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Biomedical subjects

R M Galbraith

Publications and source records attributed to R M Galbraith.

At least 55 records · Page 3Linked to original sources
PubMed1985

Accurate quantitation of native Gc in serum and estimation of endogenous Gc: G-actin complexes by rocket immunoelectrophoresis.

Complex formation between purified Gc and G-actin caused increased rocket height on immunoelectrophoresis with monospecific Gc antiserum, and artifactually high calculated Gc levels. The increase in rocket height varied in log: linear fashion with the amount of G-actin present, up to a plateau attained at equimolarity. The raw Gc values could therefore be corrected to within +/- 10% of known levels by addition of excess G-actin and use of standard plots obtained with Gc after saturation with G-actin. This also allowed quantitation of the percentage of Gc complexed with G-actin. In subsequent studies of whole human sera, comparison of normal controls with pregnant subjects and patients with liver disease showed evidence of differences both in absolute quantities of Gc and the relative proportion circulating as complex with G-actin. This appeared to be due to increased release of cellular actin into the extracellular space. These results show that rocket immunoelectrophoresis can be modified to provide accurate Gc levels, and also information concerning different molecular forms of this protein.

Actins↗
PubMed1985

Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I.

Addition of group-specific component (Gc) to G-actin with or without deoxyribonuclease I (DNAase) led to formation of binary complexes (Gc-G-actin) and ternary complexes (Gc-G-actin-DNAase) respectively. The electrophoretic mobility of ternary complexes, as shown by crossed and rocket immunoelectrophoresis, was slower than that of binary complexes, although both were faster than native Gc. In gradient polyacrylamide-gel electrophoresis, such complexes could again be resolved, apparently on the basis of relative molecular size: Gc-G-actin-DNAase (Mr approx. 131000), Gc-G-actin (Mr approx. 98000) and Gc (Mr approx. 56000). In contrast, the pI of ternary complex was indistinguishable by isoelectric focusing from that of binary complex, even though both were clearly more acidic than native Gc. The affinity of Gc for G-actin (affinity constant, Ka, 1.9 X 10(8) M-1) was not significantly altered by additional interaction with DNAase (Ka, 1.5 X 10(8)M-1), and both binary and ternary complexes still bound 25-hydroxycholecalciferol. In addition, the inhibitory effect of G-actin on DNAase activity was not discernibly affected by interaction with Gc. These results demonstrate that the various molecular forms of Gc can be distinguished by physicochemical parameters, and that Gc and DNAase bind to distinct sites on G-actin and can interact both independently and contemporaneously with this molecule.

Actins↗
PubMed1985

Polymyxin B causes coordinate inhibition of phorbol ester-induced C-kinase activity and proliferation of B lymphocytes.

Lymphocytes were found to be rich in phospholipid/Ca2+-dependent (C-kinase) activity. Addition of polymyxin B (PMB) to in vitro assays of endogenous and exogenous phosphorylation resulted in profound inhibition of C-kinase activity. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) directly activated C-kinase, leading to increased phosphorylation of the same substrates. TPA also stimulated proliferation of B cells as assessed by 3H-thymidine uptake, and PMB strongly inhibited this effect. This coordinate inhibition of TPA-induced phosphorylation and mitogenesis indicates that PMB is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.

B-Lymphocytes↗
PubMed1985

Structural studies of T lymphocyte Fc receptors. Association of Gc protein with IgG binding to Fc gamma.

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of Mr 56,000, pI 4.8-5.1 and Mr 60,000, pI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the T-cell membrane. While the Mr 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the Mr 60,000 protein remains unknown. Three other components were detected, although with less consistency; Mr 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that less than 5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of Mr 60,000, 42,000, and 20,000-25,000, complexed IgG binding to Fc gamma of human peripheral blood T-cells also becomes spatially associated with Gc protein.

Chromatography, Affinity↗
PubMed1985

Altered configuration of Gc on the plasma membrane of transformed and malignant human B lymphocytes.

Normal human peripheral blood B cells exhibit strong membrane fluorescence for Gc (vitamin D-binding protein), and this protein can form a close spatial relationship with integral membrane immunoglobulin (mIg) with evidence of codistribution in the lipid bilayer. In contrast, fluorescence for both Gc and mIg has been found in this study to be weak or absent in several B lymphoblastoid cell lines and in chronic lymphocytic leukemia B cells. Moreover, the comobility of these components, where detectable, was also impaired. In abnormal B cells, the intensity of membrane fluorescence for Gc was substantially increased after crosslinking of mIg with antibody, and the latter was also associated with increased specific radioiodination of Gc by lactoperioxidase. These results indicate that Gc can apparently become displaced under certain circumstances within or through the lipid bilayer. The altered content or membrane topography of Gc in such abnormal B cells might be associated with impaired expression and mobility of mIg.

B-Lymphocytes↗
PubMed1985

Presence of Gc (vitamin D-binding protein) and interactions with actin in human placental tissue.

The distribution of Gc protein and actin and their interactions were studied in normal full-term human placentae. Both Gc and actin were detected by physicochemical analysis of isolated trophoblast membranes. Immunofluorescence of native placental sections showed fluorescence for both proteins on smooth muscle cells lining the fetal stem vessels, intervillous fibrin, villous fibrinoid, trophoblast membrane, and cytoplasm of villous stromal cells. Binding of Gc was demonstrated by prior incubation of sections with purified Gc which led to a striking increase in intensity of Gc fluorescence, but actin fluorescence was unaffected by this procedure and by preincubation with actin. Endogenous Gc and actin could also be removed by washing of tissue sections with chaotrope--3 M KCl or 3 M NH4SCN, denaturant--6 M urea, or glycine-HCl pH 3.8, as judged by fluorescence and SDS-PAGE of the wash supernatant. Phenotypic analysis of Gc eluted from trophoblast membranes and of corresponding matched maternal and fetal cord sera by isoelectric focusing indicated that trophoblast Gc was of predominantly maternal origin. Although the roles of Gc and actin in the placenta are unknown, these results indicate that Gc may be another maternal protein for which specific binding sites are expressed on the membrane of placental trophoblast.

Actins↗
PubMed1985

Phorbol ester induces tyrosine phosphorylation in normal and abnormal human B lymphocytes.

Tumor-promoting phorbol esters have been found to bind and activate phospholipid/Ca2+-dependent or C-kinase, and several of their effects, including proliferative responses in lymphocytes, have been assumed to be related to activity of this enzyme. However, phorbol esters have also recently been found to stimulate tyrosine phosphorylation in certain other cell types, and we therefore studied tyrosine kinase activity in normal and chronic lymphocytic leukemia (CLL) peripheral blood B lymphocytes stimulated with phorbol ester. High levels of tyrosine labeling were observed in unstimulated cells with major endogenous substrates of 75K, 66K, 43K, and 28K in Triton-soluble material, and of 56K to 61K in Triton-insoluble material; this profile was essentially similar in normal and CLL B cells. Treatment with phorbol ester for time periods varying from 20 min to 48 hr led to qualitative increases in tyrosine labeling of these phosphoproteins, as measured both in vitro and in intact cells "in vivo." Although the relative abundance of tyrosine phosphorylation as a percentage of total labeling was variable due to concomitant enhancement of serine and threonine phosphorylation, exogenous peptide substrate assays confirmed the increased tyrosine kinase activity quantitatively. Enhanced tyrosine phosphorylation was succeeded or accompanied in both normal and abnormal B cells by cellular activation, as judged by increased [3H]thymidine uptake, and terminal differentiation of CLL cells. These findings provide further evidence implicating tyrosine kinases in B lymphocyte activation.

B-Lymphocytes↗
PubMed1985

Hepatic membrane antibodies: studies of prevalence and specificity.

Using a newly developed assay for hepatic membrane-associated antibodies (HMA), we studied the prevalence and specificity of HMA in a variety of acute and chronic liver diseases to evaluate the usefulness of such an assay system. Sera were examined by indirect immunofluorescence on rat liver sections previously fixed in Bouin's fluid. As compared with a prevalence of 4% in 45 healthy controls, HMA were detected in 73% of 45 patients with chronic active hepatitis (CAH) (P less than 0.001) and in all 11 patients with primary biliary cirrhosis (PBC) (P less than 0.001). HMA were also present in 38% of 40 patients with acute viral hepatitis, 47% of 27 with alcoholic liver disease and 33% of 24 with other liver diseases not thought to be immunologically mediated. In 53 patients studied with other liver, gastrointestinal or autoimmune diseases, the prevalence of HMA was not increased over controls. Absorption studies showed that seven of 14 HMA positive sera contained specificities for polymerized human serum albumin (pHSA) or liver-specific protein (LSP) or both antigens. HMA positive samples reacted occasionally with rat kidney tubular epithelial membranes but did not appear to react with other tissues. These results demonstrate that HMA are present at higher prevalence in the autoimmune liver diseases than many previously described autoantibodies and are reactive in part to specificities on pHSA and LSP.

Antibody Specificity↗
PubMed1984

Enhanced tyrosine phosphorylation in B lymphocytes upon complexing of membrane immunoglobulin.

Activation of normal human peripheral blood B-enriched lymphocytes with antiserum reacting with membrane immunoglobulin was associated with increased phosphorylation of cellular proteins, particularly in the Triton-insoluble cytoskeletal fraction. At least two proteins (56K and 60K) were labeled predominantly at tyrosine residues. These findings may indicate another specialized membrane structure which upon specific ligand binding stimulates tyrosine phosphorylation in association with cellular proliferation.

B-Lymphocytes↗
PubMed1984

Gc (vitamin D binding protein) binds to cytoplasm of all human lymphocytes and is expressed on B-cell membranes.

Peripheral blood lymphocytes were examined immunohistologically for evidence of interactions with Gc protein, a major vitamin D binding protein in serum. In the cytoplasm, binding sites for purified Gc were readily detectable in all cells, and these sites were only partially occupied by Gc. In contrast, on the membrane of viable cells, there was negligible evidence of binding of either the apo- or holoform of Gc protein, but substantial quantities of firmly bound immunoreactive endogenous Gc were detected. Separation experiments and double-label fluorescence with antisera recognizing defined phenotypic markers showed immunoreactive membrane Gc on 30-40% of unfractionated mononuclear cells and greater than 95% of monocytes or B cells. Only 5-8% of T cells were similarly reactive; these were not apparently confined to any given subset. Extraction of unfractionated cells with 6 M urea or solubilization in Nonidet P-40 released immunoreactive Gc protein, with physicochemical properties indistinguishable from those of Gc purified from serum (apparent MW 56K; pI 4.8-5.1). These findings indicate that membrane Gc may represent another surface immunofluorescence marker for B cells, and may play a role in immunocyte function.

B-Lymphocytes↗
PubMed1983

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

Chromatography, Affinity↗
PubMed1983

Membrane antigens in chronic active hepatitis: physicochemical and immunological studies of soluble liver homogenate fractions.

Since the liver membrane components termed liver-specific protein (LSP) and liver membrane antigen (LM-Ag) have been implicated as target antigens in chronic active hepatitis (CAH), we performed physicochemical and immunological analyses of these and other fractions obtained from liver homogenates. Supernatants were prepared from homogenates of human and rabbit liver by centrifugation at 105,000 g for 1 hr; after gel filtration on Sepharose 6B, the LSP fraction was obtained as the excluded peak I and LM-Ag as an included fraction, peak IIB. Comparison by immunodiffusion with reference LSP and guinea pig antisera to LSP confirmed the similarity of our fractions to reference preparations. Upon SDS-polyacrylamide gel electrophoresis (SDS-PAGE), human and rabbit peak I were found to contain at least 25 distinct protein bands with a wide range of molecular weights (14,000- greater than 100,000) and the other peaks were found to be equally heterogeneous. Thin-layer polyacrylamide gel isoelectric focusing (PAGIF) revealed 35-40 separate bands in both peaks I and IIB with a wide range of isoelectric points (3.5-10.0). These results indicate that liver homogenate fractions such as LSP and LM-Ag can no longer be considered as single protein preparations, and are in fact highly complex mixtures of multiple proteins. Further separation and characterization of such fractions will facilitate identification of liver antigens for use in in vitro immunological assays.

Animals↗
PubMed1983

Evidence of increased Gc:actin complexes in pregnant serum: a possible result of trophoblast embolism.

The molecular configuration of group-specific component Gc protein in sera from pregnant and nonpregnant individuals was compared by analytical isoelectric focusing and by print immunofixation in conjunction with known standards of Gc:actin and Gc:vitamin D3 complexes. These studies revealed that while complexes of Gc with actin and with vitamin D were detectable in small amounts in nonpregnant sera, much larger quantities of both types of complexes were consistently visualized in pregnancy. In addition, when actin was added to pregnant sera containing Gc:vitamin D3 complexes, a third anodal complex was revealed which presented the molecular configuration of actin:Gc:vitamin D3. These results demonstrate that Gc:actin complexes may be present under physiological circumstances in the circulation. Since large amounts of trophoblast enter the maternal circulation during both normal and abnormal human pregnancy, experiments were undertaken that showed that actin was released from isolated trophoblast membranes and also upon lysis of other viable cells under physiological conditions similar to those obtained in serum, and such actin complexed rapidly with Gc. Although the effects of this phenomenon upon the immunobiology of pregnancy are unknown, these findings are consistent with the concept that Gc protein may exert a "scavenger" function in mopping up actin released from damaged cells.

Actins↗
PubMed1983

Antibodies to hepatocyte membrane antigens in chronic liver disease: detection by immunofluorescence after Bouin's fixation.

Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.

Antibodies, Antinuclear↗
PubMed1983

Preliminary immunologic studies of ciguatera poisoning.

Preliminary immunologic studies of ciguatera poisoning were performed with serum samples from patients and extracts from both toxic and nontoxic fish. Counterimmunoelectrophoresis disclosed precipitin lines with toxic fish extracts and effectively discriminated between samples established as toxic or nontoxic on the basis of human toxic reactions and by an in vivo mouse bioassay. However, it is not currently possible to conclude that affected individuals had specific antibody to ciguatoxin, since putative immune and nonimmune serum samples gave equally clear precipitin reactions with toxic extracts.

Animals↗
PubMed1982

Effects of malaria (Plasmodium berghei) on the maternal-fetal relationship in mice.

Plasmodium berghei infection was more severe in pregnant than in nonpregnant mice. Infection initiated on gestation day 7 resulted in rapidly increasing parasitemia and deaths of all pregnant mice within 12 days, while some nonpregnant mice survived until day 21 postinfection. When mice were infected on gestation day 12 or 14, a proportion of mice died before parturition; but some animals survived to deliver living pups. Reduced birthweights and increased spleen weight to body weight ratios were seen in pups from infected mice as compared with pups from uninfected animals. Histopathological abnormalities of placentae from infected animals included degeneration of the normal labyrinthine architecture and thickening of the trophoblast separating maternal and fetal blood vessels.

Animals↗
PubMed1981

Combined radiolabel-binding and immunocytochemical evaluation of receptor-ligand interactions. Studies of transferrin receptors on activated lymphocytes.

A protocol that involved both immunohistological and radiolabel-binding procedures was devised for the study of transferrin-receptor interactions. This composite approach yielded considerably more information than did either technique used alone, and also provided a simple means for exclusion of several common potential sources of error.

Fluorescent Antibody Technique↗