of 70–80 % in CD47 levels, altered Rh associated glycoprotein (RhAG) mobility, reduced GPA/GPB heterodimers, and a 3 fold increase in CD44 levels as reported previously for protein 4.2 null red cells. We stored mature red cells at 4 degrees Celsius over 35 days and found that CD47 continues to be lost in microvesicles as the red cell ages, consistent with a weaker link of CD47 with the cytoskeleton. We investigated band 3 complex stability by performing co-immunoprecipitations and found that lower amounts of band 3 were co-immunoprecipitated using an anti-ankyrin antibody in Chartres red cells compared to wild type, suggesting that the association of band 3 with the cytoskeleton is severely affected. Furthermore, less band 3 was co-immunoprecipitated with an anti-RhAG antibody, consistent with a disturbance of the association of the Rh complex with band 3. We next investigated the stage during erythropoiesis at which the observed changes in band 3 macrocomplex proteins occur. To this end we expanded and differentiated erythroid progenitors from peripheral blood of wild type and the Chartres patient using a three culture system modified from Leberbauer et al. (2005). Synchronous differentiation of a pure erythroid progenitor pool (60% enucleation) demonstrated that protein 4.2 co-immunoprecipitated with band 3 early on in erythroid progenitor differentiation. However, in protein 4.2 Chartres progenitors the mutant forms of protein 4.2 were not expressed at any stage during erythropoiesis, demonstrating that both protein 4.2 mutants are unstable and rapidly degraded. Surprisingly, flow cytometry and western blot analysis revealed that CD47, RhAG, band 3, CD44, and GPA/GPB levels are all similar compared to wild type during erythroid differentiation. Thus, despite the absence of protein 4.2 throughout erythropoiesis, the final changes in the Rh/band3 complex observed in patient’s erythrocytes are not observed. Overall our results suggest that protein 4.2 Chartres is unstable probably due to specific 4.2 mutations that either cause disruption of the band 3 binding sites or an intrinsic instability of these individual mutant proteins. The association of band 3 and ankyrin also appears to be altered in protein 4.2 Chartres suggestive of a weakening of the band 3 cytoskeleton linkage, which could also contribute to the HS phenotype. Importantly, the absence of protein 4.2 not only disturbs ankyrin recruitment to band 3 but also affects association of band 3 with RhAG and disturbs GPA/GPB complexes, which demonstrates the importance of protein 4.2 in the process of band 3 complex formation. Most strikingly, our work demonstrates that the loss of CD47 and the other alterations observed in the band 3/Rh complex in protein 4.2 Chartres must occur late during red blood cell progenitor maturation, presumably after enucleation.

scholarly journals Toxoplasma gondii: infection among shelter and stray cats in Rio de Janeiro, Brazil

2018 ◽  
Author(s):  
Pâmela Figueiredo Pereira ◽  
Alynne da Silva Barbosa ◽  
Ana Leticia Carvalho Santos ◽  
Paula Forain Bolais ◽  
Marie-Laure Dardé ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Rio De Janeiro ◽  
The Other ◽  
Hemagglutination Assay ◽  
Blood Samples ◽  
Stray Cats ◽  
Serological Status ◽  
Indirect Hemagglutination ◽  
Toxoplasma Gondii Infection ◽  
The City

Abstract The aim of this study was to identify possible infection of Toxoplasma gondii among cats in a shelter and a set of condominiums in the city of Rio de Janeiro, through changes to the cats’ serological status between two different times in 2014 and 2015. One group was made up of captive cats at the municipal shelter and the other comprised stray cats that circulated in condominiums in the city. On the first occasion, cats were caught and tagged through application of microchips; in this manner, blood samples were obtained from 261 captive cats and 172 stray cats. On the second occasion, blood samples were obtained from 94 captive cats and 56 recaptured stray cats. The serological diagnosis was made by means of the indirect hemagglutination assay (IHA) and indirect immunofluorescence reaction (IFAT) (cutoff ≥ 64). The frequency of T. gondii infection among the captive cats was 24.5% and among the stray cats, 18%. With the second analysis, it was possible to verify modifications to the serological status of anti-T. gondii antibodies, in 18% of both populations of animals. The presence of seroconversion shows that infection was possibly occurring in the region at the time of the study.

Coexisting independent sodium-sensitive and sodium-insensitive mechanisms of genetic hypertension in spontaneously hypertensive rats (SHR)

2001 ◽  
Vol 79 (9) ◽  
pp. 779-784 ◽  
Author(s):  
Ibert C Wells ◽  
Alan J Blotcky
Keyword(s):  
Spontaneously Hypertensive Rats ◽  
Plasma Membranes ◽  
The Other ◽  
Erythrocyte Membranes ◽  
Hypertensive Rats ◽  
Rat Strains ◽  
Spontaneously Hypertensive ◽  
Enhanced Diffusion ◽  
Enzyme Systems ◽  
Intracellular Ca

Some essential hypertensive patients and genetic hypertensive rat strains have less than the normal levels of Mg2+ tightly bound to the plasma membranes of their erythrocytes and other cells, i.e., the magnesium binding defect (MgBD). This binding defect appears to cause increased passive permeability of the membrane to Na+ and thereby its increased intracellular concentration, particularly if the Na+-extrusion enzyme systems of the cell are also defective. The Na+-Ca2+ exchange system in the cell membrane exports Na+ and imports Ca2+, increasing the tone of the smooth muscle cell and thus producing hypertension (HTn). This HTn is Na+-sensitive. Evidence supporting this postulate was obtained by determining the intraerythrocyte total concentrations of Na+, Ca2+, K+, and Mg2+ in two strains of spontaneously hypertensive rats (SHR and SS/Jr rats, having the MgBD together with the other requisites of the Na+-sensitive pathway) and their respective controls (WKY and SR/Jr rats, in which this complete pathway is absent). The Na+ and Ca2+ concentrations in the hypertensive rats were increased, and that of K+ was decreased. The concentrations of these cations were very similar in the two hypertensive strains. The level of membrane tightly bound Ca2+ in SHR erythrocyte membranes was significantly higher than those in the other three rat strains, which were not statistically different from each other. These results support previously reported evidence of the existence of a novel HTn-generating mechanism in the SHR rat, in which the intracellular Ca2+ concentration is increased as the result of the enhanced diffusion of this ion into the cell and the accompanying deficiency of the Ca2+ extrusion enzyme systems. This pathway is therefore Na+-insensitive, i.e., Ca2+-sensitive.Key words: essential hypertension, Na+-sensitive hypertension, Na+-insensitive hypertension, Ca2+-sensitive hypertension.

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