Cytotoxic and apoptotic effects of ethanolic propolis extract on C6 glioma cells.

Propolis is a natural resinous substance obtained from beehives, and emerging evidence supports that it has antitumor, antiinflammatory, antioxidant, and antimicrobial activities. The aim of the study is to examine the cytotoxic, antioxidant, and apoptotic features of ethanolic propolis extract (PE) on C6 glioma cells. The cells were treated with ethanolic PE at various concentrations for 24 hours, after which the total antioxidant status (TAS) and total oxidant status; malondialdehyde, protein carbonyl, 8-hydroxy-2'-deoxyguanosine, and glutathione (GSH) levels; Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity; and apoptotic markers were measured. Ethanolic PE at 100, 250, and 500 µg/mL concentrations showed optimal activity on C6 glioma cells. TAS and GSH levels were significantly increased in C6 glioma cells treated with 100 and 500 µg/mL PE compared to control cells (P < .05). Similarly, the activity of Cu/Zn-SOD was higher in C6 glioma cells treated with 250 or 500 µg/mL ethanolic PE compared to control cells (P < .05), as was the caspase-3 mRNA expression level. The highest levels of caspase-8 and -9 expression were in C6 glioma cells treated with 500 µg/mL PE. Collectively, our results indicate that ethanolic PE has cytotoxic and apoptotic effects on C6 glioma cells. Furthermore, it may provide a protective role in the antioxidant defense system. PE shows potential for development as a natural antioxidant and apoptotic agent for the treatment of brain tumors.

Kruppel-Like Transcription Factor-4 Gene Expression and DNA Methylation Status in Type 2 Diabetes and Diabetic Nephropathy Patients.

BACKGROUND/AIM: Diabetic nephropathy (DN) is one of the most serious microvascular complications in diabetic patients. The kruppel-like transcription factor-4 (KLF-4) affects the expression of genes involved in the pathogenesis of DN. The present study aims to identify the KLF-4 expression and DNA methylation (DNAMe) status in patients with type-2 diabetes (T2D) and DN and to reveal the contribution of the KLF-4 to the development of DN. MATERIAL AND METHODS: The cohort study was performed with blood samples from 120 individuals; T2D group (n = 40), DN group (n = 40) and control group (n = 40). The expression level of the KLF-4 gene was analyzed using the real-time polymerase chain reaction (qRT-PCR) and the methylation profile detected using the methylation-specific PCR (MS-PCR) technique. RESULTS: According to our findings, KLF-4 mRNA expression in the T2D group was 1.60 fold lower than in the control group (p = 0.001). In the DN group, the expression of KLF-4 mRNA was 2.92-fold less than that of the T2D group (p = 0.001). There was no significant alteration in the DNAMe status among the groups. CONCLUSION: Our findings showed that regardless of the DNAMe status, KLF-4 gene expression may play a role in the development of T2D and DN. This suggests that the KLF-4 gene may be the target gene in understanding the mechanism of nephropathy, which is the most important complication of diabetes, and planning nephropathy-related treatments, but the data should be supported with more studies.

Analysis of genetic polymorphisms associated with intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a common degenerative spinal condition. Recent studies have shown that the incidence of disc herniation and disc degeneration may be explained by genetic factors.  In this study, we investigated the link between various polymorphic variants of the vitamin D receptor (VDR), matrix metalloproteinase 2 (MMP2), and insulin like growth factor 1 receptor (IGF1R) genes and IVDD in patients with IVDD, in Turkey. We examined and genotyped 199 patients with IVDD and 197 healthy individuals. Genomic DNA was isolated from the peripheral blood leukocytes of all participants, and analyzed using real-time PCR. Via melting curve analysis, VDR, MMP2, and IGF1R polymorphism variant distributions were determined. The patients with IVDD showed higher frequencies of the VDR ApaI A allele genotype as compared to the control group; however, there were no significant differences in the frequencies or allelic distributions of the IGF1R and MMP2 genotypes between the IVDD patients and the control group. The incidence of IVDD in these Turkish patients is correlated with the VDR ApaI gene polymorphism, but not with the IGF1R and MMP2 polymorphisms.

Micronuclei frequencies in lymphocytes and cervical cells of women with polycystic ovarian syndrome.

OBJECTIVE: The aim of this study was to determine micronucleus (MN) frequencies in exfoliated cervical cells and peripheral blood lymphocytes of women with polycystic ovarian syndrome (PCOS). MATERIALS AND METHODS: Fifteen patients with PCOS and 11 healthy control patients were included in the study. Cervical smears and peripheral blood were collected from all patients. Specimens were analyzed for MN frequencies and compared between the groups. In addition to MN, other nuclear anomalies connected with both genotoxicity and cytotoxicity were evaluated. RESULTS: The MN frequencies in cervical smear and peripheral blood lymphocytes were compared in patients with PCOS and normal controls. There was no statistically significant difference between the groups regarding micronucleus frequency in peripheral blood lymphocytes (p=0.239). The mean MN scores in exfoliated cervical cells of patients with PCOS and normal controls were 1.19±0.57 and 0.74±0.34, respectively. The difference regarding micronucleus frequencies in cervical cells was statistically significant between the groups (p=0.032). CONCLUSION: Although study group is small, our study results support that there is an increased micronucleus frequency in cervical exfoliated cells of PCOS patients; this is a determinant of genetic hazard in the disease.

Recurrent proximal 18p monosomy and 18q trisomy in a family due to a pericentric inversion.

Here, we report on a family with pericentric inversion of chromosome 18 [inv(18)(p11.2q21)] and two recombinants with a duplication of q21 → qter and a deletion of p11.2 → pter regions in a four-generation family. This chromosomal abnormality was inherited in our first patient from the father, while it was transmitted to the second patient from the mother. Array-CGH analysis were used to better characterize duplicated and deleted chromosomal regions and showed no genomic copy number variation (CNV) differences between these two relatives. We discussed genotype-phenotype correlations including previously reported.

Evaluation of smoking genotoxicity in Turkish young adults.

BACKGROUND: For the past few decades, it has been widely known in developed countries that tobacco is dangerous, but it is still insufficiently realized how big these dangers really are. AIMS: To determine and evaluate micronuclei (MN) frequencies of young smokers and nonsmokers in three different tissues (peripheric blood lymphoctes, buccal mucosa, and exfoliative urothelial cells) at the same time. MATERIALS AND METHODS: MN assay was performed on buccal mucosa, urothelial cells, and peripheric blood lymphocyte samples obtained from 15 healthy male smokers (>5 pack-years) and 15 healthy male nonsmoker controls who had not been exposed to any known genotoxic agent. STATISTICAL ANALYSIS USED: The statistical differences between smoker and nonsmoker groups were calculated by using student t test. The differences between smoker-group tissues were compared by ANOVA. RESULTS: It was found that MN frequency (mean value ± standard deviation) in oral mucosa cells from smokers and controls were 1.20 ± 0.22% and 0.26 ± 0.10%; in urothelial exfoliative cells, 1.29 ± 0.28% and 0.12 ± 0.08%; in peripheric blood lymphocytes, 1.53 ± 0.23% and 0.38 ± 0.12%, respectively. The mean MN frequencies in buccal mucosa, urothelial exfoliative cells, and peripheric blood lymphocytes were significantly higher in smokers than in those of controls (P<0.05). All tissues were affected from smoking, but the most destructive effect was seen in urothelial cells of smokers (P<0.05). CONCLUSIONS: Our data showed that cigarette smoke is a DNA damage causitive agent on exfoliative buccal mucosa and urothelial cells and peripheric blood lymphocytes of young smokers, but it has most destructive effect on urothelial cells.

A new approach to chromosomal abnormalities in sperm from patients with oligoasthenoteratozoospermia: detection of double aneuploidy in addition to single aneuploidy and diploidy by five-color fluorescence in situ hybridization using one probe set.

OBJECTIVE: To determine the frequencies of disomy, nullisomy, total aneuploidy, and diploidy in the sperms of infertile men. DESIGN: A controlled prospective study. SETTING: Assisted reproductive technology (ART)/IVF Unit and Department of Medical Biology and Genetics, Meram Medical Faculty, Konya, Turkey. PATIENT(S): Infertile men with oligoasthenoteratozoospermia (OAT) and normal fertile donors. INTERVENTION(S): After slide preparation from semen samples, sperm nuclei were analyzed for chromosomes 13, 18, 21, X, and Y by five-color fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): The sperm aneuploidy (disomy and nullisomy) and diploidy rates were determined according to the number of signals detected for each probe in infertile and fertile men. RESULTS: Patients with OAT had a significantly higher incidence of disomy (except chromosome 18 and XX disomy), nullisomy (except chromosome 18), and diploidy than normal fertile controls. In addition to double disomy, double nullisomy and disomy+nullisomy were observed in patients with OAT, but none of these were seen in controls. CONCLUSION(S): In this study patients with OAT had an increased rate of sperm aneuploidy and diploidy. This finding suggest that patients with OAT may be at an increased risk of producing aneuploid and triploid offsprings. For this reason, it may be very important to perform the sperm fluorescence in situ hybridization in patients with OAT. Thus, a more informative genetic counseling might be given to couples with male factor infertility who are at an increased risk of having aneuploid offsprings and triploid conceptions before intracytoplasmic sperm injection (ICSI).

Evaluation of the results of cordocentesis.

OBJECTIVE: To evaluate the results of cordocentesis carried out in our clinic at Meram Medicine Faculty of Selcuk University in Konya, Turkey. MATERIALS AND METHODS: Cytogenetic results and complication data were obtained by cordocentesis from 250 pregnancies performed in our clinic. RESULTS: Adequate amount of cord blood was taken 98% of the time, the successful culture rate was 92.8%, and none of the 18 cases in which no proliferation was detected in the culture accepted a new intervention. Cordocentesis was performed in 14 cases (5.6%), because no results were obtained from amniocentesis carried out for various indications. According to cytogenetic evaluation, chromosomal abnormality was detected in 12 cases (5.17%), including four cases of trisomy 21, four cases of trisomy 18, one case of trisomy 13, one case of triploidy (69,XXX) and two cases of chromosomal inversion. Of the 250 cordocentesis cases, there were 12 (4.8%) cases of fetal loss, including four cases of rupture of membranes, four cases of abdominal pain and vaginal bleeding and four cases of a spontaneous abortus. In 53 (21.2%) cases, cordocentesis was performed because of hydrops fetalis; and of the total 12 losses, six were in this group. The fetal loss rate was 11.32% in the hydrops fetalis group. CONCLUSION: If cordocentesis is carried out by highly skilled physicians and optimal culture conditions are available, cordocentesis is an invasive prenatal diagnostic and therapeutic procedure that is performed secondary to amniocentesis with high accuracy and safety. In cases of hydrops fetalis in which cordocentesis is carried out, fetal loss is more likely to occur.

Extremely skewed X-chromosome inactivation patterns in women with recurrent spontaneous abortion.

BACKGROUND: The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of recurrent spontaneous abortion (RSA) but the results obtained were conflicting. AIMS: We therefore investigated the XCI patterns in peripheral blood DNA obtained from 80 patients who had RSA and 160 age-matched controls. METHODS: Pregnancy history, age, karyotype, and disease information was collected from all subjects. The methylation status of a highly polymorphic cytosine-adenine-guanine repeat in the androgen-receptor (AR) gene was determined by use of methylation-sensitive restriction enzyme HpaII and polymerase chain reaction. RESULTS: Skewed XCI (> 85% skewing) was observed in 13 of the 62 patients informative for the AR polymorphism (20.9%), and eight of the 124 informative controls (6.4%) (P = 0.0069; chi2 test). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 11 (17.7%) patients, and in only two controls (P = 0.0002; chi2 test). CONCLUSIONS: These results support the interpretation that disturbances in XCI mosaicism may be involved in the pathogenesis of RSA.

A case with ICF syndrome lost to rubella pneumonitis.

The immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder characterized by variable immunodeficiency, instability of the pericentromeric heterochromatin, and facial dysmorphism. Here we report a new case of ICF syndrome who died of rubella pneumonitis. A six year-old-girl who was the first child of consanguineous parents was admitted to the hospital because of bronchopneumonia. Laboratory investigations revealed pan-hypogammaglobulinemia, lymphoperria, normal proportions of peripheral blood lymphocytes with an inverted CD4/CD8 ratio, and interstitial pneumonia with a positive serology of acute rubella infection. The ICF syndrome was diagnosed by centromeric instability in the standard cytogenetic analysis. An inclusion body was demonstrated in the lung biopsy after the death of the patient. Chromosomal investigation could be helpful along with other tests for diagnosis of variable immunodeficiency accompanied by facial dysmorphism.