Search PubMed⌕ Search

Biomedical subjects

J E Dyr

Publications and source records attributed to J E Dyr.

At least 19 recordsLinked to original sources
PubMed1997

Simultaneous isolation of protein C activator, fibrin clot promoting enzyme (fibrozyme) and phospholipase A2 from the venom of the southern copperhead snake.

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal-chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A2.

Agkistrodon↗
PubMed1997

Solid-phase extraction in malondialdehyde analysis.

Malondialdehyde (MDA) has been widely used as an index of lipoperoxidation in biological and medical sciences as well as in the food industry. A solid-phase extraction (SPE) of the condensation product of the MDA with 2-thiobarbituric acid (TBA) was developed using LiChrolut C18ec, 200 mg (Merck, Darmstadt, Germany), as a SPE cartridge and methanol as an eluent for sample pretreatment before HPLC analysis. The samples of blood plasma, platelet concentrates, or erythrocyte membranes (ghosts) were deproteinized by acetonitrile in the presence of sodium hydroxide prior to the reaction with TBA. The reaction mixture was processed using SPE. The SPE extracts (800 microL of methanol) were put to dryness and after dissolution with 100 microliters of mobile phase, 50 microliters was analyzed by RP-HPLC with fluorescence detection (excitation at 514 nm, emission at 556 nm). The mean MDA concentration in plasmas of 32 healthy donors was 0.37 +/- 0.25 mumol/L and the mean MDA concentration in normal ghosts was 8.3 +/- 4.1 pmol/microgram of protein content. In the case of a patient with a severe form of beta-thalassemia, the concentration of plasma MDA was raised to 1.22 mumol/L and the amount of MDA in erythrocytal ghosts was raised to 21.05 pmol/microgram of protein content. MDA concentration in platelet concentrates (six bags) in the first day of storage was 0.46 +/- 0.18 mumol/L and in the fifth day of storage was 0.55 +/- 0.44 mumol/L.

Blood Platelets↗
PubMed1997

[The Bernard-Soulier syndrome. A hereditary functional and structural disorder of blood platelets].

BACKGROUND: The authors submit a clinical and laboratory description of a patient with Bernard-Soulier syndrome diagnosed in this country for the first time. The investigation comprises an analysis of haemostatic functions, selected structural indicators and the detailed morphology of thrombocytes. The analysis of defined inborn functional disorders of thrombocytes is a method for studying haemostatic mechanisms. METHODS AND RESULTS: In addition to standard haemostatic methods flow cytometry in used to assess the size of platelets, and using monoclonal antibodies against glycoproteins, their presence on the surface membrane is assessed. Thrombocytic glycoproteins are further analyzed using diagonal electrophoresis on sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). The authors found medium thrombocytopaenia with the presence of giant thrombocytes which on elecronmicroscopic examination, with the occasional exception of endoplasmic membrane proliferation, do not display structural deviations. The assessed deviation of thrombocyte aggregation after ristocetin, suggesting a defect of the adhesive capacity of thrombocytes, is apparently due to reduction of the surface glycoprotein GPIb, proved by flow cytometry and SDS-PAGE. The drop of glycoprotein GPIb to 20% of normal values suggests a heterozygote type of disorder with a medium grade of haemorrhagic manifestations. CONCLUSIONS: This is the first case of Bernard-Soulier syndrome in the Czech literature. Its analysis provides evidence of the relationship of the adhesive function to the surface glycoprotein GPIb and confirm the effectiveness of the elaborated diagnostic methods.

Aged↗
PubMed1997

Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.

Adsorption↗
PubMed1996

Comparison of several mouse and rat monoclonal antibodies against human fibrinogen.

Six monoclonal antibodies raised against human fibrinogen have been characterized. Mouse monoclonal antibodies were targeted against sequential epitopes on the immunodominant D-domain of fibrinogen and they crossreacted with all molecules containing the D-domain [fibrin, fibrin(ogen)-degradation products]. Their behavior was not influenced by proteolytic degradation of fibrinogen with plasmin. Rat MoAbs were specific for the conformational epitopes on intact fibrinogen. Their reactivities were substantially lower with fibrin(ogen)-degradation products. Degradation of structures on intact fibrinogen was concomitant with the decay of rat MoAbs reactivity. Those structures were presumably on the C-terminal end of fibrinogen alpha chain and/or on the N-terminal end of fibrinogen beta chain.

Animals↗
PubMed1995

Characterization of platelet antigen for CD45RA monoclonal antibodies.

CD45RA monoclonal antibodies recognize the higher molecular weight isoforms (220 and 205 kDa) of leukocyte common antigen family (CD45), which are typically expressed on B cells and unstimulated T cells. We have found that there are at least three distinct CD45RA monoclonal antibodies which react with platelet 42 kDa (P42) intracellular protein antigen, which seems to be different from any to date described platelet proteins with similar molecular weight. This platelet antigen is a single chain protein, very likely not a glykoprotein, with isoelectrical point between 6.8 and 7.5. P42 does not seem to be a membrane protein and is not associated with platelet cytoskeleton. Results of immunofluorescence assay suggest that P42 may be redistributed to platelet surface after platelet aggregation.

Animals↗
PubMed1994

Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.

Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E. coli inclusion bodies. The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger. This procedure has an inherent advantage of combining refolding and purification procedures in one step. The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration. The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus.

Anion Exchange Resins↗
PubMed1994

Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside.

The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described. These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.

Antibodies, Bacterial↗
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↗