Biomedical subjects

J Hudecek

Publications and source records attributed to J Hudecek.

At least 19 recordsLinked to original sources

PubMed2006

Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes - similarity between human and rat systems.

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.

Animals↗

PubMed2004

[Thrombin activatable fibrinolysis inhibitor (TAFI) and its importance in the regulation of fibrinolysis].

Thrombin activatable fibrinolysis inhibitor (TAFI) also named procarboxypeptidase U (CPU), procarboxypeptidase R (CPR) and plasma procarboxypeptidase B (CPB) provides an important link between fibrinolysis and coagulation cascade. Activated TAFI (TAFIa) reduces a generation of plasmin because it cleaves off the carboxy-terminal lysine residues from partially degraded fibrin and thereby abrogates the fibrin cofactor function in the tPA-mediated catalysis of plasminogen to plasmin. TAFI is activated by thrombin-thrombomodulin complex. TAFI transformation to the activated TAFI (TAFIa) induced by thrombin supports the important role of coagulation cascade in regulation of fibrinolysis. This can be proved by a fact that the patients with a factor XI (FXI) deficiency are prone to bleeding from tissues with a high local fibrinolytic activity (urinary tract, nose, oral cavity, tonsils) that can be explained by a decreased thrombin-mediated TAFI activation. On the other hand the prothrombotic mutation of factor V (FV Leiden) associated with a resistance to activated protein C (APC-resistance) possess both mechanisms-an increased thrombin generation in coagulation cascade and a down regulation of fibrinolysis by a way of the thrombin-induced TAFI activation. For the future an inhibition of TAFI (e.g. by FXI inhibitors) offers the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders. In bleeding disorders (hemophilia A, B) the drugs with a higher efficiency of TAFI for down regulation of an increased fibrinolysis could be used.

Blood Coagulation↗

PubMed2004

[Infection and hemostasis].

Localised and following systemic inflammatory reaction accompanying progression of infection causes generation of anti-inflammatory cytokines. They activate leucocytes, endothelium, coagulation and fibrinolysis. Sepsis is usually accompanied by already decompensated disseminated intravascular coagulation which significantly affects mortality of patients with this disease. The main cause of hypercoagulation state during sepsis seems to be inhibition of fibrinolysis as a result of overproduction of plasminogen activator inhibitor-1 in later stages of the disease. Some microorganisms have specific properties which affect individual components of hemostasis and thus increase their virulence. Because natural inhibitors of coagulation have not only anticoagulation but also strong anti-inflammatory effect, they seem to be an optimum remedy for fluorid coagulopathy during sepsis. Moreover, their use usually does not increase risk of bleeding.

Blood Coagulation↗

PubMed2003

Leukemic cells and aberrant phenotypes in acute leukemia patients: a flow cytometry analysis.

The aim of this study was to evaluate the heterogeneity of immunophenotype features in acute leukemia patients and to detect the presence of leukemia-associated immunophenotypes. We prospectively investigated the phenotype of blast cells from 44 adult acute leukemia patients using a large panel of monoclonal antibodies by multiparametric flow cytometry. Thirty-three patients were classified as AML according to the FAB classification. Eleven patients were diagnosed as ALL (10 cases B-ALL, 1 case T-ALL) according to both FAB and immunnophenotyping. We found leukemia-associated phenotypes in 28 of 33 AML patients (84.8%) and in 8 of 11 ALL patients (72.7%). In 61.1% of patients more than one aberrant phenotype was observed. Linear infidelity was the most frequent aberrancy in both AML (64.3%) and ALL (37.5%) subgroups. The present study shows that MFC is a helpful method for sufficient identification of leukemic cells and for determination of blast cells immunophenotype heterogeneity. The double stain flow cytometry in our study revealed aberrant phenotypes in up to 81.8% patients.

Adolescent↗

PubMed2003

[Factor V Leiden and the Slovak population].

The resistance to activated protein C (APC-resistance) based on the presence of factor V Leiden (F V Leiden) is the most frequent thrombophilic condition in the white race population. It contributes to the origin of thrombosis especially in the venous part of blood vessels. Significant geographic differences have been detected within Europe. The aim of this retrospective study was to determine the frequency in the occurrence of F V Leiden: 1. in healthy (asymptomatic) Slovak population, 2. in their consanguineously unrelated members with thrombosis and 3. in patients with myocardial infarction (IM) without or with other known risk factors of this disease (nicotinism, obesity, hypertension, dyslipoproteinemia, diabetes mellitus), respectively. The detection of FV Leiden was made by molecular biology methods. The occurrence in a group of 152 healthy individuals was four % (6 persons) and this frequency corresponds to the geographic localization of the Slovak Republic in Europe. In a group of 349 patients with thrombosis in anamnesis, FV Leiden was detected in 103 persons (29.5%). The occurrence was higher than the usually reported incidence in these patients (20%). Likewise, in a group of 35 patients with IM without risk factors in anamnesis, the occurrence of FV Leiden (8.6%) was significantly higher in comparison with healthy population and the incidence further increased significantly in a group of 41 patients with IM and the presence of at least one risk factor (14.6%). The authors therefore suppose an active role of the Leiden mutation of FV gene in the pathogenesis of this disease.

Activated Protein C Resistance↗

PubMed2001

Differences in flexibility of active sites of cytochromes P450 probed by resonance Raman and UV-Vis absorption spectroscopy.

Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.

Animals↗

PubMed2000

Structural similarities and differences of the heme pockets of various P450 isoforms as revealed by resonance Raman spectroscopy.

Cytochromes P450 CYP102 A1, 1A2, and 3A4, all belonging to the class II type of P450 enzymes, were studied by resonance Raman spectroscopy. Spectra were measured for the oxidized substrate-free, oxidized substrate-bound, and reduced forms of each of these P450s. The analysis of the resonance Raman spectra indicates that the individual isoforms differ with respect to orientation and conformations of the heme side chains, whereas the overall porphyrin geometry is essentially the same. In the oxidized state, the vinyl groups exhibit both a coplanar and an out-of-plane orientation with respect to the heme, albeit with different relative propensities in the various isoforms. In the reduced state, both vinyl groups are forced into a coplanar orientation. In addition to the differences in behavior of the vinyl groups, the redox-linked spectral changes also include the bending mode of the propionate side chains. The spectral differences associated with the porphyrin substituents are likely to reflect subtle conformational differences in the heme pocket of various P450 isoforms which may constitute the structural basis for the known variability of their functions.

Bacterial Proteins↗

PubMed2000

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study.

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.

Animals↗

PubMed2000

Flexibility and stability of the structure of cytochromes P450 3A4 and BM-3.

The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172].

Bacterial Proteins↗

PubMed1999

[Analysis of phenotype resistance to activated protein C (APC resistance)].

The laboratory diagnosis of resistance to activated C protein (APC-resistance) involves examination of the phenotype and genotype of this thrombophilia. For examination of the phenotype coagulation and chromogenic tests are used. Their essence is examination in the presence and absence of exogenous APC. While the result of the original coagulation examination of APC-resistance which uses the APTT principle is influenced by a number of factors, the sensitivity and specificity of the modification of this examination (dilution of the examined plasma sample by FV deficient plasma before making the test) in relation to detection of FV Leiden is almost 100% and eliminates the majority of limitations of the original examination. The chromogenic assessment of APC-resistance has similar advantages, however, it cannot differentiate between the heterozygous and homozygous form of FV Leiden. During examination of the genotype of subjects with APC-resistance the mutation of FV Leiden is detected in as many as 90%. The group of subjects with the phenotype of APC-resistance comprises in particular subjects with acquired APC-resistance caused by conditions which lead to a disbalance between procoagulation and anticoagulation proteins of haemostasis which influence the reactions of the applied laboratory examinations. The acquired phenotype of APC-resistance can be also associated with an increased risk of thrombosis and the clinical manifestations of this thrombophilia resemble the classical, FV Leiden conditioned APC resistance. Rarely also congenital causes of the phenotype of APC-resistance are encountered caused by another mutation than the Leiden mutation of gene FV. The concurrent examination of the patient's plasma with the original and modified coagulation test makes it possible to assess the inborn cause of APC-resistance (positive finding also in modified examination). The presence of FV Leiden is then confirmed by examination of the genotype by the polymerase chain reaction.

Activated Protein C Resistance↗

PubMed1999

[New beta0-thalassaemic insertion mutation (CD 7/8, +G) in a Slovak family, associated with the Mediterranean haplotype IX].

The authors describe a newly identified beta0-thalassaemic mutation found in two subjects from two generations of a Slovak family. The beta0-thalassaemic allele developed by insertion of one nucleotide (+G, CD 7/8) into the first exon of the beta-globin gene. The mutation causes a shift of the open globin reading frame which leads to the development of a terminal codon in codon 22. The thalassaemic allele is associated with the mediterranean haplotype IX. The mutation has in both heterozygotes the phenotype of beta0-thalassaemia minor with a slightly elevated level of HbF.

Alleles↗

PubMed1997

[Treatment of MALT lymphoma of the digestive tract].

Mucosa-associated lymphoid tissue (MALT) is an autonomous part of the immune system with a specific function, histological structure and circulation of cells. As a result of malignant transformation of this tissue maltomas develop, peripheral extranodal non-Hodgkin lymphomas of B-origin more than two-thirds of which are found in the digestive system. The authors investigated in a retrospective study the effectiveness of different types of treatment of maltomas of the digestive tract with a low and high grade of malignity in 16 patients. It appears that splenectomy is not necessary in these patients and that the effectiveness of primary chemotherapy with a view of inducing complete remission of the disease is comparable with surgical treatment and subsequent adjuvant treatment without deterioration of the patient s life by mutilating surgery. An integral part of treatment of gastric maltomas is eradication of infection caused by Helicobacter pylori.

Adult↗

PubMed1997

[Heparin cofactor II and thrombophilic conditions].

Heparin co-factor II is in addition to antithrombin III another heparin dependent thrombin inhibitor. This glycoprotein blocks thrombin action not only in haemostasis but also in its other effects. Congenital deficiency of heparin co-factor II was found equally frequently among asymptomatic subjects and patients with thrombotic complications. Although its deficiency probably is not a serious risk factor of thromboembolic disease, it can promote its development in a significant way. The authors describe the case of a child with thrombotic complications and a congenital deficiency of heparin co-factor II.

Child, Preschool↗

PubMed1994

[Resistance to activated protein C, so-called APC resistance: the first two cases in Slovakia].

For the anticoagulant action of activated protein C (APC) in addition to protein S another newly detected cofactor is essential. Its deficiency is described as APC-resistance. This defect which is closely associated with the molecule of the factor V seems to be the most frequent cause of thrombophilia. The authors describe two patients where APC-resistance was diagnosed for the first time in Slovakia.

Adult↗

PubMed1994

[Large blood platelets--risk factors in myocardial infarction].

Thrombocytes participate in the pathogenesis of myocardial infarction. Larger platelets are more active than small ones. In patients after an acute coronary a larger number of platelets was found, a medium volume of půatelets and a similar thrombocytocrite as compared with the healthy population of comparable structure and age. The MPV in patients of advanced age is statistically significantly larger. There are no significant correlations between MPV and the risk factors investigated by the authors.

Adult↗

PubMed1991

Nanosecond fluorescence of tryptophans in cytochrome P-450scc (CYP11A1): effect of substrate binding.

Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.

Adrenal Glands↗

PubMed1989

Nanosecond fluorometry of the single tryptophan in cytochrome P-450e (P450IIB2).

Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.

Animals↗

PubMed1988

Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane.

The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.

Algorithms↗