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Biomedical subjects

V Brabec

Publications and source records attributed to V Brabec.

At least 55 records · Page 3Linked to original sources
PubMed1996

DNA adducts of antitumor trans-[PtCl2 (E-imino ether)2].

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.

Animals↗
PubMed1995

Correlation between cytotoxicity and DNA binding of polypyridyl ruthenium complexes.

The cytotoxicity of chloropolypyridyl ruthenium complexes of structural formulas [Ru(terpy)-(bpy)Cl]Cl, cis-[Ru(bpy)2Cl2], and mer-[Ru(terpy)Cl3] (terpy = 2,2':6'2"-terpyridine, bpy = 2,2'-bipyridyl) has been studied in murine and human tumor cell lines. The results show that mer-[Ru(terpy)Cl3] exhibits a remarkably higher cytotoxicity than the other complexes. Moreover, investigations of antitumor activity in a standard tumor screen have revealed the highest efficiency for mer-[Ru(terpy)Cl3]. In a cell-free medium, the ruthenium complexes coordinate to DNA preferentially at guanine residues. The resulting adducts can terminate DNA synthesis by thermostable VentR DNA polymerase. The reactivity of the complexes to DNA, their efficiency to unwind closed, negatively supercoiled DNA, and a sequence preference of their DNA adducts (studied by means of replication mapping) do not show a correlation with biological activity. On the other hand, the cytotoxic mer-[Ru(terpy)Cl3] exhibits a significant DNA interstrand cross-linking, in contrast to the inactive complexes which exhibit no such efficacy. The results point to a potential new class of metal-based antitumor compounds acting by a mechanism involving DNA interstrand cross-linking.

2,2'-Dipyridyl↗
PubMed1995

Recognition of DNA interstrand cross-links of cis-diamminedichloroplatinum(II) and its trans isomer by DNA-binding proteins.

Recognition and processing by cellular proteins of DNA modified by platinum complexes have been suggested to be relevant to the mechanism of their antitumor activity. Platinum complexes form on DNA various mono- and bifunctional adducts. It has already been described by other authors that intrastrand cross-links formed on DNA by antitumor cis-diamminedichloroplatinum(II) (cisplatin) between neighboring purine residues are recognized by several DNA-binding proteins. In contrast, these proteins do not recognize the intrastrand cross-links formed on DNA by cisplatin or its clinically ineffective trans isomer (transplatin) between nonadjacent base residues. An eventuality heretofore not addressed is that DNA interstrand cross-links (ICLs) of platinum compounds may be recognized by and bound to DNA-binding proteins. DNA probes of 110 base pairs (bp) were constructed containing five equally spaced ICLs of cisplatin or transplatin. These ICLs were formed at specific sites at which these adducts are preferentially formed in natural DNA. Gel electrophoresis mobility shift and competition assays with these probes were used to investigate the specific recognition and binding of the calf thymus HMG1 protein to the DNA ICLs of both platinum isomers. The ICL of antitumor cisplatin was recognized by and bound to the HMG1 protein with a similar affinity as the 1,2-intrastrand d(GpG) cross-link of this drug. The protein binding to the ICL is selective for the DNA modification by cisplatin, but not by chemotherapeutically inactive transplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1995

Comparison of the ankyrin (AC)n microsatellites in genomic DNA and mRNA reveals absence of one ankyrin mRNA allele in 20% of patients with hereditary spherocytosis.

Combined deficiency of ankyrin and spectrin represents the most common biochemical abnormality in hereditary spherocytosis (HS). To examine whether a decrease in ankyrin mRNA represents a frequent cause of this type of HS, we took advantage of the reported (AC)n microsatellite polymorphism in the 3' untranslated region of ankyrin cDNA. We first measured the number of AC repeats in genomic DNA encoding erythrocyte ankyrin in 36 unrelated Czech HS patients with combined ankyrin and spectrin deficiency and found 21 of these subjects (58%) to be heterozygotes for the (AC)n microsatellite size. Further analysis of reticulocyte RNA showed that ankyrin cDNA from 7 of these 21 heterozygotes (33%) contained only one of the two ankyrin alleles. We conclude that approximately 1/3 of ankyrin-deficient autosomal dominant HS is caused by reduced expression of one ankyrin allele which, in turn, is caused by either a reduced transcription of one allele of the mutated ankyrin gene or abnormal processing or decreased stability of the mutant ankyrin mRNA. Because ankyrin deficiency is detected in approximately 60% of HS subjects, this result suggests that approximately 20% of all HS is caused by a decreased expression of one ankyrin mRNA allele.

Alleles↗
PubMed1995

DNA interactions of antitumor platinum(IV) complexes.

Modifications of natural DNA and synthetic double-stranded oligodeoxyribonucleotides by cis-diamminedichloro-trans-dihydroxyplatinum(IV) (oxoplatin) were studied by means of ELISA, Maxam-Gilbert footprinting techniques, HPLC of enzymically digested DNA, and transcription assay. It was found that oxoplatin can bind DNA directly without addition of a reducing agent. In addition, the antibodies elicited against DNA modified by cisplatin were not competitively inhibited by DNA modified by oxoplatin. However, DNA containing the adducts of oxoplatin became a strong inhibitor of these antibodies, if it was subsequently treated with ascorbic acid, which is a reducing agent. These results were interpreted to mean that oxoplatin can form DNA adducts containing the platinum moiety in the quadrivalent state. The direct irreversible binding of the platinum(IV) drug is, however, slow as compared to the reaction of its platinum(II) counterpart. It was also found that oxoplatin preferentially binds to guanine residues and can form DNA intrastrand and interstrand cross-links containing platinum(IV). The DNA adducts containing platinum(IV) can inhibit in vitro transcription by a prokaryotic DNA-dependent RNA polymerase. We find that the platinum(IV) complex binds to DNA at similar sites as its platinum(II) counterpart. On the other hand, the DNA adducts containing the platinum(II) or platinum(IV) analogues differ in the number of ligands and the formal charge on their platinum center. We suggest that these differences could be responsible for distinct conformational features and stability of DNA modified by platinum(II) or platinum(IV) complexes.

Animals↗
PubMed1995

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis.

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.

Alleles↗
PubMed1995

A nonsense mutation 1669Glu-->Ter within the regulatory domain of human erythroid ankyrin leads to a selective deficiency of the major ankyrin isoform (band 2.1) and a phenotype of autosomal dominant hereditary spherocytosis.

We describe a nonsense mutation in the regulatory domain of erythroid ankyrin associated with autosomal dominant hereditary spherocytosis with a selective deficiency of the ankyrin isoform 2.1 (55% of normal), a deficiency of spectrin (58% of normal) proportional to the decrease in ankyrin 2.1, and a normal content of the other main ankyrin isoform, protein 2.2. PCR amplification of cDNA encoding the regulatory domain of ankyrin revealed a marked decreased in the ratio of ankyrin 2.1 mRNA to the ankyrin 2.2 mRNA. Sequencing of ankyrin gene in the region where the 2.1 and 2.2 mRNA differ detected a nonsense mutation 1669Glu-->Ter (GAA-->TAA) in one ankyrin allele. Only normal ankyrin 2.1 mRNA was detected in the reticulocyte RNA. Since the alternative splicing within the regulatory domain of ankyrin retains codon 1669 in ankyrin 2.1 mRNA and removes it from ankyrin 2.2 mRNA, we propose that the 1669Glu-->Ter mutation decreases the stability of the abnormal ankyrin 2.1 mRNA allele leading to a decreased synthesis of ankyrin 2.1 and a secondary deficiency of spectrin.

Abnormalities, Multiple↗
PubMed1995

[Diagnosis of beta-thalassemia on the basis of HbA2 determination].

The increased level of HbA2 is a reliable marker of heterozygous beta-thalassaemia. The levels of HbA2 measured by three different methods were compared and the ranges for the normal and for the heterozygous beta-thalassaemia were assessed. The levels of HbA2 2.76 +/- 0.47% for normal (30 blood donors) and 4.62 +/- 0.77% for beta-thalassaemia (50 patients) were obtained by the chromatographic method 2.61 +/- 0.42% HbA2 for normal (30 blood donors) and 5.82 +/- 0.89% HbA2 for beta-thalassaemia (46 patients) were assessed by electrophoresis on hydragel (Sebia) and 2.8 +/- 0.62% HbA2 for normal (30 blood donors) and 6.04 +/- 0.96% HbA2 (47 patients) were found when using cellulose acetate electrophoresis. An increased level of foetal Hb was found in nine patients with beta-thalassaemia. The diagnosis of beta-thalassaemia was confirmed by molecular genetic methods in all cases with an elevated HbA2 level, while a normal HbA2 level did not rule out heterozygous beta-thalassaemia.

Chromatography↗
PubMed1995

[Structural variants in hemoglobin occurring in the Czech Republic].

The authors present a review of clinical and laboratory findings of seven in the Czech Republic hitherto diagnosed structural haemoglobin variants. Unstable variants are found most frequently: Hb-Köln, Hb-St. Louis, Hb-Nottingham, Hb-E and Hb-Hradec Králové. The variant Hb-Hradec Králové (Hb-HK) or alpha 2 beta 2 115 (G17) Ala-Asp was newly detected. The great instability of Hb-HK chains makes classical diagnosis of Hb-pathy impossible. It was possible to identify it only at a molecular genetic level. A manifestation of Hb-HK instability is also the thalassaemic feature of the disease and the formation of Heinz bodies from free chains. The only representative of haemoglobins with a high oxygen affinity identified in this country was newly detected. It was given the name Hb-Olomouc or alpha 2 beta 2 86 (F2) Ala-Asp. This haemoglobin variant leads to erythrocytosis in father and son and the same clinical manifestations were recently described also in Japan. The last structural variant of haemoglobin found in this country is Hb-M Milwaukee or alpha 2 beta 2 67 (E11) Val-Glu which in our patients is manifested rather by haemolysis with formation of Heinz bodies than classical cyanosis. The cause of instability of Hb-M in our patients is not known. Hb-S was not diagnosed so far in the Czech Republic.

Adolescent↗
PubMed1994

EDMA 2000 as a matrix for high-performance liquid chromatography of human haemoglobin chains.

An ethylenedimethacrylate polymer-based matrix (EDMA 2000) is described that shortens the time for separation of haemoglobin (Hb) chains to 30 min, compared with the use of C4 large-pore Vydac columns, commonly used for reversed-phase HPLC of Hb variants. EDMA 2000 was successfully used for the isolation of an abnormal beta-chain of the unstable Hb recently characterized as Hb Nottingham.

Child↗
PubMed1994

Mutations in the pyruvate kinase L gene in patients with hereditary hemolytic anemia.

We have completely sequenced the introns of the human L-type pyruvate kinase (PK) gene using the published cDNA sequence. Subsequently, DNA from 12 unrelated PK deficiency (PKD) patients of Central European origin was investigated for mutations in this gene by solid-phase sequencing. We detected 10 different mutations, 9 of which result in single amino acid alterations, whereas the tenth destroys a splice site. Eight of the 10 mutations have not been described before. We found 7 missense mutations: G994-->A (Gly-332-->Ser), G1006-->T (Ala-336-->Ser), A1081-->G (Asn-361-->Asp), G1174-->A (Ala-392-->Thr), G1493-->A (Arg-498-->His), G1529-->A (Arg-510-->Gln), C1594-->T (Arg-532-->Trp), one in-frame triplet deletion (del) as well as one insertion (ins): del AAG1060-62 (del Lys-354), ins AGC after C1203 (ins Ser after Cys-401), and one splice-site mutation at the border of intron A to exon 3: g/G283-->a/G. Although the enzymatic properties are substantially changed in all PK mutations, only two affected amino acid positions are in or close to the active site. Mutations C1594-->T, G994-->A, del AAG1060-62 and the splice-site mutation g/G283-->a/G have been detected in two different patients each. Mutation G1529-->A was found in five different alleles. Haplotype analysis with the A/C polymorphism at position 1705 gave evidence for a single origin of this most frequent mutation in PKD as suggested by Baronciani and Beutler (Proc Natl Acad Sci USA 90:4324, 1993). Carrier detection and prenatal diagnosis are now feasible for the affected families.

Anemia, Hemolytic, Congenital↗
PubMed1994

Monofunctional adducts of platinum(II) produce in DNA a sequence-dependent local denaturation.

The effects on the conformation of DNA produced by the monofunctional adducts of chloro-(diethylenetriamine)platinum(II) chloride or cis-diamminemonoaquamonochloroplatinum(II) have been investigated by means of the single-strand-specific probe chloroacetaldehyde (CAA). The denatured sites to which CAA was bound and that were induced in DNA by the monofunctional adducts of the platinum complexes were characterized by means of three experimental approaches. These include measurement of the fluorescence of a plasmid fragment treated with CAA, analysis of oligonucleotides treated with CAA and cleaved by piperidine, and termination of duplex transcription on a fragment of plasmid DNA treated with CAA. The results indicate that the denaturational change preferentially occurs in the base pair containing the monoadducted deoxyriboguanosine in the trinucleotide sequence Py-deoxyriboguanosine-Py (Py is a pyrimidine deoxyribonucleoside). It was suggested that this conformational alteration facilitates in DNA the formation of minor bifunctional adducts of cis-diamminedichloroplatinum(II).

Acetaldehyde↗
PubMed1994

Hb Nottingham or alpha 2 beta 2 98 (FG5) Val-->Gly in a Czech child.

We report a fourth case of Hb Nottingham [alpha 2 beta 2 98 (FG5) Val-->Gly] observed in an 8-year-old girl in the Czech Republic with clinical and laboratory symptoms of severe hemolytic anemia. The unstable hemoglobin probably represents a de novo mutation, since the parents of the patient and the two siblings do not exhibit any hematological abnormalities. Splenectomy had a beneficial effect on the degree of hemolysis, as well as on the Hb level.

Amino Acid Sequence↗
PubMed1994

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

We describe a duplication of 10 nucleotides (2,455-2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

Red cell ferritin and iron stores in chronic granulocytic leukemia.

Basic red cell ferritin was investigated in 28 patients with different phases of chronic granulocytic leukemia (CGL). Red cell ferritin was significantly decreased in remission after busulphan treatment and significantly elevated in the blast crisis as compared to healthy controls. Bone marrow stainable iron was decreased or absent in 86% of patients in the initial phase at the time of diagnosis and in 92% of those in remission. Red cell ferritin correlated with serum ferritin, however, serum ferritin level remained above normal range during all phases of the disease. A negative correlation between red cell ferritin and hemoglobin (Hb) (r = -0.605, p < 0.001) suggested that red cell ferritin level reflected the rate of iron utilization for heme synthesis. Decreased red cell iron stores observed in the remission may be explained by regression of dyserythropoiesis and by restoration of normal Hb synthesis after busulphan treatment. A progressive dyserythropoiesis in the blast crisis may lead to an increased red cell ferritin level.

Biopsy↗
PubMed1994

[Treatment of iron overload states with oral administration of the chelator agent, L1 (Deferiprone)].

Nine iron overloaded patients were treated with L1--Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one) in daily dose 3 g (40-50 mg/kg) for 12 weeks. In 7 patients the efficiency of L 1 treatment was compared to the therapeutic effect of the same dose of desferrioxamine (Desferal). A significant increase in urinary iron excretion was observed after administration of both chelating agents. Iron excretion after L 1 treatment was approximately 65% of that obtained with Desferal. The amount of excreted iron correlated with the amount of iron stores before chelation. A significant decrease in transferrin saturation, serum and red cell ferritin was observed after treatment with Desferal, L 1 administration caused a significant decrease only in serum ferritin level. However, all the parameters reflecting iron stores remained increased when compared to normal values after 12 weeks of chelation therapy. An incomplete absorption from gut and some reutilization of chelated iron may be responsible for less potent iron chelation by L 1 in comparison to Desferal. A low tolerance of the drug together with repeated nausea and vomiting were the most frequent adverse effects observed in the course of L1 administration. L 1 treatment had to be discontinued due to repeated vomiting in one patient and due to progressive granulocytopenia and thrombocytopenia in another patient. Because of the side effects more clinical studies with L 1 are needed before its introduction in wide clinical practice.

Administration, Oral↗
PubMed1994

[Iron stores in patients with chronic kidney failure treated with recombinant human erythropoietin].

Nine patients with anemia of chronic renal failure were treated with recombinant human erythropoietin (rHuEPO) in dose 50-150 IU/kg/week. After 8 weeks the treatment was maintained with 30-50 IU/kg/week for one year. A significant increase of hemoglobin (Hb) level and red blood cell (RBC) count was observed in all patients. Administration of rHUEPO maintained Hb level higher than 100 milligrams and RBC count above 3.0 x 10(12)/l. Iron stores decreased in all patients. Parameters reflecting either the real amount of iron available for erythropoiesis or iron stores in erythroid precursors, i.e. red cell ferritin (eF), free erythrocyte protoporphyrin (FEP) and transferrin saturation (satTRF) were the most reliable tools for diagnosis of iron deficient erythropoiesis. Serum ferritin (sF) was not decreased in most patients, however, sF level was below 50 micrograms/l in all patients at the time of diagnosis of iron deficiency. Iron supplementation in a daily dose allowing absorption of 100mg of elementary iron was sufficient to cover the increased demand for iron in rHuEPO treated patients.

Anemia↗