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Biomedical subjects

F M Clarke

Publications and source records attributed to F M Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1990

Partial characterization of the PAF-induced soluble factors which mimic the activity of 'early pregnancy factor'.

Platelet-activating factor (PAF) stimulated mouse spleen cells to release soluble factors (termed S2 factors) which were capable of inducing increased rosette inhibition titres when applied to fresh mouse spleen cells in the rosette inhibition assay. In this ability the S2 factors mimic that of pregnancy serum, an action previously ascribed to 'early pregnancy factor'. The PAF-stimulated production of these S2 factors was not influenced by inhibitors of cyclooxygenase metabolism, but was completely inhibited by the lipoxygenase inhibitors, diethyl carbamazine and nordihydroguaiaretic acid. The S2 factors had a lipid-like character in that they were extractable in organic solvents. The calcium ionophore A23187 also stimulated the production of these factors which may well be products of the lipoxygenase pathway of arachidonic acid metabolism.

Animals↗
PubMed1987

Early pregnancy factor: large scale isolation of rosette inhibition test-active polypeptides from ovine placental extracts.

Protocols are described for the isolation of substantial (mg) amounts of a rosette inhibition test (RIT)-active polypeptide fraction from ovine placental extracts. The main component of the preparation is a 12K polypeptide which contains a highly reactive thiol group. Oxidation may occur during isolation with the result that the final preparation is a mixture of the 12K polypeptide and a 25K disulphide linked dimer. The highly reactive thiol group was found to be directly involved in activity expression since gentle reduction followed by iodoacetylation resulted in a complete loss of activity. Antisera were prepared and the antibodies removed all the RIT activity from fresh ovine placental extracts, indicating that molecules related to those in the isolated preparation were responsible for all the activity in crude extracts. The antibodies also removed all the RIT activity from ovine and murine pregnancy sera, obtained both before and after implantation. Since EPF is defined as an RIT activity detected in pregnancy serum, these results establish that EPF activity is due to molecules similar to those isolated from the placental extracts. The availability of the preparative protocol and antibodies should hasten the biochemical definition of the EPF phenomenon.

Animals↗
PubMed1987

Autoantibody to aldolase in acute and chronic hepatitis.

Sera from 82 patients with acute or chronic hepatitis and 40 chronic carriers of hepatitis B were examined by ELISA and immunoblotting for reactivity with the glycolytic enzyme aldolase. The results of the ELISA tests, expressed as a percentage of a positive control, were compared to those obtained with sera from 39 patients with rubella, 11 with cytomegalovirus infection and 74 healthy subjects. The ELISA reaction with sera, expressed as mean +/- standard deviation was, for 15 patients with hepatitis A, 58.3 +/- 20.5%; 15 with hepatitis B, 59.5 +/- 42.18; 23 with hepatitis non-A, non-B 51.1 +/- 34.4%; 11 with HBsAg positive chronic active hepatitis, 70.1 +/- 31.5%; and 17 with autoimmune chronic active hepatitis, 66.8 +/- 21.4%. All values were significantly (p less than 0.05-p - less than 0.001) higher than those obtained with sera from carriers of hepatitis B surface antigen, 25.6 +/- 27.2%; rubella, 21.1 +/- 20.0%; cytomegalovirus infection, 19.2 +/- 27.8%; or healthy subjects, 20.9 +/- 16.2%. In two randomly selected sera, reactivity with aldolase by ELISA was neutralized by absorption with the enzyme. Selected sera showing reactivity by ELISA reacted by immunoblotting with aldolase. The findings suggest that acute or chronic liver damage may provoke the production of autoantibodies to aldolase.

Acute Disease↗
PubMed1986

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice.

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.

Animals↗
PubMed1985

Antibody to G-actin in different categories of alcoholic liver disease: quantification by an ELISA and significance for alcoholic cirrhosis.

Autoantibodies to smooth muscle (ASMA), and to actin which is a major determinant of such reactivity, were measured in the serum of 94 patients with three defined categories of alcoholic liver disease, fatty liver, alcoholic hepatitis, and alcoholic cirrhosis, and in controls matched individually by age and sex with the patients. Autoantibody to monomeric G-actin was detected by an ELISA and autoantibody to polymeric F-actin by immunofluorescence staining of fibroblast stress fibers in cultured cells. Values for the ELISA were expressed as a percentage of the value for a strongly reactive standard serum. The mean value for antibody to G-actin in 40 patients with alcoholic cirrhosis (70 +/- 33%) was significantly greater than that for the matched controls (28 +/- 18%), but the mean value for the 30 patients with alcoholic hepatitis (46 +/- 16%) and the 24 with fatty liver (42 +/- 24%) did not differ significantly from the controls. High levels of reactivity with G-actin correlated significantly with HLA B7. ASMA was demonstrable to low titer in 11 of the 94 sera, and positive ASMA reactions by immunofluorescence correlated with high binding values to G-actin in the ELISA. Antibody to F-actin was found in only one serum and no controls. Thus in different liver diseases the reactivity of antibodies to monomeric G-actin and polymeric F-actin may differ, presumably because of specificity for different determinants of the actin molecule. Reactivity to G-actin may distinguish a group of alcoholic subjects in whom a predisposition to autoimmune reactivity is one of the determinants of progression of liver damage to cirrhosis.

Actins↗
PubMed1984

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart.

Perfused rat hearts show a markedly increased binding of phosphofructokinase and fructose-bisphosphate aldolase as a consequence of ischaemia, but little change in binding of pyruvate kinase, lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase. After 10 min ischaemia over one quarter of the phosphofructokinase and three quarters of the aldolase are bound. The effect of anoxia is less well marked in its influence on binding with only aldolase showing a significant increase in binding. These results suggest that one factor involved in the increased binding during ischaemia is the fall in pH of the heart. Binding studies with isolated myofibrils confirm that the affinity and stoichiometry of aldolase binding are considerably increased as the pH is lowered over a range comparable to that which occurs in ischaemic heart. The low level of binding of glyceraldehyde-3-phosphate dehydrogenase in perfused rat hearts correlates with the relatively low affinity of this enzyme for binding to rat or rabbit cardiac myofibrils. There are species differences in the enzyme binding response to ischaemia. Sheep hearts show rapid and large increases in the binding of glyceraldehyde-3-phosphate dehydrogenase in addition to changes in aldolase and phosphofructokinase binding. The greater binding of glyceraldehyde-3-phosphate dehydrogenase reflects the greater affinity of sheep cardiac myofibrils. It is suggested that the altered metabolic demands of ischaemia are satisfied by changes in glycolytic enzyme organisation as the enzymes shift from the soluble to the particulate phase of cardiac muscle.

Animals↗
PubMed1983

ELISA assay for IgG autoantibody to G-actin: comparison of chronic active hepatitis and acute viral hepatitis.

An ELISA was developed to test for IgG autoantibody to rabbit skeletal muscle G-actin in liver disease. To express results of the ELISA, we used a single reference serum with known high anti-actin activity, and assessed binding of test sera as a percentage of that displayed by the reference antiserum. Antibody to G-actin was measured in 40 sera from patients with chronic active hepatitis (CAH), 39 from acute viral hepatitis A (Hep A), 46 from acute viral hepatitis B (Hep B), 23 from non-A, non-B hepatitis (Hep non-A, non-B), 31 from systemic lupus erythematosus (SLE), 21 from scleroderma and 93 from normal persons. The results were compared with those obtained using indirect immunofluorescence tests with frozen sections of rodent stomach or fibroblast monolayers as substrates. Anti-G-actin activity of serum was significantly higher (P less than 0.001) in CAH (mean 63% +/- 23, range 18-110) than in controls (mean 15% +/- 12, range 0-51), systemic lupus erythematosus (mean 25% +/- 16, range 8-76), scleroderma (mean 23% +/- 13, range 3-59), Hep A (mean 24% +/- 11, range 6-25), Hep B (17% +/- 8, range 2-36), or Hep non-A, non-B (mean 23% +/- 11, range 7-53). There was no significant difference (P greater than 0.05) in the occurrence of anti-G-actin activity of serum in Hep A, Hep B or non-A, non-B compared with controls. At an ELISA reading of greater than or equal to 40% (mean + 2 s.d. of controls) the assay detected autoantibodies to G-actin in 85% of CAH, compared to 80% on fibroblast monolayers and 70% on rodent stomach. The ELISA described in the present study is a simple, sensitive and quantitative assay for autoantibody to G-actin. It should prove useful in assessing subspecificities of actin antibodies in liver diseases, and in differential diagnosis, particularly CAH from acute viral hepatitis.

Actins↗
PubMed1983

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein.

Actins↗
PubMed1983

Early pregnancy factor is an immunosuppressive contaminant of commercial preparations of human chorionic gonadotrophin.

Early pregnancy factor (EPF) is a pregnancy associated substance detected in human serum and urine throughout the first and second trimesters of pregnancy. It has also been detected in several commercial preparations of human chorionic gonadotrophin (hCG). The various molecular weight forms of EPF which occur in human pregnancy serum, urine and commercial hCG preparations have been partially characterized and found to be similar to each other but distinct from hCG. Further evidence is presented which suggests that it is EPF rather than hCG which is responsible for the immunosuppressive activity of some crude hCG preparations.

Chaperonin 10↗
PubMed1982

Actin in human spermatozoa.

An actin-like material was extracted from human spermatozoa. The material comigrated with actin on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, and showed specific affinity for myosin. Purified anti-actin antibodies were used to localize actin to the posterior region of the sperm head and to the connecting piece, midpiece and mainpiece of the tail. Determination of the function of sperm actin requires further investigation.

Actins↗
PubMed1981

The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation.

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The binding of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to the greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.

Animals↗
PubMed1980

Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle.

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 x 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 x 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

Actins↗
PubMed1980

Interaction of aldolase with actin-containing filaments. Structural studies.

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.

Actins↗
PubMed1980

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.

Animals↗