Transcriptomic expression levels of the VHL, TIMP-3, and RASSF1A genes in renal tumors.

OBJECTIVE: In this study, we aimed to investigate the relation between the mRNA expression levels of VHL, TIMP-3 and RASSF1A genes, and the histopathological and clinical characteristics of patients with renal tumors. PATIENTS AND METHODS: Radical nephrectomy specimens of cases presented without neoadjuvant treatment were confirmed to be cancerous, non-cancerous, benign, and healthy after removal from separate localizations. A total of 69 patients with kidney tumors (138 tissue samples) were included in the study group. RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative real time PCR (qPCR) were performed, and the GAPDH gene was used to normalize mRNA levels. RESULTS: In the RCC cancerous tissue, TIMP-3 levels increased 1.3 times and RASSF1A levels increased 1.4 times compared to the corresponding levels in non-cancerous tissues, and there was no statistically significant difference in these values. On the other hand, VHL gene expression levels in cancerous tissue were 2.8 times higher than in matched adjacent non-cancerous tissues (p < 0.05). In the case of oncocytomas, TIMP-3 levels were found to be 3.2 times higher, RASSF1A levels 3.8 times higher, and VHL levels 2.2 times lower than the corresponding levels in healthy tissues (p < 0.05). CONCLUSIONS: The roles of VHL, TIMP-3, and RASSF1A mRNA expression in contributing to the development of renal tumors could not be clearly established. Further studies are therefore required to elucidate the mechanisms underlying renal tumors.

Treatment with cucurbitacin B alone and in combination with gefitinib induces cell cycle inhibition and apoptosis via EGFR and JAK/STAT pathway in human colorectal cancer cell lines.

The epidermal growth factor receptor (EGFR) associated with signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), plays an important role in colorectal cancers (CRCs). Gefitinib (Gef) is an orally active inhibitor targeting the adenosine tri phosphate-binding domain of EGFR, and cucurbitacin B (CuB) is a selective inhibitor of JAK/STAT signaling with potent antitumor activity via suppression of STAT3 phosphorylation, but the underlying mechanism is not clear. We aimed to investigate the apoptotic and antiproliferative effects of CuB as a single agent and in combination with Gef on both HT-29 and HCT-116 cell lines. Cell proliferation, cell cycle distribution, and apoptosis were evaluated using viability assay, fluorescent microscopy, cytotoxicity assay, proliferation, DNA fragmentation, and cleaved caspase 3 levels. Real-time polymerase chain reaction and Western blot analyses were performed to determine the expression of relevant genes and proteins including antiapoptotic, proapoptotic, and cell cycle regulation. EGFR, phosphorylated EGFR (pEGFR), STAT3, and pSTAT3 proteins were evalutaed with Western blot analysis. Our results showed that, compared to CuB alone, CuB plus Gef treatment caused a significant growth and cell cycle inhibition and induced apoptosis in both cell lines. Also CuB plus Gef treatment decreased DNA synthesis rate more effectively than CuB alone. Treatment with CuB alone and in combination with Gef decreased the expression levels of B-Cell CLL/Lymphoma 2 (Bcl-2), BCL2-like 1 (BCL2L1), cyclin D1, pSTAT3, and pEGFR and increased the expression levels of Bcl-2-like protein 4, Bcl-2 homologous antagonist/killer, Bcl-2-associated death promoter, Bcl-2-like protein 11, and p27kip1 levels. Our results suggest that treatment with CuB alone and more likely in combination with Gef may be a considerable alternative therapeutic approach for CRC, at least in vitro.

The DNA methyl transferase inhibitor, 5'-aza-2-deoxycitidine, enhances the apoptotic effect of Mevastatin in human leukemia HL-60 cells.

Statins induce antiproliferative effects and apoptotic response in various cancer cell types. Moreover, they also sensitize tumor cell lines from different origins to many agents. We aimed to investigate possible effects of Mevastatin (Mev) alone and sequential treatment of 5'-aza-2-deoxycitidine (DAC) and Mev on HL-60 cell line using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay, lactate dehydrogenase release assay, flourescence microscopy, DNA fragmentation analysis, determination of DNA synthesis rate, and active caspase-3 assay. Messenger RNA (mRNA) expression of apoptotic and antiapoptotic genes were also evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for BAX, BCL2, and XIAP genes and quantitative Real-time PCR for CASP3, CASP8, and CASP9 genes. We showed that treatment with Mev alone and DAC followed by Mev resulted in apoptotic response in a time- and dose-dependent manner. We also found that pretreatment with DAC sensitized HL-60 cells to Mev and caused more apoptotic cell death than Mev-alone treatment via caspase-3 activation and DNA fragmentation. Moreover, sequential addition of Mev after DAC diminished DNA synthesis rate more effectively than Mev-alone treatment. Furthermore, DAC pretreatment significantly increased CASP3 and CASP9 mRNA expression even with lower doses of Mev. BAX, BCL2, and XIAP gene mRNA levels were also found to be changed in the presence of DAC and Mev. Determination of the exact molecular effects of statins and DAC would allow us to identify new molecular targets to develop more effective treatment regimens for cancer.

Auricular cartilage repair using cryogel scaffolds loaded with BMP-7-expressing primary chondrocytes.

The loss of cartilage tissue due to trauma, tumour surgery or congenital defects, such as microtia and anotia, is one of the major concerns in head and neck surgery. Recently tissue-engineering approaches, including gene delivery, have been proposed for the regeneration of cartilage tissue. In this study, primary chondrocytes were genetically modified with plasmid-encoding bone morphogenetic protein-7 (BMP-7) via the commercially available non-viral Turbofect vector, with the aim of bringing ex vivo transfected chondrocytes to resynthesize BMP-7 in vitro as they would in vivo. Genetically modified cells were implanted into gelatin-oxidized dextran scaffolds and cartilage tissue formation was investigated in 15 × 15 mm auricular cartilage defects in vivo in 48 New Zealand (NZ) white rabbits for 4 months. The results were evaluated via histology and early gene expression. Early gene expression results indicated a strong effect of exogenous BMP-7 on matrix synthesis and chondrocyte growth. In addition, histological analysis results exhibited significantly better cartilage healing with BMP-7-modified (transfected) cells than in the non-modified (non-transfected) group and as well as the control.

The effects of resveratrol on cyclooxygenase-1 and -2, nuclear factor kappa beta, matrix metalloproteinase-9, and sirtuin 1 mRNA expression in hearts of streptozotocin-induced diabetic rats.

Resveratrol (RSV) has a beneficial role in the prevention of diabetes and alleviates some diabetic complications, such as cardiomyopathy. We investigated cyclooxygenase-1 (COX-1), COX-2, nuclear factor κB (NF-κB), matrix metalloproteinase-9 (MMP-9), and sirtuin 1 (SIRT1) mRNA expression levels in heart tissue after RSV treatment in streptozotocin (STZ)-induced diabetic rats. After induction of chronic diabetes with STZ, 10 mg RSV/kg per day was administered to DM and DM+RSV groups for four weeks. At the end of the experiment, all rats were sacrificed and heart tissues were stored at -80°C; mRNA expression levels of COX-1, COX-2, NF-κB, MMP-9, and SIRT1 genes were analyzed with quantitative real-time PCR. We did not find any significant effect of RSV on MMP-9, COX-1, COX-2, or NF-κB mRNA levels among the groups. However, SIRT1 mRNA levels decreased in the DM group compared to controls and increased in the DM+RSV group when compared to the DM group. SIRT1 is activated by RSV treatment in diabetic heart tissue. Activation of SIRT1 by RSV may lead to a new therapeutic approach for diabetic heart tissue. We conclude that RSV treatment can alleviate heart dysfunction by inhibiton of inflammatory gene expression such as SIRT1.

Cytogenetic results of amniocentesis materials: incidence of abnormal karyotypes in the Turkish collaborative study.

The experience on prenatal chromosome diagnosis of four Turkish centers participating in a collaborative study on 6041 genetic amniocentesis performed during a 4-8 years period were reviewed. 5887 (97.5%) patients had strong clinical indications for prenatal chromosome studies and 154 (2.5%) were referred because of maternal anxiety and a bad history of previous gestations. The main indication groups were: advanced maternal age (3197 cases), positive serum screening (2011 cases), ultrasound-identified anomaly (492 cases), previous fetus/child with chromosomal aberrations (103 cases), a history of a previous abnormal and/or mentally handicapped child (70 cases) and a parental chromosome rearrangement (14 cases). The average maternal age was 33.9 years and average gestational age was 18 weeks. A total of 179 affected fetuses were detected in this collaborative study (3%) of which 133 were unbalanced (74.3%). Among the 124 (69%) numerical aberrations, 102 (82.3%) were autosomal aneuploidies, 20 (16.1%) were gonosomal aneuploidies and 2 (1.6%) were poliploidies. Among the 55 (31%) structural aberrations, balanced translocation was the most common (63.6%) and 11 cases of inversion, four cases of unbalanced translocation, two cases of marker chromosome and three cases of other abnormalities were found. The overall culture success rate was 99.7%. Pregnancy termination that is permitted by legal authorities was accepted by 94.7% (126/133) with parents at unbalanced cytogenetic result announcement.

The effect of PUVA treatment on sister chromatid exchange (SCE) values in psoriasis vulgaris patients.

BACKGROUND: Psoralen+ultraviolet A (UVA) (PUVA) is used successfully in the treatment of several skin diseases including psoriasis. PUVA has been reported to cause skin cancers, especially when used in high doses. In vivo and in vitro effects of different mutagens and carcinogens on DNA may be detected by sister chromatid exchange (SCE). OBJECTIVE: To investigate the mutagenic effects of different PUVA doses on DNA with SCE analysis. MATERIALS AND METHODS: Forty-two psoriasis patients under PUVA treatment were included in the study as the study group. The control group consisted of 22 psoriasis patients who did not receive PUVA treatment. The study group was divided into three groups according to PUVA doses. SCE/cell values were compared in the study and control groups and in the three dose-dependent groups of the study group. RESULTS: Mean SCE/cell values of the three dose-dependent patient groups were significantly higher than the control group (p<0.001), whereas there was not a statistically significant difference in themselves. CONCLUSION: PUVA treatment seemed to increase SCE values; however, there was not a correlation between PUVA doses and SCE frequencies.

Population data of five genetic markers in the Turkish population: comparison with four American population groups.

In this study, the phenotype and allele frequencies of five enzyme systems were determined in a total of 611 unrelated Turkish individuals and analyzed by using the exact and the chi 2 test. The following five red cell enzymes were identified by cellulose acetate electrophoresis: phosphoglucomutase (PGM), adenosine deaminase (ADA), phosphoglucose isomerase (PGI), adenylate kinase (AK), and 6-phosphogluconate dehydrogenase (6-PGD). The ADA, PGM and AK enzymes were found to be polymorphic in the Turkish population. The results of the statistical analysis showed, that the phenotype frequencies of the five enzyme under study are in Hardy-Weinberg equilibrium. Statistical analysis was performed in order to examine whether there are significant differences in the phenotype frequencies between the Turkish population and four American population groups. This analysis showed, that there are some statistically significant differences between the Turkish and the other groups. Moreover, the observed phenotype and allele frequencies were compared with those obtained in other population groups of Turkey.

Detection of Helicobacter pylori in dental plaque and gastric biopsy samples of Turkish patients by PCR-RFLP.

Helicobacter pylori is regarded as an important pathogen playing a key role in the pathogenesis of peptic ulcer. Different studies about the mode of transmission of the microorganism report conflicting results about dental plaque as the source of H. pylori infection. In the present study we aimed to detect the presence of H. pylori in dental plaque of Turkish patients by polymerase chain reaction (PCR) and if any to do typing by restriction fragment length polymorphism (RFLP) analysis. Fifty dyspeptic patients, to whom upper gastrointestinal endoscopy was performed, were included in the study. Dental plaques were obtained before endoscopic examination. Both dental plaque and gastric biopsy samples were amplified with Ure A and Cag A gene primers. There were no positive dental plaque samples even in the 23 patients whose gastric biopsy specimens were positive. Our findings showed that there is not a correlation between dental presentation of the microorganism and H. pylori gastritis.

PCR and RFLP analysis for identification and typing of Helicobacter pylori strains isolated from gastric biopsy specimens.

Helicobacter pylori (H. pylori) infection is the most common gastrointestinal tract infection which plays an important role in the ethiopathogenesis of peptic ulcer and gastritis. In recent years, molecular biological methods have been presented for detection of H. pylori in addition to histopathological and microbiological methods. Among these methods, polymerase chain reaction (PCR) and following restriction fragment length polymorphism analyses (RFLP) are highly sensitive methods for diagnosis and follow up of patients. In this present study our aim was to amplify H. pylori urease A and B genes by PCR and perform RFLP analysis. Gastric biopsy specimens from 17 female and 18 male patients were included in the study. Amplified PCR products were subjected to RFLP analysis and typing of the bacteria in pre and posttreatment specimens were performed. H. pylori urease A and B gene amplification was observed in 32 pretreatment samples and in 8 of 21 posttreatment specimens. As a result, PCR is a sensitive method to determine the H. pylori infection. RFLP, which is another effective method in order to demonstrate the reinfection of H. pylori.

The in vitro effect of beta-carotene and mitomycin C on SCE frequency in Down's syndrome lymphocyte cultures.

Down's syndrome (DS) has the highest incidence among chromosomal disorders and is a predisposing factor in acute leukemia pathogenesis. DS patients are sensitive to both physical and chemical inducers at the DNA level. Studies on beta-carotene, an antioxidant, suggested the there is a relationship between high beta-carotene diet and reduced tumor incidence in humans indicating that beta-carotene is a chemopreventive agent against cancer. Sister chromatid exchange (SCE) is known as a sensitive parameter among the genotoxicity tests. In this study, we aimed to investigate the in vitro effect of beta-carotene on SCE frequencies in 7 DS patients and 7 healthy controls aged between 0-16 years. A direct leukomogenic agent Mitomycin-C (MMC) was used as a powerful SCE inducer. Addition of MMC to the cultures alone resulted in a significant enhancement of SCE frequencies in both groups when compared to the spontaneous values. In the study, beta-carotene seemed to decrease MMC induced mean SCE/cell values, but did not have an effect on unstimulated cells. As this is a limited study, it is hard to conclude that beta-carotene is a chemopreventive agent in DS patients, although our results seem to support other investigators' reports.

Interactions of DNA with fluorescent dyes: by scanning tunneling microscopy.

Genomic DNA was obtained from peripheral blood samples of healthy volunteers and interacted with two fluorescent dyes (i.e. Hoechst 33,258 and ethidium bromide) in aqueous media. These media containing DNA-dye complexes deposited on the gold coated mica surfaces. Then, STM images were obtained in which the STM was operated in air at atmospheric pressure with a tip-to-substrate bias voltage of 250-1000 mV (sample positive) and the tunneling currents in the range of 10-20 pA by using etched tips of Pt/Ir, in constant current mode. Both dyes from molecular clusters on DNA. While, the Hoechst molecules were observed on the DNA chains at regular distances, the ethidium bromide molecular clusters did not.

Detection and typing of human papillomavirus DNAs by restriction endonuclease mapping of the PCR products.

Human papillomavirus (HPV) has been implicated strongly with human cervical, anal and penile cancers. The polymerase chain reaction (PCR) was used to detect HPV in cervical specimens of 88 women working at the public whorehouse. Using consensus primers which encode the L1 region of the HPV genome, the presence of HPV DNA was demonstrated in 2 specimens. Restriction endonuclease digestion of the amplified products was carried out for accurate typing. Samples which were positive by L1 PCR were digested with Hae III, BstN I and Dde I restriction enzymes. The patterns produced by digestion were identified as HPV types 6b and 16.

Gene BR140, which is related to AF10 and AF17, maps to chromosome band 3p25.

The genes AF10 and AF17 have been identified as the basis of the t(10;11) and t(11;17) translocations, events that result in their fusion to the MLL/HRX gene in acute myeloid leukaemias. AF10 and AF17 bear significant homology to each other within their putative zinc finger and leucine zipper domains, although they are diverged outside these regions. The BR140 gene encodes a 140 kDa protein of unknown function that contains a putative zinc finger domain, a leucine zipper region, and, in addition, a bromo domain. The zinc finger and leucine zipper domains of BR140 have significant homology to those of AF10 and AF17, suggesting that it belongs to this newly described gene family and, therefore, could be a target for chromosome translocation. To assess the potential involvement of BR140 in chromosome translocations in leukaemia, the chromosomal location of the BR140 gene has been determined by using several independent methods. A combination of Southern analysis, polymerase chain reactions (PCR) on monochromosomal cell hybrids, and fluorescence in situ hybridisation (FISH) has been used to show that the BR140 gene maps to chromosome band 3p25.

The distribution of the HLA-DQ alpha alleles and genotypes in the Turkish population as determined by the use of DNA amplification and allele-specific oligonucleotides.

Allele and genotype frequencies for the HLA-DQ alpha locus were determined for use in forensic analyses in Turkey. The polymerase chain reaction and the reverse dot blot format were employed to detect six different HLA DQ alpha alleles, which were detected among 150 unrelated individuals with allele frequencies ranging from 7% to 26.7%. The distribution of the observed genotypes was in Hardy-Weinberg equilibrium. The discrimination power of this system in the Turkish population sample was 0.94, and the allelic diversity was 0.81. This study provides support for the validity of the HLA-DQ alpha procedure for typing forensic samples.

The effect of the male contraceptive agent gossypol acetic acid on mouse bone marrow cells in vivo: micronuclei and mitotic index.

In this work the genotoxic effect of gossypol acetic acid (gossypol) was evaluated by determining the frequency of micronuclei and mitotic index in male mouse bone marrow cells in vivo. Bone marrow cells were collected at 24th hour after the single intraperitoneal (20, 40, and 80 micrograms/g) administration of gossypol. Polychromatic erythrocytes (PCEs) in the bone marrow were then evaluated with respect to micronuclei frequency. The dose-dependent increase in the micronuclei frequency was observed. However, when compared with the control group, the increase was not found to be significant (P > 0.05). Also the mitotic index values were not found to be different from those control values (P > 0.05). The results suggest that gossypol is not a clastogenic and mutagenic agent in mouse bone marrow cells in vivo.

A chemical modification approach to the olfactory code: vapor phase labeling using photoaffinity odorants.

A photoaffinity labeling technique was used to study the receptors involved in the discrimination of odorants. Aromatic azides, 1-azidonaphthalene (AzN) and 1-azido-4-nitronaphthalene (AsNN), were found to be pleasant-smelling compounds and produced good responses, giving standard EOG's (electro-olfactogram) of the kind observed for normal odorants. Following irradiation of the frog olfactory mucosa with light during constant stimulation with one of the azides vapor, there was a specific partial inhibition of the receptors for that odorant. The extent of reduction in amplitude of the EOG responses to AzN and AzNN varied between 40 to 60% of the original amplitude.

[Salmonella tester strain TA104 for the detection of mutagenic and carcinogenic chemicals in our environment]. / Cevremizdeki mutajenik ve karsinojenik maddelerin Salmonella typhimurium TA104 susu ile saptanmasi.

We used the Salmonella mutagenicity test for detecting chemical carcinogens as mutagens in the Salmonella typhimurium tester strain TA104 . The mutagenicity of several compounds was assessed by induction of histidine revertants in the TA104 . In each experiment we routinely included positive mutagenesis controls using three different concentrations of known mutagens. The mutagenic chemicals such as sodium azide, hydrogen peroxide and hydroxylamine were found to be mutagenic to TA104 at very low concentration (10(-4) mg/ml). Their mutagenic activity decreased while their concentrations were increased. The effect of acridine orange, 2, 4, 6-trinitrobenzene sulphonic acid, 2- phenylnaphthalene and 20- methylcholanthrene were also found to be mutagenic to TA104 at the concentration of 10(-2) mg/ml. The mutagenicity of other materials such as hair dyes, meat- broth preparations+ and cigarette smoke condensates were also tested, and all of them were found to be mutagenic to TA104 . The highest mutagenic activities were observed at the concentration of 10 mg/ml for two different hair dyes and of 1 mg/ml for cigarette smoke condensates.