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Biomedical subjects

J E Dyr

Publications and source records attributed to J E Dyr.

At least 37 records · Page 2Linked to original sources
PubMed1992

[Fibrinogen seen as an independent cardiovascular factor from the molecular aspect].

The blood protein fibrinogen is one of the main independent cardiovascular risk factors and it is very likely that an increased fibrinogen level is not the consequence of cardiovascular disease but its direct cause. Fibrinogen participates actively in many processes in the organism and undergoes various changes of the molecule but it is not known what is the molecular background, why even a slight increase of the fibrinogen level is so dangerous as regards increased risk of cardiovascular attacks. In the present work the authors compared, using monoclonal antibodies, the rate of release of fibrinopeptides A and B (FpA, FpB) by the action of thrombin from fibrinogen in solution and from fibrinogen adsorbed to a solid surface. The authors revealed that the rate of FpB breakdown from sorbed fibrinogen, contrary to fibrinogen in solution is comparable to the release rate of FpA and does not depend on the release of FpA (except for competitive inhibition). The release of FpB from sorbed fibrinogen thus takes place, contrary to fibrinogen in solution, at a significant recordable rate from the very onset of thrombin action. Fibrinogen adsorption to a solid surface is associated with conformation changes which cause this effect and which lead to the formation of a two-dimensional formation formed by fibrinogen after its interaction with the surface of activated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular Diseases↗
PubMed1992

[New methods of isolating recombinant proteins].

Recombinant proteins are isolated from very complex protein mixtures present in the producing cell. The isolation process involves in general four mutually interconnected stages: 1. release of the recombinant protein from the cellular environment, 2. preparation of the specimen for separation, 3. separation, 4. qualitative and quantitative analysis of the preparation. Each of these stages is formed by a complex series of methods which destroy the cellular wall, solubilize the specimen and involve the use of suitable precipitation, chromatographic, electrophoretic, immunochemical and other techniques for separation and analysis. In the submitted paper the authors describe a relatively simple isolation of recombinant peptide used for the preparation of a diagnostic kit for AIDS. From knowledge of the sequence of nucleotides in cDNA antisense peptides can be derived which have a high affinity with the isolated protein and they can be also used for affinity chromatography. An effective isolation technique is the use of mimetic ligands on the basis of textile dyes. By their combination and possible modification it is also possible to achieve separation of the required protein from contaminating substances. The mentioned highly specific methods can be combined with classical chromatographic techniques. During every step individual chromatographic fractions are tested by SDS electrophoresis in polyacrylamide gel incl. possible use of immunoblotting a specific staining.

Escherichia coli↗
PubMed1991

Comparison of rat and human major platelet glycoproteins.

1. Using electrophoretic techniques combined with various detection methods we ascribed rat platelet glycoproteins (GPs) related to human GPIb, GPIIb and GPIIIa. 2. Rat GPIIb and GPIIIa crossreacted with rabbit polyclonal antibodies against human GPIIb and GPIIIa. 3. Species differences in glycosylation of GPs were shown using various lectins. 4. Molecular mass of rat major GPs was determined by SDS-PAGE (unreduced, reduced, kDa): GPIb (200, 166/26), GPIIb (140, 120/32) and GPIIIa (96, 106). 5. Isoelectric points of rat GPIIb and GPIIIa are shifted to the alkaline region as compared to human related GPs.

Animals↗
PubMed1991

[Hemostasis disorders in monoclonal gammopathies].

The authors investigated haemostatic parameters of 43 patients with monoclonal gammapathy with principal diagnosis of multiple myeloma (MM-35), non-Hodgkin's lymphoma (NHL-3), M. Waldenström (3) and monoclonal gammapathy of undetermined significance (MGUS-2). Primary haemostasis defect was found in 24 patients. With the exception of 2 thrombocytopenic patients, the defect of aggregation, procoagulant activity and retraction is supposed to be caused by paraprotein. The examination of the 14C 5-HT release and TX B2 synthesis in thrombocytes showed, that platelet activation is unaffected and the paraprotein interferes with interactions of thrombocytes or with coagulation system. In plasma coagulation system explicit abnormalities were found only in thrombin time in 12 patients. The more detailed examination disclosed, that the defect resulted from paraprotein interference with fibrin monomer polymerisation. The thrombin proteolytic activity remained unaffected. In 3 patients shortened euglobulin lysis time was observed. Laboratory haemostasis defect was found in 26 patients (60%), however, the bleeding symptoms manifested in 5 cases (11%) only. The analysis of study results showed, that the most important abnormalities leading to overt bleeding are thrombocytopenia or combined haemostasis defect. Isolated laboratory defects remained silent in most cases.

Female↗
PubMed1990

[Monoclonal antibodies against fibrinogen and fibrin and against their proteolytic degradation products].

The level of fibrinogen (FDP) and fibrin (fDP) degradation products is one of the surprisingly few unequivocal indicators of the activity of the coagulation and fibrino (geno) lytic system. Today it is beyond doubt that, similarly as the FDP/fDP level is of unequivocal importance, traditional methods of their assessment are very equivocal. Fibrin and FDP/fDP are molecules derived from fibrinogen, which are formed from the latter as a result of a series of reactions during which structures are formed or are made available which are not present in the fibrinogen molecule. New antigenic determinants are formed, in relation to the original molecule so-called neoepitopes, neoantigens. The great predominance of monoclonal antibodies prepared in the meantime reacts with fibrinogen as well as with fibrin and their degradation products. By means of specific approaches and original methods it proved, however, possible to prepare some specific monoclonal antibodies which were successfully used in diagnostic tests in vitro; their use for detection of thrombi in vivo is tested as well as their use for an increasingly effective thrombolytic therapy after their conjugation with plasminogen activators. It may be expected that this problem will be intensely developed in the near future.

Antibodies, Monoclonal↗
PubMed1990

The action of a fibrin-promoting enzyme from the venom of Agkistrodon contortrix contortrix on rat fibrinogen and plasma.

The action of a fibrin-promoting enzyme isolated from the venom of A.c.contortrix was investigated. The ratio of fibrinopeptides A and B released was similar in isolated human and rat fibrinogen and human plasma, fibrinopeptide B always being released preferentially. However, no clotting occurred in rat plasma, and no fibrinopeptides were released even after prolonged incubation. The results suggest strong, fast-acting irreversible neutralization of the enzyme activity in rat plasma.

Animals↗
PubMed1990

[Alloimmune neonatal thrombocytopenia due to anti-P1A1 thrombocyte antibodies].

The authors describe three patients with neonatal thrombocytopenia where they detected in the maternal serum, using the immunofluorescence and immunoenzymatic test, antibodies against thrombocytes and thus confirmed the alloimmune nature of thrombocytopenia. By molecular characterization of the immunoreactivity of the thrombocytic antibody in the mother of neonate no. 1 evidence was provided that the antibody is against glycoprotein IIIa which carried antigen P1A1. In the reference laboratory in Amsterdam the anti-P1A1 specificity of this thrombocytic antibody was confirmed. The authors obtained and tested the first type anti-P1A1 serum in Czechoslovakia which can serve to detect P1A1 antigen negative subjects.

Antibodies↗
PubMed1989

Conversion of fibrinogen to fibrin induced by preferential release of fibrinopeptide B.

Fibrin clot-promoting enzyme preferentially releasing fibrinopeptide B from fibrinogen was isolated from the crude venom of Agkistrodon contortrix and its mode of action was studied in detail. A purification procedure involving affinity chromatographies on immobilized lectin and arginine removed plasmin-like and kallikrein-like activities towards low-molecular-weight chromogenic substrates. Fibrin-promoting enzyme cleaved off only fibrinopeptides A and B from fibrinogen. The initial relative rate of release of fibrinopeptide B/fibrinopeptide A depended strongly on the presence of Ca2+. In the absence of Ca2+ the amount of fibrinopeptides released by fibrin-promoting enzyme at the gel point was greater. Fibrinopeptide B was no longer released before fibrinopeptide A from the non-polymerizing N-terminal disulphide knot of fibrinogen. Catalyzed by activated factor XIII, complete gamma-dimer and alpha-polymer formation was observed in fibrin from which only 23% of fibrinopeptide A, but 100% of fibrinopeptide B was released by fibrin-promoting enzyme. gamma-dimer formation markedly preceded alpha-polymer formation. These results strongly imply a similar overall arrangement of monomer molecules in this fibrin when compared with a thrombin-induced fibrin in which fibrinopeptide A is released before fibrinopeptide B. These observations support the concept that on fibrinopeptide B release from fibrinogen polymerization sites are exposed which reinforce the sites exposed on the release of fibrinopeptide A.

Calcium↗
PubMed1989

Distortion of the electrophoretic titration curves of some proteins.

The electrophoretic titration curves of complex mixtures of vitamin K-dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3-10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM-2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM-2 membrane without incubation with albumin.

Animals↗
PubMed1989

Fibrinopeptide-releasing enzymes in the venom from the southern copperhead snake (Agkistrodon contortrix contortrix).

Protein fractionation techniques utilizing the different properties of the sample (size, charge, sugar moiety) were employed to characterize the crude A. c. contortrix venom. Gel filtration chromatography resolved about six to eight peaks, high performance liquid ion exchange chromatography and chromatofocusing about 12-14 peaks exhibiting hydrolytic activity. Two fibrin clot-promoting enzymes--both releasing fibrinopeptides A and fibrinopeptides B, but with different fibrinopeptide A/fibrinopeptide B relative rates were observed. The two enzymes (or enzyme isoforms) were serine proteinases with essentially the same hydrolytic activity towards low molecular chromogenic substrate for thrombin. They were of approximately the same size (one peak of 68,000 relative mol.wt on gel filtration chromatography), had apparent isoelectric points of about 6.4 and 5.4, respectively, and were glycoproteins.

Chromatography, Gel↗
PubMed1988

Evaluation of individual variability in the composition of Agkistrodon contortrix contortrix venom by means of HPLC and two-dimensional PAGE.

Qualitative and quantitative differences of coagulant enzymes in venom samples from individuals of the Agkistrodon c.c. were studied by means of 2D-PAGE and high performance anion-exchange chromatography. A great diversity was found among individual venoms. The two most similar venoms had an identical composition in only about 90%, the least similar ones in about 45% spots. All venoms studied contained more than two fractions with fibrinolytic activity. In three out of the nine analysed venoms two different thrombic proteases were present, one venom contained only one of these enzymes, whereas in five venoms no thrombic activity was detectable.

Animals↗
PubMed1988

Assessment of a complex mixture of interacting proteins in the coagulant active venom from Agkistrodon contortrix contortrix.

The crude venom of Agkistrodon contortrix contortrix was characterized by means of 2D-PAGE (using various separation principles in the respective directions) and high performance gel filtration chromatography. It was found that the venom presents a rich and remarkably stable mixture of proteins, mostly glycoproteins, which may interact each other. High stability of the venom in spite of the presence of many proteolytic enzymes, must most likely be attributed to the sugar moieties of venom proteins. Carbohydrate composition also causes considerable heterogeneity in charge and the presence of wide range of charge isomers. The intricate complexity of the venom makes it a real difficult-to-separate mixture.

Animals↗
PubMed1987

Organ distribution of fibrinogen-bound methotrexate in mice with Gardner lymphosarcoma.

The distribution of fibrinogen-bound 3H methotrexate was investigated in Gardner lymphosarcoma bearing mice. 3H labeled methotrexate (3H MTX) was covalently bound by means of aminopropyl carbodiimide to bovine and mouse fibrinogen (FBG). The preparations as well as the free 3H MTX were applied i.v. in a single dose to three groups of C3H mice on day 6 after the inoculation of Gardner lymphosarcoma. 3H MTX level was determined in the blood, spleen, tumor and liver. Sufficient amounts of MTX were released by proteolysis of FBG-MTX derivatives to induce chemotherapeutical effects. Protracted accumulation of MTX applied in the form of FBG-MTX derivatives was found in the spleen and in the liver, in contradistinction to free drug application, suggesting the proteolytic degradation as a directing step responsible for the prolonged persistence of FBG-MTX derivatives in the organs. In the tumor the highest amount of MTX was released from mouse FBG supporting the view of ready uptake of homologous FBG by tumors.

Animals↗