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Biomedical subjects

A Koj

Publications and source records attributed to A Koj.

At least 73 records · Page 4Linked to original sources
PubMed1985

Inhibition of horse leucocyte proteinases by eglin, a proteinase inhibitor from leeches.

Interaction of eglin c with three neutral proteinases (1, 2A and 2B) from horse leucocytes was investigated using synthetic and protein substrates. With N-tert-butyloxycarbonyl-L-alanine-p-nitrophenyl ester as substrate inhibition of proteinase 1 and 2A was practically complete at equimolar inhibitor concentrations (Ki below 1 nMol/l). The complex with proteinase 2B showed a dissociation constant of approximately 25 nMol/l. The latter proteinase was only partly inhibited also in the presence of azocasein, whereas almost linear inhibition was observed for all 3 proteinases with fibrinogen as substrate. The inhibition rate constants (kon) for horse leucocyte proteinases with eglin were in the range of 8 to 13 X 10(5) M-1 S-1.

Animals↗
PubMed1984

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.

Acute-Phase Proteins↗
PubMed1984

Human keratinocytes and monocytes release factors which regulate the synthesis of major acute phase plasma proteins in hepatic cells from man, rat, and mouse.

Human keratinocytes and activated monocytes produces factors which can stimulate the proliferation of thymocytes. The same activity has also been implicated in regulating the expression of plasma proteins in liver cells during the acute phase reaction. To assess whether factors produced by such cells can directly influence liver cells to change the production of acute phase plasma proteins, we studied in tissue culture the response pattern of hepatic cells from three species: human hepatoma cells ( HepG2 cells), and primary cultures of rat and mouse hepatocytes. Conditioned media from the squamous carcinoma COLO-16 cells, normal epidermal cells, and activated peripheral monocytes were able to stimulate the synthesis of specific acute phase plasma proteins: alpha 1-antichymotrypsin in HepG -2 cells, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, alpha 1-acute phase protein, and alpha 2-macroglobulin in rat hepatocytes, and alpha 1-acid glycoprotein, haptoglobin, and hemopexin in mouse hepatocytes. Only in rat cells, dexamethasone was found to have further enhancing effect. The increased production of plasma proteins could be explained by an elevated level of functional mRNA. Comparing thymocyte-stimulating activities with the effects on plasma protein production, we found some difference both between the conditioned media of epidermal cells and monocytes, and between the responses of the three hepatic cell systems. Furthermore, gel chromatography of conditioned media resulted in partial separation of activities regulating liver cells and thymocytes. Since there is no strict correlation between thymocyte- and hepatocyte-stimulating activities, the presence of different sets of specific factors is assumed.

Acute-Phase Proteins↗
PubMed1984

Synthesis and turnover of prothrombin during experimental inflammation in rats.

The response of prothrombin to inflammatory reactions was investigated in rats. Inflammation was induced by the administration of either subcutaneous turpentine or intraperitoneal endotoxin, and its effects were studied 24 h and 48 h later. Albumin and alpha 1-acute-phase globulin served as the controls. There were only insignificant changes in plasma prothrombin concentration during inflammation which contrasts sharply with a decrease in circulating albumin by approximately 25% and an increase in alpha 1-acute-phase globulin by 300-400%. These changes were paralleled by similar changes in the incorporation of [3H]lysine into these proteins during the incubation of liver slices from rats that had been pretreated with the phlogistic agents. Prothrombin catabolism, studied using 131I-prothrombin, was increased by approximately 20%; albumin turnover, studied simultaneously with 125I-albumin, was not significantly affected, though the capillary transfer rate of albumin was significantly elevated 48 h after the induction of inflammation. It is concluded that rat prothrombin is not an acute-phase protein.

Animals↗
PubMed1983

Synthesis and secretion of some plasma proteins by tissue slices of Morris hepatoma 7777 and liver from control and turpentine-injected rats.

Slices of Morris hepatoma 7777 or rat liver isolated from control or turpentine-injected rats were incubated for 2 h with 14C-leucine. Radioactivities incorporated into albumin, alpha-fetoprotein, fibrinogen, alpha 1-AP-globulin, haptoglobin and alpha 1-acid glycoprotein were determined after the proteins had been isolated from the incubation medium or tissue homogenate by immunoprecipitation with monospecific antisera. It was found that hepatoma synthesizes fibrinogen, alpha 1-AP-globulin and alpha 1-acid glycoprotein in the amounts comparable to rat liver, whereas formation of albumin and haptoglobin is reduced 5- to 10-fold. Local inflammation elicited by injection of turpentine to tissue donors increased formation of acute-phase protein in liver slices but had no effect on synthesis of these proteins in preparations of Morris hepatoma, although certain ultrastructural changes in the Golgi complex were observed not only in the liver but also in the tumour.

Animals↗
PubMed1983

Kinetics and stoichiometry of inactivation of some pancreatic and leucocyte serine proteinases by rabbit alpha 1-proteinase inhibitors F and S.

Purified rabbit alpha 1-proteinase inhibitors F and S were incubated with bovine trypsin, chymotrypsin or horse leucocyte neutral proteinases in order to determine the stoichiometry of inhibition, inactivation rates of the enzymes and dissociation constants of the complexes. Trypsin reacted with the two forms of alpha 1-PI with different velocities but in the molar ratio of 1:1 and yielding stable complexes. Chymotrypsin reacted very fast with the two forms of alpha 1-PI but required a three-fold molar excess of alpha 1-PI-S for almost complete inhibition, and again the dissociation constants were low. Leucocyte proteinases also needed certain molar excess of the inhibitor but the reaction was complicated by instability of the complexes, especially with Z-Ala-ONp as substrate. We conclude that the interaction of rabbit alpha 1-PI with leucocyte proteinases, and particularly alpha 1-PI-S with chymotrypsin, includes not only complex formation but also some inactivation of the inhibitor.

Animals↗
PubMed1983

Ethionine-dependent inhibition of acute-phase plasma protein synthesis in the rat.

Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins.

Animals↗
PubMed1982

Changes in the blood level and affinity to concanavalin A of rat plasma glycoproteins during acute inflammation and hepatoma growth.

Plasma concentrations of ten individual proteins were measured by electroimmunoassay in young male Buffalo rats following injection of turpentine oil or implantation of Morris hepatoma 7777. The highest relative responses to inflammation and tumour growth were found for alpha 2-macroglobulin, alpha 1-acute-phase globulin and alpha 1-acid glycoprotein. As shown by crossed immuno-affinoelectrophoresis the concanavalin A-reactive fractions of the latter two glycoproteins were predominantly increased in plasma from injured and tumour-bearing rats.

Animals↗
PubMed1982

Isolation of rat alpha-fetoprotein from Morris hepatoma 7777.

1. Tissue slices from Morris hepatoma 7777 were incubated for 3 hr in Krebs-Ringer-bicarbonate solution and the cell-free supernatants were used for isolating alpha-fetoprotein. 2. Purification procedure included salting out with ammonium sulphate, affinity chromatography (either Concanavalin A-Sepharose or Cibacron-Blue-Sepharose), hydrophobic chromatography and preparative polyacrylamide gel electrophoresis (PAGE). 3.alpha-Fetoprotein was obtained with a yield of approx. 10% and in pure form, as judged from crossed immunoelectrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Animals↗
PubMed1981

Ontogeny and tissue distribution of alpha-1-antitrypsin of the mouse.

alpha 1-Antitrypsin is the second most abundant proteinase inhibitor in plasma. The fact that it is a globular glycoprotein of relatively small size (Mr 53 500) allows it access to a wide variety of fluids and tissue sites. alpha 1-Antitrypsin has been purified from mouse plasma by affinity chromatography and ion exchange. The purified protein exhibits homogeneity on polyacrylamide electrophoresis, but electrophoretic heterogeneity on crossed immunoelectrophoresis. Mouse and rat alpha 1-antitrypsin show strong crossreactivity and the half-life for mouse alpha 1-antitrypsin is 15.5 h. Fetal levels are 15% of adult and it requires 25--30 days before adult levels are reached in the neonate. Maternal levels remain unchanged throughout pregnancy and at parturition. The inhibitory is presented in a number of body fluids including serum, breast milk, gastrointestinal washing, lung washings and bile. The source of alpha 1-antitrypsin for all of these fluids appears to be the liver.

Animals↗
PubMed1981

Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S.

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.

Animals↗
PubMed1981

Interaction of horse plasma antithrombin III and alpha 1-proteinase inhibitor with some serine proteinases.

Antithrombin III and alpha 1-proteinase inhibitor isolated simultaneously from horse citrated plasma were tested for inhibitory activity against bovine trypsin and chymotrypsin, as well as elastase-like neutral proteinases from horse leucocytes. The stoichiometry of reaction and kinetic parameters (kass, Ko) were estimated and related to the protein pattern obtained after exposure of these proteinases to horse inhibitors as analyzed by polyacrylamide gel electrophoresis (PAGE and PAGE-SDS). As shown by fast reaction rates and low values of dissociation constants the two inhibitors effectively inactivate trypsin. On the other hand, AT III is completely inactive against chymotrypsin or leucocyte elastases with alpha 1PI only partly inhibits these enzymes.

Animals↗
PubMed1980

Synthesis and catabolism of rabbit alpha 1-antitrypsins F and S.

The metabolic relationship between the two major forms of rabbit alpha 1-antitrypsin, F and S, was investigated by using labeling techniques in vivo and in vitro. After the injection of [14C]leucine, the S/F specific-radioactivity ratio showed characteristic changes with time: at 1 h, the ratio was high (1.2-1.4), but by later times (5-7h) it decreased to a value of approx. 1.1. Two different techniques were used to purify alpha 1-antitrypsin for labelling with iodine. The half-lives of the differentially labelled and simultaneously injected F- and S-forms were 68.1 (+/- 7.6 S.D) and 55.3 (+/- 8.1 S.D)h respectively. Combined electrophoretic and gamma-spectrometric studies provided no evidence for metabolic interconversion of the alpha 1-antitrypsin forms in the circulation. These observations suggest that rabbit alpha 1-antitrypsins F and S are, despite their close chemical composition and immunological identity, metabolically independent proteins. Therefore the possibility is raised that alpha 1-antitrypsin synthesis in rabbits is controlled by two autosomal genes or two sets of such genes.

Animals↗
PubMed1980

Comparison of synthesis and secretion of plasma albumin, fibrinogen and alpha 2-macroglobulin by slices of Morris hepatomas and rat liver.

Slices of rat liver or Morris Hepatomas 5123 GVI and 7777 were incubated for 2 h with 14C-leucine. Newly synthesized labelled proteins were determined independently in the medium and tissue homogenate, total protein after precipitation with trichloro-acetic acid, and albumin, fibrinogen and alpha 2-macroglobulin after immuno-precipitation with monospecific antisera. In comparison with control rat liver, synthesis and secretion of albumin were reduced four- to five-fold in the 2 heptomas examined. On the other hand, synthesis and secretio of fibrinogen and alpha 2-macroglobulin in Hepatomas 5123 GVI annd 7777 were similar to rat liver, especially to the liver isolated from rats with turpentine-induced local inflammation.

Animals↗