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Biomedical subjects

R M Galbraith

Publications and source records attributed to R M Galbraith.

At least 37 records · Page 2Linked to original sources
PubMed1988

Evidence of a novel association of unsaturated fatty acids with Gc (vitamin D-binding protein).

The strong sequence homology described recently between Gc (Vitamin D-binding protein) and albumin, and the ability of the latter, to bind 2-p-toluidinylnaphthylene sulfonate (TNS) promoted similar binding studies with Gc. TNS bound to native Gc as demonstrated by fluorescence, but chloroform:methanol extraction of Gc and gas chromatography revealed that large amounts of unsaturated fatty acids - 16:1, 18:1, 18:2 and 20:4 were also associated with Gc. In addition, TNS fluorescence was abolished by delipidation of Gc, and restored upon subsequent reconstitution with fatty acid. Finally, 70-80% of [3H]-arachidonic acid added to whole serum bound to Gc. These results demonstrated that TNS fluorescence of the native molecule reflects associated lipid, and suggest a novel role for Gc as a reservoir for a circulating pool of unsaturated fatty acids.

Animals↗
PubMed1988

Proportion of circulating Gc (vitamin D-binding protein) in complexed form: relation to clinical outcome in fulminant hepatic necrosis.

Alterations in circulating levels of vitamin D-binding protein (Gc) and the percentage of Gc in complexed form were further studied in normal subjects and in patients with fulminant hepatic necrosis in relation to clinical outcome. Levels of Gc were markedly reduced in all 7 patients studied, particularly in nonsurvivors. The percentage of Gc in complexed form was generally less than 10% in normal subjects. However, complexes were increased in all patients, and the percentage correlated strongly with clinical outcome, being 22% +/- 7.3% in survivors and 72% +/- 7.5% in those who died (p less than 0.001). These results provide further evidence that Gc plays an important role in complexing and clearance of cellular actin released during tissue necrosis.

Hepatic Encephalopathy↗
PubMed1988

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.

Actins↗
PubMed1987

Altered conformation of Gc (vitamin D-binding protein) upon complexing with cellular actin.

Complexing of serum Gc (Vitamin D-binding protein) with cellular actin can occur in the extracellular space as a result of cell turnover, and particularly cell necrosis. The clearance of such complexes is significantly more rapid than that of Gc alone, and several tissues are involved in their uptake, but the mechanisms involved are unknown. We present evidence here that interaction with actin results in alteration of certain physicochemical properties of Gc. Fluorescence of the hydrophobic probe 2-p-toluidinylnaphthylene-6-sulfonate was abolished by complex formation with actin. In addition, isoelectric focusing of complexes between Gc, and actin from different tissues, revealed that complexes were generally more acidic than either protein individually. These findings indicate that complexing of Gc with actin results in altered conformation.

Actins↗
PubMed1987

Reaction of T lymphocytes with anti-T3 induces translocation of C-kinase activity to the membrane and specific substrate phosphorylation.

Reaction of the T cell membrane with monoclonal antibodies to T3 can initiate cellular activation, and this is associated with increased intracellular Ca2+ and inositol-trisphosphate (IP3) release. We therefore studied the possible involvement of Ca2+/phospholipid-dependent kinase (C-kinase) in these phenomena. Quantitative assays of exogenous substrate phosphorylation in unstimulated cells showed Ca2+/phospholipid-dependent kinase activity in the cytosol, but no comparable activity in the particulate fractions corresponding to membrane and cytoskeleton material. At concentrations of soluble anti-T3 that partially activate T cells in the absence of macrophages, there was a 50 to 60% decrease in C-kinase activity in the cytosol, with a comparable increase in activity in the membrane fraction. A similar transfer of activity was also induced with the known C-kinase activator, 12-O-tetradecanoyl-phorbol-13-acetate, although redistribution was more rapid in onset, more complete, and more sustained. Redistribution of enzyme activity was additionally confirmed by qualitative assays of endogenous substrate phosphorylation. Labeling of intact cells followed by immunoprecipitation analysis with anti-T3 indicated signal-dependent phosphorylation of two components of the T3 complex and an unidentified 94,000 substrate that was resistant to reduction and alkylation. These findings are consistent with an important role for C-kinase in transduction of membrane events by the T3-Ti complex.

Antibodies↗
PubMed1987

Molecular signaling mechanisms in T-lymphocyte activation pathways: a review and future prospects.

Understanding of the molecular mechanisms which drive the complex activation responses of T lymphocytes was previously limited. However, current studies in lymphocytes, and in other cells, have indicated the involvement of several secondary messenger or signal systems, and recent progress in elucidating the relevant pathways has been extraordinarily rapid. This review therefore attempts to provide an overview of these processes--including the effects of Ca2+, hydrolysis of phospholipids, arachidonic acid, Ca2+/phospholipid-dependent and tyrosine kinases, calmodulin, cyclic nucleotides, ion channels, and adherent cells--and their roles in weaving a subtle and highly responsive web of regulatory signals.

Calcium↗
PubMed1987

High levels of group-specific component (vitamin-D-binding protein) in the cerebrospinal fluid of infants aged less than 2 months.

Because of the possible involvement of group-specific component (Gc) or vitamin-D-binding protein in the immunological functions of mononuclear cells and the increased risk of central nervous system infections in early infancy, we studied Gc levels in the cerebrospinal fluid (CSF) of children. CSFs were examined for the Gc concentration using ELISA and rocket immunoelectrophoresis, with purified Gc as standard. The results showed a significant inverse correlation (p less than 0.05) between the age of the patients and CSF Gc levels. Gc levels in the CSF were significantly increased in infants less than 2 months of age (12.5 micrograms/ml), as compared to infants greater than 2 months (1.7 micrograms/ml, p less than 0.0028). In children greater than 2 months of age a significant correlation was found between Gc levels and those of other CSF proteins (albumin, IgG and total protein, p less than 0.002). However, no significant correlation between Gc levels and those of other CSF proteins was apparent in infants less than 2 months of age, indicating the possibility that the concentration of Gc in the CSF may be selectively increased in this age group.

Aging↗
PubMed1987

Correlation between extent of liver damage in fulminant hepatic necrosis and complexing of circulating group-specific component (vitamin D-binding protein).

Increased complexing of circulating group-specific component (Gc, vitamin D-binding protein) has been found in humans under conditions of presumed increased actin release, and particularly fulminant hepatic necrosis (FHN). We therefore used a hamster model of acetaminophen-induced FHN to further investigate this phenomenon. Liver damage was monitored by aspartate aminotransferase (AST) activity and by histologic examination, and serum Gc was analyzed by polyacrylamide gel electrophoresis (PAGE) and transblotting with anti-Gc. In controls and treated animals displaying slight liver damage, greater than 90% of Gc was of mobility corresponding to native purified hamster Gc, whereas with more severe liver damage up to 100% of Gc was found in one of two cathodal configurations that appear to correspond to complexes with actin. Densitometric quantitation of complexed Gc demonstrated a strong correlation with the severity of liver damage. These results show PAGE to be a useful method of estimating the percentage of Gc complexed in serum, and suggest that cell damage may be followed by the appearance in the circulation of cellular actin that complexes with Gc.

Actins↗
PubMed1986

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin.

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.

Actins↗
PubMed1986

Gc (vitamin D-binding protein) binds the 33.5 K tryptic fragment of actin.

Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5.

Actins↗
PubMed1986

Translocation of phospholipid/Ca2+-dependent protein kinase in B-lymphocytes activated by phorbol ester or cross-linking of membrane immunoglobulin.

Stimulation of peripheral-blood B-lymphocytes with phorbol ester or anti-immunoglobulin demonstrated intracellular translocation of phospholipid/Ca2+-dependent protein kinase (C-kinase) activity from cytosol to membrane fractions. This phenomenon, which was dose- and time-dependent, was found in both normal and chronic-lymphocytic-leukemia B-cells. This suggests that C-kinase-dependent protein phosphorylation may be related to membrane receptor occupation and may therefore be important in B-lymphocyte responses.

Antibodies, Anti-Idiotypic↗
PubMed1986

Plasmodium berghei: histology, immunocytochemistry, and ultrastructure of the placenta in rodent malaria.

The pathological changes associated with malarial infection in pregnancy were studied in rats and mice infected with Plasmodium berghei at different stages of gestation. Histopathological and ultrastructural studies of infected placentae near term in both species revealed disruption of architecture with gross thickening and necrosis of cells in the labyrinthine zone and fibrosis of the trilaminar trophoblast separating the maternal and fetal circulations. In the mouse, the extent of histopathological alterations in infected placentae ranged from the presence of immature erythrocytes in the fetal circulation in low grade maternal infection, to the marked deposition of fibrinoid material on the trilaminar trophoblast and inflammatory masses in severely infected placentae. In the rat, histopathological aberrations in the placentae were marked by placental stroma edema, fibrosis, and cellular infiltration. Immunohistological studies of cryostat sections of placentae from infected animals showed more parasites and pigment in infected mouse placentae than in the corresponding rat organ, but in both species parasites and pigment were largely confined to the maternal blood spaces and were only occasionally found in necrotic areas of trophoblast. No clear differences were observed between infected and control placentae in terms of the amount of IgG, IgM, or IgA which were each present in various amounts. These observations and the rarity of congenital malaria in the animals indicate that the placenta constitutes a major barrier to infection of the fetus. However, the pathological aberrations in the infected placentae may impose a biochemical stress upon the fetus which may account for the low birthweight, the increased frequency of abortion, and the greatly increased maternal and fetal death rates observed in malaria.

Animals↗
PubMed1986

Inhibition of T3 mediated T-cell proliferation by Ca2+-channel blockers and inhibitors of Ca2+/phospholipid-dependent kinase.

The potential roles of Ca2+ ions in the response of T lymphocytes to stimulation with monoclonal antisera to the T3 antigen were investigated by means of pharmacological agents that predominantly inhibit the flux of Ca2+ ions into cells (verapamil, nifedipine) or the activity of Ca2+-dependent kinases (trifluoperazine, polymyxin B). As assessed by uptake of [3H]thymidine, proliferation induced with anti-T3 +/- recombinant IL-2 at 72 h was inhibited by greater than 80% in the presence of nifedipine at 50 microM, and almost completely arrested (greater than 95% inhibition) with the other agents at the same concentration. Further quantitative assays of the effects of polymyxin B and trifluoperazine on C-kinase labelling of exogenous substrate showed a major reduction with both agents, but inhibition was substantially greater with polymyxin B that with trifluoperazine (IC50 = 14 and 70 microM respectively). These results were confirmed by qualitative assessment of Ca2+/phospholipid-dependent phosphorylation of endogenous substrates, which demonstrated major phosphoproteins of MW 56,000, 52,000, 43,000, and 20,000, and dose-dependent reduction in labelling in the presence of polymyxin B. Similar results were obtained under more physiological conditions in intact cells labelled with 32P orthophosphate. These findings indicate several possible roles for Ca2+ in T-cell activation, and several possible levels of activity, including modulation of calmodulin-dependent kinases and effects on Ca2+/phospholipid-dependent kinases and Ca2+ channels.

Antibodies, Monoclonal↗
PubMed1985

Identification of a major endogenous substrate for phospholipid/Ca2+-dependent kinase in pancreatic acini as Gc (vitamin D-binding protein).

A major 56 kDa substrate for phospholipid/Ca2+-dependent kinase (C-kinase) in pancreatic acinar cells is physicochemically and immunologically indistinguishable from the vitamin D-binding protein, Gc or group-specific component. Cellular Gc was also phosphorylated in intact cells following treatment with carbachol as a physiological stimulus. These findings indicate the potential usefulness of Gc as a defined substrate for further studies of the biological role of C-kinase activity in pancreatic acini and possibly in other cells.

Animals↗