Viability of Human Arterial Grafts Monitored by Comet Assay.

An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

Serum levels of matrix metalloproteinases 2 and 9 in patients with acute myocardial infarction.

The myocardial extracellular matrix plays an important role in maintaining the structural and functional integrity of the heart and is centrally involved in post-myocardial infarction repair processes. We analysed some genetic and proteomic aspects that could play an important role in the development of myocardial infarction. Matrix metalloproteinases are enzymes that contribute strongly to the degradation of extracellular matrix components. In this study the serological levels of MMP-2 and MMP-9 were investigated using immunological testing in 34 patients with myocardial infarction and 34 matched control subjects. The serum levels of MMPs were determined by ELISA. Changes in serum levels were characterized within 24 h and after 6 months post myocardial infarction. Significantly higher levels of MMP-2 (299.47 ± 117.61 ng/ml) and MMP-9 (93.56 ± 53.74 ng/ml) were determined in patients with myocardial infarction compared to the controls, in both cases P < 0.001. MMP-9 levels decreased significantly in the 6 months after cardiac event, whereas the levels of MMP-2 were almost equal to the post-infarction ones. While comparing the results from four patients that died of cardiovascular cause within 6 months we found significantly higher MMP-2 (435.00 ± 55.83 ng/ml, P = 0.003) and MMP-9 (166.25 ± 41.07 ng/ml, P = 0.018) values. Microarray analysis was used to determine the gene expression of selected genes for MMPs and their regulators from peripheral blood. The selected genes did not show satisfactory results that could have a potential implication for diagnostics of tissue degeneration.

DNA analysis of ancient skeletal remains.

Non-Governmental Organization Archaia (http://www.archaia.cz) carried out the rescue archaeological research at Knezeves near Prague in 1998. Most of dating objects in Knezeves come from the period of Late and Final Bronze Age. The approximately 3,000 years old set, which included 11 human remains from three settlement features, was collected for the study. First, gender was determined according to anthropological characteristics. Ancient DNA from bones was extracted by the phenol-chloroform procedure and N-phenacetylthiazolum bromide reagent. Polymerase chain reaction amplification of AMEL XY, part of amelogenin gene, with subsequent polyacrylamide gel electrophoresis and Short Tandem Repeats analysis followed. DNA profiles of skeletal remains were obtained by the fragmentation analysis of autosomal short tandem repeat markers. Genetic profiles showed us whether individuals from Knezeves were in mutual relationship (parent - descendant). The congruence of results in sex determination supported reliability of genetic methods, which are suitable for sex determination of fragmental and subadult skeletal remains.

[Genetic background of mitochondrial diabetes]. / Genetické priciny mitochondriálního diabetu.

Diabetes mellitus type 2 represents a heterogenous disease characterized by impaired glucose homeostasis. The disorder clusters in families suggesting genetic disposition, however the mechanism underlying is unknown. Many studies show more frequent maternal transmission of diabetes in the families. One of huge range of explanation is exclusively maternal transmission of mitochondria. Mitochondria are power organelles which produce ATP molecules by oxidation-reduction reactions via the respiratory chain. They contain their own genome which codes subunits of the respiratory chain and proteosynthetic apparatus for proteins encoded by this genome. Pathogenic mutations of mitochondrial DNA can affect the activity of the respiratory chain and result in various phenotypes. Mitochondrial diabetes is commonly associated with neuromuscular disorders and often presents with nonautoimmune beta cell failure. Although mitochondrial mutations are associated with diabetes, their low frequency does not explain reported more frequent maternal transmission of diabetes mellitus type 2.

Male dominance rank, mating and reproductive success in captive bonobos (Pan paniscus).

In the recent past, application of DNA genotyping techniques has enabled researchers to more accurately test relationships between dominance rank (DR), mating success (MS) and reproductive success (RS). Paternity studies often reveal that reproductive outcome does not always correlate with male DR and/or MS and thus open room for discussion and interpretation of alternative reproductive tactics of both sexes. In this study, we analysed male DR, MS and RS in a group of bonobos at Twycross Zoo (UK). Genetic relationships were determined using 8 tetrameric microsatellite loci. Despite clear and asymmetric dominance relationships, analysed using normalised David's scores based on a dyadic index of dominance among the group's 3 mature males, we found that the most dominant male did not sire the most offspring. In fact, both infants conceived during the observation period were found to be sired by the lower-ranking males. Although the alpha male had almost exclusive mating access to one of the females during the time she was showing a maximal anogenital swelling, her infant was sired by the lowest-ranking male who mostly mated with her when outside the maximal swelling period. This result suggests that either sperm competition operates and/or ovulation is decoupled from the phase of maximal anogenital swelling which could allow greater female choice.

[PCR-multiplex system for analysis of Y-chromosomal microsatellite polymorphisms DYS449, DYS456, DYS458, and DYS464]. / PCR-multiplex systém pro analýzu Y chromosomálních mikrosatelitních polymorfismu DYS449, DYS456, DYS458 a DYS464.

DNA profiling--inclusive sex determination-- with microsatellite markers is currently a commonly used genetic method of studying humans. An efficient technique of producing the genetic data is amplification of multiple microsatellites in a single PCR reaction. Here we introduce a novel PCR-multiplex system for analysis of four polymorphic Y-STRs. Specifically, these are DYS449, DYS456, DYS458, and DYS464. These loci were chosen because of their reported high diversity in Euroamerican population (10), as well as their absence in the commercial analytical kits at the time of beginning of this study. Our objective was to design this PCR-multiplex for use of fragmentation analysis by electrophoresing samples on a capillary semi-automated genetic analyzer applying only one fluorescent dye. The PCR system we propose, may be notably used in fields such as forensic and human population genetics.

[Determination of AB0 blood group system from degraded blood stains on serological and molecular genetic level]. / Urcování krevního systému AB0 z tepelne degradovaných krevních stop na sérologické a molekulárne genetické urovni.

The AB0 blood group system typing remains one of the basic laboratory tasks in a forensic practice. However, problems arise when the analysed samples are seriously degraded. We took blood samples from six volunteers (three men, three women) and made blood stains on pieces of sterile cotton cloth. Blood stains were incubated at three different temperatures (22 degrees C, 37 degrees C, 56 degrees C) for various periods of time (1 day, 1 week, 14 days, 1 month, 3 months, 6 months, 1 year). For blood stains degraded at 22 degrees C we also analysed the samples after 3.5 hours of incubation. Moreover, we tried to determine the AB0 blood group system after thermal degradation at high temperature, accurately at 200 degrees C for 10 min. For the AB0 blood group system typing a Polymerase Chain Reaction method was used to amplify glycosyltransferase gene, when DNA had been isolated from artificially created blood stains, followed by their subsequent artificial thermal degradation. For serological AB0 typing the mixed agglutination and the Therkelsen method were used. The DNA analysis seemed to solve problems with seriously degraded blood stains but we found out that classical serological methods were even better in some cases.

[B0 aberrant genotype of AB0 blood group system]. / Aberantní genotyp B0 v krevním systému AB0.

The AB0 blood group system typing remains one of the basic laboratory tasks in a forensic practice. However, problems arise when the analysed samples are seriously degraded. The DNA analysis promised to solve this but an unexpected complication was encountered. For the AB0 blood group system typing a Polymerase Chain Reaction method was used to amplify glycosyltransferase gene, when DNA had been isolated from artificially created blood stains, followed by their subsequent artificial thermal degradation. In the B0 genotype an aberrant genotype was suprisingly found and its structure was confirmed by sequencing. This meant that a newly formed B00 (not the original BO) genotype was present. Such a finding, to our best knowledge, had not been observed yet and we were unable to find any references in the professional literature. The explanation of this result thus remains unclear.

[Effect of Stopangin mouth spray on blood alcohol levels measured by the Alcotest 7410 analyser made by Drager]. / Ovlivnení hodnoty hladiny alkoholu merené analyzátorem Alcotest 7410 fy Dräger sprayem Stopangin.

During a check-up of a driver by a police patrol in the Czech Republic by means of a breath analyzer of Dräger Co. an alcohol blood level of 0.50 g/kg was assessed. Later the driver reported that before the test he used at 30- and 5-minute intervals the drug Stopangin spray. In the Institute of Forensic Medicine the authors made an experiment which revealed that 22 minutes after administration of the drug the apparatus gave a negative result. Agreement with these conclusions was expressed also by a representative of Dräger Co. and the results were published as an expert opinion with a recommended procedure for the police of the Czech Republic in the South Moravian region.

[Molecular genetic analysis of Alzheimer's dementia in the Czech population. The APP-717 mutation in the gene for amyloid protein precursor]. / Molekulárne genetická analýza Alzheimerovy demence v Ceské populaci. Mutace (APP-717) v genu pro amyloidní proteinový prekurzor (APP).

BACKGROUND: Although locus mutations in the gene for the amyloid precursor protein were already described in patients with Alzheimer's disease, there still are some patients where this mutation was not found and no link was found with other possible genetic loci on chromosomes 14 and 19. Therefore a group of subjects with Alzheimer's disease was subjected to tests for the presence of a mutation in the APP gene (in position 717). METHODS AND RESULTS: In a selected group of subjects with Alzheimer's disease (AD) in the gene for amyloid precursor protein in position 717 mutations of its transmembraneous region are found. The authors analyzed the genome DNA of cerebral tissue of Czech subjects for the presence of this mutation by means of the polymerase chain reaction with subsequent verification by sequencing analysis. In every subject genetic analyses from cerebral areas of the frontal lobe, temporal lobe, parietal lobe and hippocampus were performed. The methods used were the polymerase chain reaction (PCR) and sequencing. From the total number of 18 subjects with confirmed Alzheimer's disease and six non-related subjects without histopathological signs of Alzheimer's disease after the age of 90 years, three sequencing changes were found in position 717 of exon 17 of the transmembranous region of the precursor of beta-4 amyloid glycoprotein. In the first case it was substitution of thymin for adenine in codon 717, in the second case substitution of cytosine for thymine, in the third case a sporadic mutation of guanine for thymine in codon 717 was found. CONCLUSIONS: It was revealed that codon 717 could be a so-called hot spot site preferred for the preferential development of mutations in codon 717 in the gene for the amyloid precursor protein (APP).

[Mutation in the beta3-adrenergic receptor gene (Trp64Arg) does not influence insulin resistence, energy metabolism, fat distribution and lipid spectrum in young people. Pilot study]. / Mutace beta3-adrenergního receptorového genu (Trp64Arg) nemá vliv na inzulinovou rezistenci, energetický výdej, distribuci tuku a lipidové spektrum u mladistvých--pilotní studie.

UNLABELLED: A missence mutation Trp64Arg in the beta3-adrenergic receptor gene is associated with obesity, insulin resistance, a lower metabolic rate and the earlier onset of NIDDM but the published results are controversial. We investigated the effect of this mutation on insulin resistance (euglycemic hyperinsulinemic clamp), on fat mass and fat distribution (anthropometry, bioimpedance, CT) and resting metabolic rate (indirect calorimetry), lipid spectrum and other metabolic disturbances in Czech juveniles recruited from juvenile hypertensives (H, n = 68) and controls (C, n = 81). The frequency of this mutation (determined by digestion of 210 bp PCR product with MvaI) was double in H than in C (14.7%, vs. 7.4%) and the carriers of Arg64 allele had sig. higher fasting glucose (H: p = 0.002. C: p = 0.025). Four Trp64/Arg64 and six Trp64/Trp64 men (age 23 +/- 4.2, vs. 22.5 +/- 1.9 y, BMI 26 +/- 5.5, vs. 22.9 +/- 5.1 kg/m2) took part in a detailed pilot study. But no signif. differences (Horn's method) in fasting glucose (4.6 +/- 0.6, vs. 4.9 +/- 0.4 mmol/l), in parameters of insulin resistance (M-value150-180 min. 9.1 +/- 1.1, vs. 8.9 +/- 1.5 mg glucose/kg.min(-1)), resting metabolic rate/lean body mass (RMR/kg LBM: 78.6 +/- 4.6, vs. 85.6 +/- 23.2 kJ/kg), lipid spectrum and other screened parameters were found. The lowest resting metabolic rate (RMR/kg LBM 55.4; 62.6 kJ/kg) was found in brothers (both C, Trp64/Trp64) who highly differ in body constitution (BMI 19.0 resp. 32.4 kg/m2). We suppose that in this case the energy metabolism is probably determined by other genetic loci and does not correlate with body fat mass. CONCLUSION: Our pilot study does not confirm the influence of Trp64Arg mutation in heterozygous carriers on insulin resistance, energy metabolism and lipid spectrum.

[Ret proto-oncogene mutation found in the Czech population and its predictive value for offspring of patients with medullary carcinoma of the thyroid gland]. / Mutace nalezené v Ret protoonkogenu v ceské populaci a jejich prediktivní prínos pro potomky pacientu s medulárním karcinomem stítné zlázy.

The authors completed a total of 23 pedigrees with the clinical diagnosis of medullary thyroid carcinoma (5 MEN 2A pedigrees, 11 FMTC pedigrees and 7 MTC pedigrees). Using the method of polymerase chain reaction (PCR), it was possible to define the rate of the most frequent mutations in exons 10, 11 and 16 of Ret-protooncogene present in the Czech population. The most frequent hereditary mutation found in MEN 2A and FMTC groups is substitution of thymine for cytosine in position 2095 of the transmembranous domain of the Ret-tyrosine kinase gene. Another six types of known mutations were tested.

[Non-insulin-dependent diabetes mellitus--strategies and problems in genetic research]. / Non-inzulin dependentní diabetes mellitus--strategie a problémy genetického výzkumu.

Non-insulin dependent diabetes mellitus (NIDDM) is at present one of the most serious and widespread diseases with a mass incidence and its treatment is very costly. In the genesis and development of the disease genetic factors participate along with environmental factors. Genetic research of NIDDM is difficult, as ensues from the very essence of this heterogeneous disease. Research concentrates on the identification of specific genetic determinants which influence the predisposition of the individual to glucose intolerance which, if revealed, would make more effective prevention and treatment possible. Intensive research is focused on different candidate genes. Although some "minority" genes were identified, the primary diabetogen was not revealed so far and partial positive results must be confirmed on a larger population sample. The article reviews the contemporary state of genetic research of the disease.

[Restriction fragment length polymorphisms in the families of patients with Down's syndrome (trisomy 21)]. / Polymorfismy délek restrikcních fragmentu v rodinách pacientu s Downovým syndromem (trisomie 21).

Using the method of polymorphisms of lengths of restriction fragments (RFLP), the authors compare agreement and differences in the normal healthy Czech population and in families with a patient suffering from Down's syndrome. 2-alpha-satellite DNA probes were used which are weighed in the pericentromeric area of heterochromatin of the long arms of chromosomes 13 and 21. These probes contain a number of repetitive sequences, most frequently represented in human heterochromatin of the majority of chromosomes. By hybridization with an alpha-RI-6 probe multiallelic polymorphisms were obtained in families with Down's syndrome in five restrictive endonucleases (Bsp RI, Eco RI, Pst I, Taq I and Xba I). Restrictions with enzymes Bam HI and Hind III were non-polymorphous. Hybridization with the alpha-RI-IB probe revealed polymorphism with restrictive endonuclease Taq I. Enzymes Bam HI, Eco RI, Hind III, Pst I and Xba I (3) were non-polymorphous. The difference of the two probes in the centromeric area of chromosomes 13 and 21 was confirmed by hybridization in situ, using 3H-labelled thymidine triphosphate (TIP) in quantitative experiments on short-term cultures of lymphocytes of healthy subjects (4).

[DNA diagnosis in families with autosomal dominant hereditary polycystic kidney disease]. / Problémy DNA diagnostiky v rodinách s autosomálne dominantne dedicnou polycystickou chorobou ledvin.

In 1990-1992 110 patients with polycystic kidney disease adult type (ADPKD) were contacted. Forty-nine families were examined by Southern's RFLP method (standard probe 3'HVR and Pvu II restrictase). Of 424 members of these families only 25 (5.9%) refused the examination. So far 337 examinations were completed, 62 are under way. In 32 cases of 109 subjects at risk of 50% ADPKD the affection was proved with a probability higher than 95%, in 77 subjects (and 6 subjects with a 25% risk) were eliminated with an equal probability. In two families successful prenatal examinations of the foetus were accomplished. Evaluation of attitudes to DNA diagnostics revealed in our patients a greater willingness to attend postnatal examinations (up to 98%) and a smaller willingness to have prenatal examinations (21%), as compared with data published abroad.

[Linkage analysis in pedigrees with Alzheimer's dementia]. / Vyuzití vazebné analýzy v rodokmenech s Alzheimerovou demencí.

Linkage analysis with three specific DNA markers was carried out on the Alzheimer's dementia pedigrees. The DNA markers used in our study, D21S1/D21S11, D21S13 and D21S16 in the 21q11.2-21q21 regions are localized. The results from different brain DNA samples and from peripheral blood samples of AD families were evaluated by two-point linkage analysis. The linkage between DNA markers and one AD pedigree was observed (D21S1/S11 and D21S13).

[An alpha-satellite DNA sequence, alpha-RI-6, specific for human chromosomes 13 and 21, detected using the RFLP technic with digoxigenin labelled probes]. / Alfa-satelitní DNA-sekvence, alfa-RI-6, specificka pro lidské chromosomy 13 a 21, detekovaná pomocí RFLP-techniky s digoxigeninovým znacením sondy.

The authors compared two at present most widely used techniques for labelling DNA probes: a) radioactive labelling by means of the radioisotope 32P; non-radioactive labelling using the hapten digoxigenin for the visualization of the hybridization process on nylon membranes. Then sensitivity of the technique of non-radioactive labelling of heterochromatin probes was equivalent to the radioactive method.

[In situ hybridization of chorionic villi cells]. / Hybridizace in situ na bunkách choriových klku.

Two cloned alpha fragments of satellite repetitive DNA sequences were used to modify the method of DNA hybridization in situ (HIS) for tissue cells of human chorionic villi. Techniques for the cells of short-term lymphocytic cultures were used to optimize the conditions of HIS in chorionic villi cells with regard to a more specific localization of cloned repetitive DNA sequences in the target homologous chromosomes. A total of 262 specimens were prepared using the HIS method. As the results show, both cloned fragments are well applicable for routine cytogenic practice, the fragment p YAM 10-40 for the number of chromosomes, and fragment p YAM 9-72 for chromosomes 18.