Band 42, Heft 2

Background Microarray technology, aims to measure the amount of changes in transcripted messages for each gene by RNA via quantifying the colour intensity on the arrays. But due to the different experimental conditions, these measurements can include both systematic and random erroneous signals. For this reason, we present a novel gene expression index, called multi-RGX (Multiple-probe Robust Gene Expression Index) for one-channel microarrays. Methods Multi-RGX, different from other gene expression indices, considers the long-tailed symmetric (LTS) density, covering a wider range of distributions for modelling gene expressions on the log-scale, resulting in robust inference and it takes into account both probe and gene specific intensities. Furthermore, we derive the covariance-variance matrix of model parameters from the observed Fisher information matrix and test the performance of the multi-RGX method in three different datasets. Results Our method is found to be a promising method regarding its alternatives in terms of accuracy and computational time. Conclusion Multi-RGX gives accurate results with respect to its alternatives, with a reduction in computational cost.

Introduction Association between Murine double minute 2 T309G polymorphism and lung cancer risk has been investigated in several populations, but results of these studies are inconsistent. We aimed to investigate the effect of Murine double minute 2 T309G polymorphism on development of lung cancer in a Turkish population. Methods Total 200 subjects including 100 patients and 100 controls were analyzed and used polymerase chain reaction and restriction fragment length polymorphism methods for genotyping analysis of the polymorphism. Results We found that smokers compared with non-smokers have approximately eight fold higher lung cancer risk [p=0.0001, OR=8.27 (4.02–16.9)]. Frequency of GG genotype was higher in patients than in controls, but this ratio was not significant (χ 2 =3.5, p=0.17). Genotype distribution of the polymorphism was not different neither patients with non-small cell lung cancer nor patients with small cell lung cancer (χ 2 =2.89, p=0.57). We analyzed together with demographic feature (except smoking habit), clinicopathological findings and genotype frequencies of this polymorphism, and any association with lung cancer risk was not obtained. Conclusion No correlation between Murine double minute 2 T309G polymorphism and lung cancer risk was detected in this Turkish population.

Objective As a result of adverse effects including drug-resistance, toxicity and low bioavailability, there has been a crucial need for novel anticancer agents. In this present study, some novel 2,5-disubstituted 1,3,4-oxadiazole derivatives bearing pyridine moiety were synthesized and their potential cytotoxic activities were examined. Materials and methods A series of seven new compounds of 2-[(5-(3-(pyrimidin-2-yl)thio)propyl)-1,3,4-oxadiazol-2-yl)thio)]-1-(4-substituted)ethan-1-one derivatives were synthesized by reacting 5-[(3-(pyrimidin-2-yl)thio)propyl]-1,3,4-oxadiazole-2-thiol and 4-substituted phenacyl bromide derivatives in acetone with potassium carbonate. The structures of the obtained compounds were elucidated using FT-IR, 1 H-NMR and MS spectral data and elemental analyses. In vitro cytotoxic activity of the compounds was evaluated by MTT assay. Results Among the tested compounds, compound 4a was found to be the most active cytotoxic agent against A549 cells, in compared with cisplatin as standard drug. Conclusions It was determined that some of synthesized compounds had considerable anticancer activity against the A549 cell lines. Compound 4a including phenyl moiety was the most active compound against the A549 cell line and was identified as a lead moiety. Besides, compound 4c including 4-metoxy phenyl moiety exhibited cytotoxic activity against A549 cells. Consequently, compounds possess phenyl and 4-methoxy phenyl moieties have been determined to be important for cytotoxic activity of these compounds.

Objective The purpose of this study is to investigate the role of dax1 , which causes adrenal hypoplasia congenita (AHC) with associated hypogonadotropic hypogonadism (HH) and dosage-sensitive sex reversal (DSS) during carp embryogenesis and gonadal development. Methods The dax1 gene was cloned from ovaries of adult carp using homologous cloning and RACE methods. Expression patterns during embryogenesis and various adult tissues were analyzed using RT-PCR (QRT-PCR) and in situ hybridization (ISH). Results Expression of dax1 was detected at beginning of the blastula stage and peaked at 80 days post hatching (dph), while expression in the ovary was stable. Expression of dax1 was detected in males and females, and showed sexually dimorphic pattern with high level expression in juvenile male liver and gonad. Adimorphic expression was observed in adult male gonad and heart, but was higher in liver of adult females. Whole-mount in situ hybridization revealed dax1 expression in the developing diencephalon at hatching. Later, at approximately 5 dph, a novel expression of dax1 was observed in the developing hindbrain of carp larvae. Conclusion These preliminary findings suggest that dax1 is conserved during vertebrate evolution and involved in a wide range of developmental processes including neurogenesis and sex differentiation in vertebrates.

Objective Restoration of catalytic activity of Isa2 from Solanum tuberosum (Stisa2) through restoration of conserved catalytic residues by site directed mutagenesis. Methods The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed in E. coli Rosetta 2 under optimized conditions. Evaluation of debranching activity on starch, amylopectin and β-limit dextrin was carried out by measuring the amount of glucose equivalents released using the bicinchoninic acid assay. Results Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity. Conclusions These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

Objective Certain cancer types have been shown to be associated with vitamin D deficiency. The aim of this study was to appraise the relationship between the vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) of VDR Fok1 and Bsm1 with serum vitamin D, calcium and phosphorus levels among patients of lung, colon, breast and pancreatic cancer patients. Materials and methods Groups; lung, colon, breast and pancreatic cancer patients (n=212) and 58 age-matched healthy controls. Serum levels of vitamin D were measured by immunochemiluminesence method and Fok1 and Bsm1 genotypes were assessed with Real-Time Polymerase Chain Reaction. Results VDR Fok1 and Bsm1 genetic polymorphisms have a significant difference between lung cancer and control group subjects (p=0.042, p=0.040, respectively). VDR Bsm1 genetic polymorphism has a significant association between breast cancer and control group according to the logistic regression model (p=0.038). Vitamin D levels were found significantly lower in all cancer groups (p<0.01). Phosphorus levels of lung cancer and calcium levels of pancreatic cancer patients were statistically significantly lower than control group (p<0.02, p<0.01). Conclusion This study indicates that VDR genetic polymorphisms, calcium, phosphorus and vitamin D status of individuals were associated with certain cancer species.

Objective(s) The synthesis of new N′ -arylidene-4-[(1-phenyl-1 H -tetrazole-5-yl)thio]butanoylhydrazide derivatives ( 1–26 ) and investigation of their potential anticholinesterase (AChE), butyrylcholinesterase (BuChE) enzyme inhibition activities and also cytotoxic properties on mouse embryonic fibroblast cells (NIH/3T3) were aimed in this work. Materials and methods The target compounds were prepared by a three step synthetic procedure using 1-phenyl-1 H -tetrazole-5-thiol and ethyl 4-chlorobutanoate as starting materials. The structures of the obtained compounds were elucidated by IR, 1 H-NMR, 13 C-NMR spectra and elemental analysis data. The enzyme inhibition and cytotoxic activities were determined according to Ellman and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] methods, respectively. Results Compounds 14 , 15 and compound 18 exhibited the highest inhibitory activity on AChE and BuChE enzymes. Additionally, compounds 4, 5, 8 and 16 exhibited the lowest cytotoxicity against NIH/3T3 cells. Conclusion Compounds 14, 15 and 18 bearing 2-nitro, 3-nitro and 3-hydroxy substituents have showed selective enzyme inhibitory activities.

Objective A genetic polymorphism of SOD1 A251G ( rs2070424 ) is in the 3rd intron region of the SOD gene. The aim of this study was to determine the frequencies of the polymorphisms of the SOD1 A251G in a Turkish population, including 494 healthy individuals. Methods The 494 Turkish individuals were genotyped for polymorphisms of SOD 1 gene. The distribution of SOD1 A251G polymorphisms in this population was examined using a PCR-RFLP method. Genotype and allele frequencies were estimated by counting. Hardy–Weinberg equation between expected and observed genotype distributions was assessed using the X 2 test. Results In the present study, the distribution of SOD1 A251G polymorphisms in a Turkish population including 494 (females: 278, 56.3% and males: 216, 43.7%) healthy individuals was examined. The mean age of the study population was 38.4±16.6 years (males, 39.8±17.1; females, 37.3±16.1). The observed genotype frequencies of SOD1 A251G were 86.2, 13.4 and 0.4% for AA, AG and GG , respectively. Conclusions This study provides basic information about the allele and genotype frequency distributions of polymorphisms in the SOD1 A251G gene studied. These frequencies may be useful parameters as a reference for future studies on genetic basis of various diseases and cancer susceptibility.

Introductıon We aimed to determine the serum levels of transactive response of DNA - binding protein 43 (TDP-43) and ubiquitin C-terminal hydrolase-L1 (UCH-L1), which are ubiquitin-proteasome pathway related molecules and have not been investigated so far, in children with attention-deficit/hyperactivity disorder (ADHD). Methods The study group was composed of thirty children aged between 6 and 10. They were diagnosed with ADHD according to DSM-IV criteria. They were the subjects who applied to Dicle University, Faculty of Medicine, and Department of Child Psychiatry in Diyarbakır, Turkey. Children with ADHD were assessed via Turgay DSM-IV Based Child and Adolescent Behavior Disorders Screening and Rating Scale and Stroop test. Serum TDP-43 and UCH-L1 levels were analysed with enzyme-linked immunosorbent assay. Results The TDP-43 and UCH-L1 serum levels of children with ADHD were found to be statistically significantly higher than those of controls. On the other hand, we found that serum levels of TDP-43 correlated with interference effect and hyperactivity–impulsivity in children with ADHD. Conclusıon Imbalances in serum UCH-L1 and TDP-43 levels, and the correlation of TDP-43 levels with clinical parameters in children with ADHD may suggest that ubiquitin-proteasome pathway alterations are associated with ADHD. Deterioration of this pathway may cause intracellular TDP-43 aggregation.

Introduction Connexins (Cx) play essential roles in cellular homeostasis by forming gap junctions and non-junctional hemichannels. In vitro characterization of Cx26 mutations causing keratitis-ichthyosis-deafness (KID) syndrome, were shown to form leaky hemichannels. The molecular/cellular mechanisms affected by aberrant hemichannels have recently been elucidated. Here, we further wanted to characterize Cx26 KID syndrome mutations, D50A and A88V, which were shown to form aberrant hemichannels and remained unaddressed in the literature. Methods Neurobiotin uptake assay in HeLa and N2A cells transfected with Cx26-WT, D50A or A88V verified the presence of aberrant hemichannels and immunofluorescent staining with fluorescent microscopy determined cellular localization of Cx26. Finally, intracellular calcium content was examined by using calcium indicator, Fluo-3AM, and flow cytometer. Results Cx26-D50A and A88V mutations prevented the formation of gap junction plaques at cell-cell appositions and mutant proteins were observed to localize to the Golgi apparatus. Further, comparison of intracellular calcium content showed an increase in calcium amount in cells containing Cx26-D50A and A88V relative to Cx26-WT. Conclusion Retention of Cx26 in the Golgi apparatus and alteration in the intracellular calcium content due to KID syndrome mutations may influence various cellular processes that might contribute to development of epidermal phenotypes.

Introduction The Gaussian Graphical Model (GGM) is one of the well-known probabilistic models which is based on the conditional independency of nodes in the biological system. Here, we compare the estimates of the GGM parameters by the graphical lasso (glasso) method and the threshold gradient descent (TGD) algorithm. Methods We evaluate the performance of both techniques via certain measures such as specificity, F-measure and AUC (area under the curve). The analyses are conducted by Monte Carlo runs under different dimensional systems. Results The results indicate that the TGD algorithm is more accurate than the glasso method in all selected criteria, whereas, it is more computationally demanding than this method too. Discussion and conclusion Therefore, in high dimensional systems, we recommend glasso for its computational efficiency in spite of its loss in accuracy and we believe than the computational cost of the TGD algorithm can be improved by suggesting alternative steps in inference of the network.

Objective Erythropoietin (EPO) is widely used for treatment of anemia associated with different diseases; however, its adverse effects limit its use in clinical practice. Therefore, understanding the effects of EPO at the molecular and cellular level is crucial to adjust treatment regimes, and to develop non-hematopoietic EPO derivatives. In this study, we used a proteomics approach to identify how EPO treatment modifies the cellular proteome. Methods SH-SY5Y neuroblastoma cells were used as the model system to analyze the effects of EPO treatment at different time points (24 h and 48 h). Proteomic analysis revealed changes in 74 proteins after EPO treatment. Following proteomics analysis, Reactome pathway analysis were carried out to identify the affected cellular pathways. Results According to results, EPO alters the levels of 74 protein species (40 were increased, 34 were decreased). The levels of 35 proteins were changed by 24 h EPO incubation, whereas 17 protein species were altered by 48 h EPO incubation. Levels of 22 protein species were altered by both of the incubation periods (24 h and 48 h). Conclusion Overall, our results suggest that EPO mainly affects protein species in glucose metabolism, protein and RNA metabolism, cytoskeletal proteins, and mitochondrial protein species.

Objective Indole-3-acetic acid (IAA) activation has been suggested as a new strategy for cancer therapy. It has been reported that ultraviolet B (UVB) radiation can activate IAA. In the present study, we investigated whether UVB-irradiated IAA (IAA UVB ) can induce apoptosis of G361 human melanoma cells and examined the apoptotic pathway involved. Methods DNA fragmentation was measured to examine apoptosis. IAA UVB -induced signaling pathways were investigated by Western blot analysis. Results Our results show that IAA UVB reduced cell viability of G361 human melanoma cells, and induced DNA fragmentation, a hallmark of apoptosis. We also found that c-Jun NH 2 -terminal kinase (JNK) and p38, which are activated by IAA UVB , are not associated with this cell death. We further investigated the IAA UVB -mediated apoptotic pathway after pretreatment with NS398, vitamin C, and N-acetylcysteine (NAC). Although NS398, an inhibitor of cyclooxygenase-2, was not protective, vitamin C and NAC ameliorated IAA UVB -mediated cell death. In addition, when cells were pretreated with a caspase inhibitor, IAA UVB -induced apoptosis was inhibited. Conclusions These results suggest that free radicals generated from IAA by UV irradiation may cause apoptosis, and IAA UVB induces apoptosis of G361 human melanoma cells by activating caspases.

Aim The development of calixarene based phenol biosensor. Methods This study describes the application of a calixarene derivative, 5,17-diamino-25,27-bis(3-thiol-1-oxypropane)-26,28-dihydroxycalix[4]arene (HS-Calix-NH 2 ) which has both amino and thiol functionalities, in the practical surface modifications for biomolecule binding. The structure of HS-Calix-NH 2 allows easy interaction with Au surface and one-step biomolecule immobilization. Self-assembled monolayers (SAMs) of p-amino-functionalized mercaptoalkylcalixarene (HS-Calix-NH 2 ) were formed onto the Au electrode. Then, Laccase (Lac) enzyme was immobilized onto the modified surface by crosslinking with glutaraldehyde (GA). Resulted electrode (HS-Calix-NH 2 /Lac) was used for the electrochemical analysis of phenolic compounds at −50 mV. Results The linearity was observed in the range of 0.1–100 μM and 1.0–100 μM for catechol and phenol, respectively. The potential use of the biosensor was investigated for phenol analysis in artificial samples which simulate the industrial waste water, which is highly acidic and composed of concentrated salt, without needing any sample pre-treatment step. Conclusion The prepared Lac biosensor has a potential for rapid, selective and easy detection of phenolic contaminations in samples.

Oxidative stress is a quite popular subject among biochemists. With a key word search of Thomson Reuters Web of Science, a large number of entries were found starting from 1970. By using animal tissues, cell cultures or human subjects, scientists try to establish a relationship between reactive oxygen species and various situations including cancer and aging, and the protective effects of antioxidants in these cases. Some clinical trials of antioxidants are also on their way. There are several oxidative stress markers such as malondialdehyde, thiol status of the cells, superoxide dismutase, catalase and glutathione peroxidase. Although these markers are widely and continuously investigated in various laboratories, they still are not in the repertoire of a routine clinical laboratory as a biomarker of any disease. In this paper, various aspects of “oxidative stress” and antioxidants are investigated and a requirement of a radical change in the area is pointed out.

Physical transfer of viable tumor cells from one organism to another is known as transmissible cancer, which is observed in dogs, Tasmanian devils, Syrian hamsters, and some soft-shell clams. Tasmanian devil facial tumor disease is transmitted like an infectious disease between individuals through biting and other close contact. This extinction type is quite different from the other extinction types such as ecological factors. Transmissible cancers’ cellular metabolism is also different from the both normal cellular metabolism and other types of cancers’ metabolism. The lack of an immune response against the Tasmanian devil facial tumor cells is the one of the key points in the transmission of the cancerous cells. The differentiated cellular metabolism and absence of immune reaction may be due to the organisms’ enzymes. Cells may have altered surface proteins by altering enzymatic activities that cannot be recognized by both the innate and adaptive responses. The promiscuity of the key enzymes may be associated with unwanted side effects, such as cannot recognize molecular patterns on the transmitted cell or hypomethylation of DNA by altering catalytic properties enzymes or altered matrix metalloproteinases or cathelicidins.