ABSTRACT
We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.
Subject(s)
Polymorphism, Genetic , Prions/genetics , Scrapie/genetics , Sheep/genetics , Animals , Codon/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Predisposition to Disease , Genotype , Molecular Sequence Data , Nucleic Acid Denaturation , Polymerase Chain Reaction , Sequence Analysis, DNA , SlovakiaABSTRACT
The first confirmed evidence of scrapie in Slovakia was demonstrated in one sheep of the autochthonous Merino breed from the southeastern part of the country. The reported scrapie was diagnosed during compulsory transmissible spongiform encephalitis (TSE) screening of sheep over 9 months of age assigned for consumption. The positive ewe was 5-year-old, which did not show any clinical signs of scrapie. The presence of the proteinase-resistant prion protein (PrP) in brain was proved independently by two laboratories using two different immunochemical screening systems, namely the Prionics Check (Western blot analysis) and Enfer TSE enzyme-linked immunosorbent assay (ELISA). In addition, the genotyping analysis of PrP gene demonstrated the presence of PrP genotype from the high risk group R4. The affected sheep was homozygous for the allele PrP(ARQ) (ARQ/ARQ) coding for alanine (A), arginine (R) and glutamine (Q) at three most relevant codons (136, 154 and 171, respectively). The healthy sister of the positive ewe was heterozygous in the PrP locus and carried alleles ARQ/ARR.
Subject(s)
Brain/metabolism , PrPSc Proteins/genetics , PrPSc Proteins/pathogenicity , Scrapie/diagnosis , Amino Acid Sequence , Animals , Female , Genotype , Molecular Sequence Data , Polymorphism, Genetic , PrPSc Proteins/metabolism , Prions/classification , Prions/genetics , Scrapie/metabolism , Sheep, Domestic , SlovakiaABSTRACT
Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Drug Resistance, Multiple/genetics , Glycogen/metabolism , Leukemia L1210/genetics , Oligosaccharides/metabolism , Polysaccharides/metabolism , Animals , Base Sequence , Cell Survival/drug effects , Concanavalin A/toxicity , DNA Primers , Leukemia L1210/pathology , Mice , Phenotype , Polymerase Chain Reaction , RNA, Messenger/genetics , Tumor Cells, CulturedABSTRACT
In a worldwide majority of sheep breeds an excessive susceptibility to scrapie associated with the PrP gene alleles coding for valine (V; at the 136 codon) and glutamine (Q; at the 171 codon) (e.g., VRQ/VRQ, VRQ/ARQ, or ARQ/ARQ) was demonstrated. Particularly the PrPVRQ allele is closely associated with the high-risk development of the disease; the PrPARQ allele can also fulfill this function but under certain limited conditions. Polymorphism in the PrP gene sequences (conclusively related to the increased susceptibility of sheep to scrapie) of improved Valachian sheep from two Slovak regions, Orava and Spis, was determined. Examination of 735 sheep showed that ARR/ARQ was the most frequent genotype (45.2%). High-risk genotypes were determined in 32.4% of sheep (ARQ/ARQ 19.3, ARR/VRQ 9.0, ARR/VRQ 3.5, VRQ/VRQ 0.3, ARR/VRR 0.3). Low-risk genotypes were found in 67.7% of sheep (ARR/ARQ 45.2, ARR/ARR 10.9, ARR/AHQ 5.7, ARQ/ARQ 4.9, AHQ/AHQ 0.7, ARR/AHR 0.3). Despite the geographically distant flocks of improved Valachian sheep investigated no difference in the occurrence of individual PrP genotypes was observed.
Subject(s)
Genetic Predisposition to Disease , Polymorphism, Genetic , Prions/classification , Prions/genetics , Scrapie/genetics , Sheep Diseases/genetics , Alleles , Animals , Base Sequence , Breeding , Female , Genotype , Male , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Sheep , SlovakiaABSTRACT
Several metal complex agents have already been introduced into clinical tumor therapy and others are subject of antitumor studies. In this study we focused on the tetraaza macrocyclic copper complex (Cu(TAAB)Cl(2)). We studied the influence of the substance on cell growth, cell cycle, membrane integrity, necrosis, apotosis and glutathione level on the leukemic cell line L1210 in 1-day (22 h) and 3-day (72 h) experiments. The metal complex shows a dose-dependent antiproliferative effect, without affecting cell cycle phases. The present results confirm that copper complex can damage plasmatic membranes and trigger apoptosis, and that after treatment of leukemic cells with the copper complex, glutathione levels were increased.
Subject(s)
Antineoplastic Agents/pharmacology , Copper , Leukemia L1210/drug therapy , Organometallic Compounds/pharmacology , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Division/drug effects , Cell Membrane/drug effects , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Flow Cytometry , Glutathione/metabolism , Leukemia L1210/metabolism , Leukemia L1210/pathology , Mice , Oxidative Stress , Tumor Cells, Cultured/drug effectsABSTRACT
Drug resistance is a prominent problem of cancer therapy. Differences in quantity and quality of many metabolites in normal and malignant cells and their changes after treatment by anticancer drugs can be detected by nuclear magnetic resonance (NMR) both in vivo and in vitro. The results of in vivo and in vitro 1H, 13C, 19F and 31P NMR spectroscopy and their correlation with the degree of resistance to anticancer drugs are discussed. Monitoring of treatment and development of drug resistance by this non-invasive method could be useful not only in cancer research related to drug resistance but also in clinical medical oncology.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Magnetic Resonance Spectroscopy , Amino Acids/analysis , Animals , Antimetabolites, Antineoplastic/pharmacology , Breast Neoplasms/chemistry , Breast Neoplasms/pathology , Carbon Isotopes , Creatinine/analysis , Drug Resistance, Multiple , Energy Metabolism , Female , Fluorine , Humans , Isotopes , Lactates/analysis , Lipids/analysis , Mice , Mice, Nude , Neoplasm Proteins/analysis , Phosphocreatine/analysis , Phosphorus Isotopes , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/drug effectsABSTRACT
The use of conformation isomers of pBR322 DNA in the study of interactions of Pt complexes with DNA provided for a good monitoring of induced changes in the structure of DNA by gel electrophoresis. On the basis of characteristic changes in the gel electrophoretic mobility of platinated isomers of pBR322 DNA we detected the presence of Pt-DNA adducts representing both intra- and interstrand bifunctional binding of Pt complexes to DNA. Also, this method made it possible to distinguish between DNA modifications induced by the therapeutically active cis-diamminedichloroplatinum (II) (cis-DDP) alone, and those induced by its therapeutically inactive trans-isomer (trans-DDP). The electrophoretically detected DNA modifications were more effective if the interaction of the Pt complex took place with heat-denatured DNA. This process, as compared to that performed with native DNA, ran 100 times faster.
Subject(s)
Cisplatin/pharmacology , DNA/drug effects , Nucleic Acid Conformation , DNA Replication/drug effects , Electrophoresis , Formaldehyde/pharmacology , Hot Temperature , Nucleic Acid Denaturation , Stereoisomerism , Transcription, GeneticABSTRACT
L1210 cells with in vitro induced drug resistance against cis-diamminedichloroplatinum(II) (cis-Pt(II] were inoculated in mice and several times transplanted. Then the effect of cis-Pt(II) on drug-sensitive and drug-resistant L1210 cells in mice was investigated. While the DNA and protein synthesis in drug-sensitive cells after in vivo cis-Pt(II) treatment was inhibited by 50%, that of drug-resistant cells remained virtually unaffected. The content of platinum in drug-sensitive cells was approximately three times higher in comparison with drug-resistant L1210 cells.
