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Biomedical subjects

E J Victoria

Publications and source records attributed to E J Victoria.

At least 37 records · Page 2Linked to original sources
PubMed1981

Some properties of alkali-extracted red cell ghost membranes.

The properties of integral membrane proteins obtained by dilute alkali extraction of red cell ghosts were examined. A variety of conditions promoted the disulfide-mediated aggregation of integral membrane proteins, principally band 3. Procedural modifications which minimized aggregation were the use of EDTA and S-alkylation. Integral membrane proteins were solubilized under non-denaturing conditions using Brij 36T, a lauryl polyoxyethylene ether with an NMR-determined average chain length of 8.2 (oxyethylene) units. Detergent gel filtration revealed a chromatographic shoulder due to aggregated band 3 when membrane proteins were not alkylated. Analyses of the column profile also revealed a discrete peak for sialoglycoproteins and two phosphate peaks, an early one due to phospholipid and a later one not identified, but probably due to phosphoinositide.

Cell Aggregation↗
PubMed1981

Anti-Rho(D) IgG binds to band 3 glycoprotein of the human erythrocyte membrane.

Alkali-extracted erythrocyte ghost membranes from Rho(D)-positive and Rho(D)-negative donors were incubated with human immune anti-Rho(D) IgG and nonimmune IgG. After sensitization with IgG, the integral membrane proteins were solubilized in Brij 36T nonionic detergent and chromatographed by gel filtration. There was a distinct resolution of IgG into free and membrane-complexed forms. The IgG-complexed membrane proteins were isolated by the use of a staphylococcal protein A affinity support. The protein A-bound complexes were examined for polypeptide composition by gel electrophoresis after elution. Only Rho(D)-positive membrane proteins incubated with immune anti-Rho(D) IgG revealed intact band 3. Control Rh-negative membrane proteins that had reacted with immune anti-Rho(D) IgG and the Rh-positive membranes that had reacted with nonimmune IgG showed only low molecular weight fragments of band 3 that bound nonspecifically to IgG. Arguments are presented supporting a band 3 localization for the Rh antigen.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1980

Immunoelectron microscopy of Kell and Cellano antigens on red cell ghosts.

The ultrastructural arrangement of Kell (K1) and Cellano (K2) antigens on red cells was studied using ferritin-conjugated rabbit antihuman IgG to stain ghosts derived from antibody-sensitized intact red cells. Estimates of the number of sites under equilibrium binding were obtained by counting both ferritin grains and ferritin clusters on electron micrographs. Values for Kell sites on heterozygous red cells ranged from 2300 to 5900 and for Cellano sites from 2000 to 5000 with no discernible dosage effects. Both the Kell and Cellano antigens were clustered, which was due to conjugate staining of the ghost membranes and does not reflect the topological arrangement of these antigens on the native membrane. The ferritin grain patterns found with the Kell and Cellano antigens was similar to those observed with the Rh antigens except for the markedly reduced antigen site density.

Antigens, Surface↗
PubMed1980

Quantitative immunoferritin microscopy of Fya, Fyb, Jka, U, and Dib antigen site numbers on human red cells.

The Fya, Fyb, Jka, U, and Dib antigen site numbers and ultrastructural distribution patterns on the human erythrocyte membrane were determined using quantitative immunoferritin microscopy. For homozygous antigen-positive red cells, the average number of determinants per red cell was about 14,000 for Jka, 17,000 for Fya and Fyb, 19,000 for Dib, and 23,000 for the U antigen, assuming that the equilibrium binding observed represented 80% saturation of the accessible antigen sites. The site numbers for this group of antigens were less than that for the Rh antigens, but considerably more than the Kell and Cellano antigens. The technique used was capable of demonstrating a twofold difference in antigen density between heterozygous and homozygous Fy (a+) red cells. More than 85% of the Fya and Fyb antigen sites were lost following pretreatment of the red cells with papain, consistent with the serologic lability of the Fy antigens following proteolysis. The ferritin distribution observed following conjugate staining of antibody-sensitized ghost membranes was similar for all five antigens studied and showed a random, clustered ferritin pattern. Although the quantitative estimates are valid, the remarkable similarity in antigen distribution pattern for this diverse group of antigens, as well as other considerations, suggest that the findings with ghose membranes probably do not reflect faithfully the antigen arrangement on the intact red cell membrane.

Animals↗
PubMed1975

The role of antigen mobility in anti-Rh0(D)-induced agglutination.

Intact human erythrocytes were cross-linked with glutaraldehyde (GA) or dimethyladipimidate (DMA) and tested for their ability to bind [125I]-IgG anti-Rh0(D) and to undergo antibody-mediated hemagglutination. There was no decrease in antibody binding after treatment with GA concentrations up to 1.25% and DMA concentrations up to 1%. Red cells treated with these concentrations of GA and DMA did not agglutinate. The techniques employed to induce agglutination of the cross-linked red cells involved "incomplete" IgG anti-Rho (D) in albumin, "complete" IgM anti-D Rho (D) in saline, and the antiglobulin (Coombs) reaction. The agglutinability of the chemically modified red cells was inversely correlated with the extent of fixation. The dissociation of antibody binding from agglutinability in cross-linked erythrocytes suggests that Rho (D) antigen mobility is required for red cell agglutination. Antigen mobility was manifested by the transition from a relatively monodisperse distribution pattern of Rho (D) antigen sites to one of large aggregates or clusters when agglutination was induced by IgM anti-Rho (D), IgG anti-Rho (D) agglutination of protease modified red cells, and by anti-IgG agglutination of IgG anti-Rho (D)-coated red cells. Antigen clustering was not as prominent in red cells agglutinated by IgG anti-Rho (D) in the presence of albumin. Even though antigen mobility is a prerequisite for antibody-mediated hemagglutination, clustering does not appear to be an absolute requirement. The degree of antigen clustering differs with varying types of agglutination.

Binding Sites, Antibody↗
PubMed1975

Enzymes of phospholipid metabolism in the plasma membrane of Acanthamoeba castellanii.

Phospholipase A, lyophospholipase, acyl CoA hydrolase, and palmitoyl CoA synthetase are present in the plasma membrane of Acanthamoeba castellanii. The first three of these enzymes also occur in other cell fractions but in concentrations too low for the activities in the plasma membrane fraction to be accounted for by contamination by any other cell fraction. Palmitoyl CoA synthetase is restricted almost entirely to the plasma membrane and microsomal fractions; the microsomal activity is too low for the plasma membrane activity to be due to contamination by microsomes. Acyl COA:lysolecithin acyltransferase is predominantly localized in the microsomal fraction, but the activity of the plasma membrane is probably too great to be accounted for by microsomal contamination. CDPcholine:1,2-diglyceride cholinephosphotransferase is restricted almost entirely to the microsomal fraction. Phospholipase C was not detected in any cell fraction or in the growth medium.

Acyltransferases↗
PubMedDate not supplied

Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta2GPI and suppression of an anti-beta2GPI immune response.

LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta2GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta2GPI antibodies and to function as a B cell toleragen. Domain 1 was expressed in Pichia pastoris, and the N-terminus was site-specifically modified by a transamination reaction converting the N-terminal glycine to a glyoxyl group. A tetravalent platform was synthesized with linkers that terminate in aminooxy groups. This was accomplished by preparing an ethylene glycol-based heterobifunctional linker that contains both a Boc-protected aminooxy group and a free primary amine. The linker was used to modify a tetravalent platform molecule by reacting the amino groups on the linker with 4-nitrophenyl carbonate esters on the platform to provide a linker-modified platform, and the Boc protecting groups were removed to provide a tetravalent aminooxy platform. Glyoxylated domain 1 was attached to the platform to provide LJP 993 by formation of oxime bonds. The protein domains of LJP 993 retain activity as evidenced by the ability of LJP 993 to bind to anti-beta2GPI antibodies. Dissociation constants (Kd) for domain 1 and LJP 993 bound to immobilized affinity-purified anti-beta2GPI antibodies from autoimmune thrombosis patients were determined using surface plasmon resonance. An immunized mouse model was developed to test the ability of LJP 993 to act as a toleragen. A thiol containing domain 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 and maleimide-derivatized keyhole limpet hemocyanin (KLH). Mice were immunized with the KLH conjugate, and spleen cells were harvested from the immunized mice. The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiation. The hosts were then boosted with the KLH-domain 1 conjugate, and after 7 days their antibody levels were measured. Host mice receiving cells that were treated with LJP 993 produced significantly lower amounts of anti-domain 1 antibodies than controls which received untreated cells, indicative of B cell tolerance.

Amino Acid Sequence↗
PubMedDate not supplied

Multivalent thioether-peptide conjugates: B cell tolerance of an anti-peptide immune response.

Antibodies which bind beta2-glycoprotein I (beta2GPI) are associated with antiphospholipid syndrome. Synthetic peptide mimotopes have been discovered which compete with beta2GPI for binding to selected anti-beta2GPI. A thiol-containing linker was attached to the N-terminus of two cyclic thioether peptide mimotopes, peptides 1a and 1b. The resulting peptides, with linker attached, were reacted with two different haloacetylated platforms to prepare four tetravalent peptide-platform conjugates to be tested as B cell toleragens. The linker-containing peptides were reacted with maleimide-derivatized keyhole limpet hemocyanin (KLH) to provide peptide-KLH conjugates. Peptides 1a and 1b were also modified by acylation with 3-(4'-hydroxyphenyl)propionic acid N-hydroxysuccinimidyl ester. The resulting hydroxyphenyl peptides were radioiodinated and used to measure anti-peptide antibody levels. The KLH conjugates were used to immunize mice to generate an anti-peptide immune response. The immunized mice were treated with the conjugates or saline solution and boosted with the appropriate peptide-KLH conjugate. Three of the four conjugates suppressed the formation of anti-peptide antibody. The stabilities of the conjugates in mouse serum were measured, and the relative stabilities did not correlate with ability to suppress antibody formation.

Animals↗
PubMedDate not supplied

Antigen site densities and ultrastructural distribution patterns of red cell Rh antigens.

The ultrastructural distribution pattern and antigen site density of the major Rh antigens (C, c, E, e, and D) on red blood cell ghosts sensitized with WITH IgG Rh antibodies were determined using electron microscopy and ferritin conjugated rabbit anti-human IgG. The ferritin particle distribution pattern of all the Rh antigens studied was random and ranged from relatively isolated monodispersed clusters containing less than eight ferritin particles to large localized aggregates or masses of 20 or more ferritin particles. Evidence is presented to indicate that most of the antigen clustering was due to the conjugate during staining of the cell bound IgG on the stroma preparations. The conjugate-induced antigen clustering was influenced by both the titer of the conjugate and the staining time. Whether a similar degree of antigen mobility exists in the native membrane remains to be determined. Estimates of the number of cell bound IgG molecules (number of antigenic determinants) based on ferritin particle scoring of IgG Rh antibody sensitized red blood cells under nonsaturating conditions were in the range of 20,000 to 32,000 for each of the Rh antigens. These findings are consistent with the view that the Rh antigen complex is associated with a single membrane component.

Antibody Formation↗
PubMedDate not supplied

Estimation of anti-D IgG in red blood cell eluates using the specific radioactivity of 125I-labeled IgG: effect of unlabeled, cytophilic IgG.

The specific radioactivity of conventionally prepared 125I IgG anti-D eluates is significantly less (from 1/5 to 1/20) than that of the 125I IgG fraction used to prepare the eluate. This discrepancy is due to the release of unlabeled, cytophilic IgG from normal red blood cells during eluate preparation and does not represent an underestimation of the eluate anti-D IgG content. Cytophilic IgG content of eluates plays an important role in reducing the nonimmunologic binding of labeled antibody IgG. The results justify the assumption used in numerous studies that the specific radioactivity of 125I IgG fractions can be used to provide a valid estimate of the anti-D IgG content of eluates.

Cytotoxicity, Immunologic↗