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Biomedical subjects

D L Emerson

Publications and source records attributed to D L Emerson.

34 records · Page 2Linked 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

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↗
PubMed1984

The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA.

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a 'pseudoligand' for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.

Alleles↗
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

Interaction of alpha 2-HS-glycoprotein with immobilized triazine dyes.

We studied the interaction of alpha 2-HS-glycoprotein with immobilized Cibacron blue F3-GA (Blue A) and Procion red HE-3B (Red A). When whole plasma was applied on the Blue A, alpha 2-HS-glycoprotein remained unbound, together with other plasma proteins. In contrast, when this fraction was applied on the Red A, alpha 2-HS-glycoprotein was shown to bind tightly and was eluted with a linear sodium chloride gradient between 0.5 and 0.8 M. This proved to be a useful two-step technique for the purification of alpha 2-HS-glycoprotein. Further characterization revealed that the protein appeared homogeneous by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis with yields greater than 30%. A small (less than 5%) but significant fraction of alpha 2-HS-glycoprotein with a same molecular weight as the native protein was consistently found in the wash of the Red A column, and may correspond to alpha 2-HS-glycoprotein bound to a yet unidentified ligand.

Blood Proteins↗
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

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↗
PubMedDate not supplied

Decreased serum group-specific component protein levels and complexes with actin in fulminant hepatic necrosis.

Monomeric G-actin has recently been shown to form high-affinity complexes with group-specific component protein (Gc), and this process might be expected to occur in vivo when hepatocyte necrosis with release of actin takes place. We therefore measured serum Gc levels and searched for evidence of actin-containing complexes of Gc in 147 sera from 21 normal subjects and 126 patients with acute and chronic liver diseases. Sera were examined for Gc concentration using an ELISA with purified Gc as standard. Gc levels were greatly diminished (less than 10 mg per dl) in 5 of 37 patients with acute hepatitis and 1 of 3 patients with subacute hepatic necrosis, and the mean values for these groups overall as well as those for chronic active hepatitis patients were significantly less than that observed in the normal controls (mean, 35.5 mg per dl). No significant alteration in Gc level was observed in the chronic disease categories studied. In contrast, Gc concentrations less than 10 mg per dl were found in 12 of 14 patients with fulminant hepatic necrosis, and the mean for this group (9.7) was significantly reduced (p less than 0.001). Sequential studies in 12 patients with acute hepatitis of moderate to marked severity but without a fulminant phase showed significant diminutions in initial Gc levels when these were compared with Gc levels obtained during recovery. Using analytical isoelectric focusing with print immunofixation, variable amounts of Gc:G-actin complexes could be demonstrated in virtually all sera examined, but the proportion of total Gc present as complex with G-actin appeared greater in fulminant hepatic necrosis sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMedDate not supplied

Diminished serum Gc (vitamin D-binding protein) levels and increased Gc:G-actin complexes in a hamster model of fulminant hepatic necrosis.

Evidence for increased plasma levels of complexes containing Gc (vitamin D-binding protein) and cellular actin has been previously reported during fulminant hepatic necrosis in man. In order to study this process in more detail, we produced liver injury in hamsters using increasing doses of acetaminophen, with serial collection of sera for up to 168 hr after acetaminophen injection. Hamster Gc was purified using a three-step procedure and was shown to resemble closely human Gc. Polyclonal antihamster Gc was prepared and used in rocket immunoelectrophoresis and radial immunodiffusion studies for quantitation of total serum Gc and the percentage of Gc complexed with actin. Serum Gc levels were depressed in animals having liver damage, and the extent of depression 42 hr after acetaminophen correlated with the extent of elevation of AST. The proportion of the total Gc that was present in the complexed form increased in relation to the severity of the liver disease. In serial studies, diminution in Gc level preceded the rise in AST and increase in the percent complexed. These changes closely resemble observations in man and suggest that the hamster-acetaminophen hepatotoxicity model may be of value in further study of interactions of Gc with intracellular actin components and its role in actin homeostasis in conditions of massive tissue necrosis.

Acetaminophen↗
PubMedDate not supplied

Antibodies to polymerized human serum albumin in acute and chronic liver disease.

Since antibodies to polyalbumin have been noted to occur in patients with hepatitis or cirrhosis, we investigated sera from 219 patients with a variety of acute and chronic liver diseases with and without HBsAg using an ELISA. The prevalence of circulating polyalbumin antibodies was shown to be significantly increased over that of healthy controls (6.7%) in patients with autoimmune chronic active hepatitis (67%), acute viral hepatitis (41%) and fulminant hepatic necrosis (38%), but such antibodies were absent in chronic hepatitis B patients and other liver diseases. Serial studies in acute hepatitis showed evidence of antibodies early in the course of illness with disappearance prior to full recovery. In acute hepatitis B, the presence of polyalbumin antibodies was significantly associated with female sex (p less than 0.01), 3-fold higher transaminase levels and shorter duration of clinical illness (less than 4 weeks in all cases). Polyalbumin antibodies appear to be associated with diseases characterized by active hepatocyte necrosis. Since they are evident early in acute hepatitis B when complexes of polyalbumin and virus are likely, these antibodies may play an adjunctive role to other hepatitis B-related antibodies in the clearance of hepatitis B virus infection.

Antibodies↗
PubMedDate not supplied

Linkage between surface immunoglobulin and cytoskeleton of B lymphocytes may involve Gc protein.

The membrane immunoglobulin (MIg) of B lymphocytes is thought to have an important role in antigen recognition and cellular activation. In common with many membrane glycoproteins, MIg moves extensively in the lipid bilayer, and after binding of specific antisera displays lateral mobility with patch and cap formation. This phenomenon appears to involve the cytoskeleton, particularly the actin that is present in the cell membrane of B lymphocytes and aggregates beneath capped immunoglobulin. Recently, it has been reported that the isolation of MIg results in co-purification of actin and an unknown protein of molecular weight (MW) approximately 56,000 (refs 7, 8). We now demonstrate that this component displays physicochemical and immunological properties indistinguishable from those of Gc (group-specific component). In addition, evidence is presented which suggests that this vitamin D3-binding protein is involved in the linkage between MIg and actin, and may therefore be important in signal transduction.

Actins↗
PubMedDate not supplied

Inhibition of lymphocyte activation by antisera to embryonic antigens shared with human placental trophoblast.

In order to further examine the inhibition of lymphocyte activation reported with conventional antisera to human trophoblast membranes, we studied their effects on cells stimulated by antigen in mixed lymphocyte culture (MLC) and by the mitogen phytohemagglutinin. The results were compared with the effects of antisera known to recognize intrinsic membrane determinants on activated T cells (DR antigens and placental alkaline phosphatase) and with those of antisera to normal human serum components such as transferrin which may bind to the activated lymphocyte membrane. The results indicated that antibodies to placental alkaline phosphatase, which constitutes the predominant specificity of conventional trophoblast membrane antisera, caused inhibition both of MLC response and, to a lesser extent, of activation induced by mitogen. Similar inhibition was obtained with antisera to human DR antigens, while antisera to normal human serum and transferrin were not suppressive. These findings, together with time course studies and immunocytochemical studies of the homologous antigens, suggest that these antisera mediate their inhibitory effects in part through binding to antigens which appear on cells as they undergo activation.

Alkaline Phosphatase↗