Effect of Annona muricata aqueous leaf extract on reactive oxygen and nitrogen species.

OBJECTIVE: Annona muricata extracts are used in traditional medicine due to their significant biological effects. Verification and elucidation of their mechanisms is beneficial in terms of the usefulness of these extracts in everyday life or in the context of disease treatment or prevention. MATERIALS AND METHODS: The effectiveness of the extract was assessed from dried A. muricata leaves available for direct consumption. It is targeted against reactive oxygen and nitrogen species such as superoxide (O2•-), hydroxyl (•OH), nitric oxide (NO) radicals, and peroxynitrite anion (ONO2-) at concentrations of 5, 10, 25, 50, 100 µg.ml-1. RESULTS: No significant inhibitory activity was measured against O2·- at the assessed concentrations of the extract. Conversely, substantial antioxidant properties were found towards ·OH. Moreover, very efficient uptake was recorded at low concentrations of the extract, 5 µg.ml-1 (53.91%) and 10 µg.ml-1 (45.3%). The antioxidant effect decreased with increasing concentration. By indirect determination of NO oxidation derivatives it was found that, as the extract concentration increased, the nitrite concentration decreased. In contrast, even at low concentrations, the extract causes an increase in the peroxynitrite concentration. CONCLUSIONS: The results themselves show that the effects of A. muricata leaf extract are mainly mediated by the activity against •OH, as well as the consequences of increased ONO2- formation.

Preliminary results of ethnic divergence of G1181C (rs2073618) and C290T (rs9525641) OPG gene polymorphisms in groups of postmenopausal Slovak women.

OBJECTIVE: In this study, we focused on observation of the genetic polymorphisms of the OPG genes G1181C (rs2073618) and C290T (rs9525641), their interactions with biochemical markers and anthropometric parameters in groups of postmenopausal Slovak women (Roma and non-Roma, n = 311). PATIENTS AND METHODS: Genomic DNA was extracted and purified from peripheral blood leukocytes by the kit Ultraclean® Blood non-spin® (Carlsbad, CA, USA) using a standard protocol. Genotyping was performed by the TaqMan SNP genotyping assay. Biochemical markers were measured by the Cobas e411 (Roche Diagnostic, Tokyo, Japan) and Cobas Integra400 plus (Roche Diagnostic, Rotkreuz, Switzerland) analysers. RESULTS: We recorded a higher frequency of the T allele in the C290T polymorphism of the non-Roma control group (53.846%), in Roma groups: control (T - 56.618%) osteoporotic (T - 51.471%). In the G1181C polymorphism, the CC genotype occurred more in the osteoporotic group (34.286%) compared to the control group (27.885%). In the group of postmenopausal Roma women, a statistically significant difference (p<0.05) was found between osteoporotic and control in the biochemical parameters' osteocalcin, C-terminal telopeptide I, and age. Statistically significant differences (p<0.0001) were also found in bone mineral density and T-score. The high odds ratio suggests the association of G1181C with osteoporosis. A close relationship was found between haplotypes, BMD, T-score, and IL-6 in control; and BMI, WHR, T-score, and osteocalcin in osteoporotic groups of Roma and non-Roma women. CONCLUSIONS: The results point to differences in the occurrence of genotypes and associations of haplotypes with the manifestation of osteoporosis in Roma and non-Roma women. However, a larger number of samples is needed to determine whether or not there are differences between the Roma and non-Roma populations.

WNT10A variants in relation to nonsyndromic hypodontia in eastern Slovak population.

Nonsyndromic hypodontia is a congenital absence of less than six permanent teeth, with a most common subtype maxillary lateral incisor agenesis (MLIA). Mutations in several genes have been described in severe tooth agenesis. The aim of this study was to search for the variants in wingless-type MMTV-integration site family member (WNT10A), paired box 9 (PAX9) and axis inhibitor 2 (AXIN2) genes, and investigate their potential role in the pathogenesis of non-syndromic hypodontia. Clinical examination and panoramic radiograph were performed in the cohort of 60 unrelated Slovak patients of Caucasian origin with nonsyndromic hypodontia including 37 MLIA cases and 48 healthy controls. Genomic DNA was isolated from buccal swabs and Sanger sequencing of WNT10A, PAX9 and AXIN2 was performed. Altogether, we identified 23 single-nucleotide variants, of which five were novel. We have found three rare nonsynonymous variants in WNT10A (p.Gly165Arg; p.Gly213Ser and p.Phe228Ile) in eight (13.33%) of 60 patients. Analysis showed potentially damaged WNT10A variant p.Phe228Ile predominantly occurred only in MLIA patients, and with a dominant form of tooth agenesis (odds ratio (ORdom) = 9.841; P = 0.045; 95% confidence interval (CI) 0.492-196.701;ORrec = 0.773; P = 1.000; 95% CI 0.015-39.877). In addition, the WNT10A variant p.Phe228Ile showed a trend associated with familial nonsyndromic hypodontia (P = 0.024; OR= 1.20; 95% CI 0.97-1.48). After Bonferroni correction, these effects remained with borderline tendencies. Using a 3D WNT10A protein model, we demonstrated that the variant Phe228Ile changes the proteinsecondary structure. In PAX9 and AXIN2, common variants were detected. Our findings suggest that the identified WNT10A variant p.Phe228Ile could represent risk for the inherited nonsyndromic hypodontia underlying MLIA. However, further study in different populations is required.

Historical sketch of Slovak Haban (Hutterite) population based on autosomal STR analysis.

According to the Hutterite chronicles, the Habans arrived from Austrian Tyrol, Switzerland, and northernmost Italy and stayed in four regions of Slovakia (Sobotiste, Vel'ké Leváre, Moravský Svätý Ján, Trencín). There are some communities in western Slovakia that retained their Haban cultural identity and still identify themselves as descendents of the Hutterite population with their own specific customs. Slovak Habans are typical founder population with significant social isolation for which high degree of inbreeding is typical. Present study investigated STR polymorphisms as a powerful genetic tool for population genetic studies. The aim was to perform a comparative, population genetic study based on 15 STR loci widely used in forensic genetics, of the Haban population, the Slovak majority population and the population of Tyrol. We analyzed allele frequencies and other statistical parameters in three selected populations in order to identify groups of specific ethnic origin and establish their genetic relationship. The data set included 110 unrelated Habans and 201 unrelated individuals from the Slovak majority population, as well as allelic frequencies for the population of Austrian Tyrol available in the literature. Population pairwise FST values used as a short term genetic distance between populations showed significant differentiation between the Habans and both reference populations (FST=0.0025 and 0.0042 for comparison with the Slovaks and Austrians, respectively; p<10(-3)). The Slovak Hutterites were demonstrated to be genetically distinct and more closely related to their geographic neighbors than to their historical ancestral population, which may be at least partially explained by gene flow between neighboring Haban and Slovak populations.

DNA analysis of Y chromosomal AZF region in Slovak population with fertility disorders.

BACKGROUND: The Y chromosome is characterized by a low number of functional genes, relatively high number of repetitive sequences and the ability of recombination purely by short arms of telomeres PAR1 and PAR2. The long arm contains an AZF region with genes participating in spermatogenesis. Microdeletions of three subregions, namely AZFa,b,c and their mutual combinations are responsible for male infertility and the resulting azoospermia and oligospermia. OBJECTIVES: The aim of this study based on evaluating 822 patients during a period of ten years was to analyse types of microdeletions in men with fertility disorders in Slovakia. METHODS: For detecting the microdeletions in Y-chromosomal AZF region and for identifying the Y-specific sequences we used PCR while using three different sets of sY sequences. REPORTS: We reported 38 cases of deletions in AZF region, namely 18 cases when using the first set of sequences, 12 cases when using the second set, and finally 8 cases when using the third set. When using the last set of sequences according to the European Academy of Andrology and European Molecular Genetics Quality Network, we detected deletions only in patients with azoospermia. In addition to deletions in each of AZF a,b,c subregions we recorded also a complete deletion of the whole AZF region. In the AZFa subregion, we recorded a deletion of sequence sY86. CONCLUSION: The study has confirmed that the detection of microdeletions of AZF region is significant from the diagnostic and prognostic views (Tab. 5, Ref. 21). Full Text in free PDF www.bmj.sk.

[DNA analysis on Y chromosomal AZF region deletions in Slovak population]. / DNA analýza AZF oblasti Y chromozómu u Slovákov s poruchou plodnosti.

OBJECTIVE: Study on Y chromosomal AZF region deletions in Slovak population, application of DNA technique. DESIGN: Genetic-prospective study. SETTING: Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava. METHODS: For detecting microdeletions in the Y-chromosomal AZF region in men with fertility disorders and for identifying Y-specific sequences we used the method of polymerase chain reaction (PCR) with using three different sets of sY sequences. For a verification of the specific type of deletion we used also fluorescently labeled kit. RESULTS: Diagnoses of referred patients were divided into 2 groups: azoospermia, oligospermia. In the followed-up group of 822 patients there were 349 patients with azoospermia, 473 patients with oligospermia. Globally we reported 38 cases of deletions in the AZF region of the Y chromosome, i.e. 4.62%. 24 patients with deletion are from the group of patients with azoospermia, i.e. 6.88%, 14 patients are from the group of patients with oligospermia (2.95%). Considering particular types of deletions we recorded deletions in each region, AZFa, AZFb and AZFc, combinated AZFbc deletion, but also a complete deletion of the whole AZF region. CONCLUSION: The study confirmed that detection of microdeletions of the AZF region is significant from diagnostic and prognostic view and it pointed out the importance of selection criteria for selecting patients.

Genetic variation analysis of 15 autosomal STR loci in Eastern Slovak Caucasian and Romany (Gypsy) population.

The genotype polymorphism studies were carried out on two different populations: Eastern Slovak Caucasian (138) and Romany (Gypsy) (138), both from the town of Presov, at 15 highly polymorphic short tandem repeats (STRs) loci. The selected kit PowerPlex 16 system (Promega) included amelogenin, two penta-nucleotide repeats and 13 tetra-nucleotide repeats. The comparison of the allele frequencies between Eastern Slovak Caucasians and Romanies has shown significant differences in the majority of the focused loci. The P-values of exact test for Hardy-Weinberg equilibrium probabilities, observed and expected heterozygosity, matching probability, power of discrimination and exclusion, polymorphic information content, typical paternity index, genetic diversity and the other population-genetic indices were calculated.

Detection of Philadelphia chromosome in patients with chronic myeloid leukemia from the Presov region in Slovakia (1995-2004).

Philadelphia chromosome (Ph) is a characteristic chromosomal marker that is associated with chronic myelogenous leukemia (CML). Philadelphia chromosome in bone marrow cells in patients with suspected diagnosis of CML in the Presov region (1995-2004) was detected in 94.4 % of cases. In one patient a complex translocation involving the chromosomes 8, 9 and 22 was identified. One patient has showed extra numerical and structural chromosomal aberrations. The mosaic karyotype of Ph chromosome was found in 5.9 % of cases. The conventional cytogenetic analysis remains the standard method for the purpose of diagnosis and monitoring of the therapeutic response and minimal residual disease in patients with chronic myeloid leukemia (Tab. 1, Fig. 1, Ref. 18). Full Text (Free, PDF) www.bmj.sk.

Prenatal cytogenetic analysis in the Presov region (Slovakia) in 1999-2004.

Prenatal genetic diagnostics is a part of prenatal care. Prenatal karyotyping is used to identify major genetic and congenital abnormalities in a developing fetus. In the Presov region (Slovakia) in 1999-2004 370 amniotic fluid samples were analysed by G-banding. Abnormal karyotypes were detected in 3.8% of samples. A karyotype using classical banding methods is the only fully informative method able to detect all chromosomal abnormalities. Identification of fetal abnormal chromosomes in high risk pregnancies allows proper pediatric and obstetric managment of the cases as well as genetic counselling (Tab. 1, Fig. 2, Ref. 5).