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First paraben substituted cyclotetraphosphazene compounds and DNA interaction analysis with a new automated biosensor.

Cancer, as one of the leading causes of death in the world, is caused by malignant cell division and growth that depends on rapid DNA replication. To develop anti-cancer drugs this feature of cancer could be exploited by utilizing DNA-damaging molecules. To achieve this, the paraben substituted cyclotetraphosphazene compounds have been synthesized for the first time and their effect on DNA (genotoxicity) has been investigated. The conventional genotoxicity testing methods are laborious, take time and are expensive. Biosensor based assays provide an alternative to investigate this drug/compound DNA interactions. Here for the first time, a new, easy and rapid screening method has been used to investigate the DNA damage, which is based on an automated biosensor device that relies on the real-time electrochemical profiling (REP™) technology. Using both the biosensor based screening method and the in vitro biological assay, the compounds 9 and 11 (propyl and benzyl substituted cyclotetraphosphazene compounds, respectively), have resulted in higher DNA damage than the others with 65% and 80% activity reduction, respectively.

Overexpression of peptide deformylase in breast, colon, and lung cancers

BackgroundHuman mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression.MethodsThe mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay.ResultsPDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines.ConclusionsThis is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Prevalence and Characterization of Salmonella Isolated from Chicken Meat in Turkey

This study was conducted in a Turkish province to investigate the presence of Salmonella spp. in 150 chicken meat samples using 2 phenotyping techniques: classic culture technique (CCT) and immunomagnetic separation (IMS). For the confirmation of the isolates at molecular levels, invA gene was detected in these isolates. The presence of invA, class 1 (Cls1) integrons, and integrase (Int1) genes was demonstrated by PCR assay; and the resistance of the isolated Salmonella spp. strains to antibiotics was determined by disk diffusion test. All the cultural and PCR results were evaluated together; Salmonella spp. were detected in a total of 64 (42.66%) chicken meat samples. Contamination rate was higher in carcasses (53.33%, n = 75) than in meat pieces (32%, n = 75). When results of standard culture were compared with IMS technique, IMS (n = 54) showed a clear superiority over the CCT (n = 38). A very high resistance rate (≥ 89.28%) to vancomycin, tetracycline, streptomycin, or nalidixic acid was found. Trimethoprim-sulfamethoxazole resistance was present in 32.14%. Relatively lower incidence of resistance (≤ 8.33%) to gentamicin, chloramphenicol, ampicillin, and ceftriaxone was observed. Concurrent resistance to at least 4 antibiotics was detected in 92.85% of the isolates. Cls1 integrons and Int1 were positive in 80.95% and 95.23% of the isolates, respectively. However, Int1 alone was detected in 15.47% (n = 13). In conclusion, the high prevalence of Salmonella spp. in chicken meat may pose a potential public health risk, and the presence of antibiotic-resistant Salmonella spp. isolate together with Cls1 integron and/or integrase might play an important role in horizontal antibiotic gene transfer.

A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections.

Mycoplasma gallisepticum is the primary agent of chronic respiratory disease causing important economic losses in the poultry industry. Serological monitoring is essential to maintain mycoplasma-free breeder flocks and often complicated by the cross-reactions between different mycoplasma species. To overcome serological cross-reactions, a large fragment of the M. gallisepticum PvpA cytadhesin, species-specific surface-exposed protein, was produced in E. coli as a recombinant protein (rPvpA336) and used as a potential diagnostic antigen. The rPvpA336 protein possesses 336 mycoplasma-specific amino acids with relative molecular weight of 44 kDa. A deletion region of 37 amino acids was identified when compared to the wild-type PvpA protein. Immunoreactivity of the rPvpA336 protein has been demonstrated by Western blot analysis with M. gallisepticum-positive and -negative chicken sera. Furthermore, an enzymatic rapid immunofiltration assay (ERIFA) prototype based on the rPvpA336 protein has been developed and its species-specific detection capability has been demonstrated by using M. gallisepticum and/or M. synoviae-positive and -negative chicken sera. In addition to its species-specificity, the ERIFA prototype presents certain advantages such as rapidity, field-applicability and cost-effectiveness. Therefore, these advantages would make the prototype a species-specific rapid diagnostic tool of choice in the field and limited laboratory conditions for screening M. gallisepticum infections.

Prevalence and Characterization of Coagulase Positive Staphylococci Isolated from Salted Anchovy

A total of 100 salted anchovy samples were used to investigate the prevalence of S. aureus and other coagulase positive Staphylococci (CPS) as well as to determine the methicillin (MR) and antibiotic resistance (AR) profile, the presence of Panton-Valentine leukocidine (PVL) toxin gene (lukS/F-PV), slime factor properties (SFP), and the genotypic relatedness of the isolates. Agar disc diffusion assay (ADDA) and microdilution broth susceptibility test (MDBST) were applied to compare the specificity and sensitivity of the MR detection methods. A total of 41 CPS isolates were detected at the 102 and 103 CFU/g levels in contrast to S. aureus. The 16S rRNA (genus specific) was detected in all the isolates in contrast to nuc (species-specific) and lukS/F-PV genes. A total of 16/41 isolates were found to be MR by using the three methods. Polymerase chain reaction (PCR) assay was a more sensitive and reliable method for the detection of MR isolates. The antibiotic resistance rates were 75.60, 73.17, 51.21, 31.70, 12.19, and 4.87% to penicillin, ampicillin, tetracycline, erythromycin, ciprofloxacin, and clindamycin, respectively. All the isolates were sensitive to gentamicin and vancomycin. The SFP were determined in all the isolates by using Congo Red agar, and 20 different genotypes were determined by using randomly amplified polymorphic DNA (RAPD)-PCR assay.

In Vitro Effects of Ivermectin and Sulphadiazine on Toxoplasma gondii

OBJECTIVE Ivermectin and sulphadiazine were tested individually to determine their in vitro effects on Toxoplasma gondii grown in human epidermoid larynx carcinoma (Hep-2) cell culture. STUDY DESIGN In-vitro study. MATERIAL AND METHODS Toxoplasma growth was quantities by an enzyme immunoassay performed directly on the fixed cultures, using a rabbit anti-T. gondii immunoglobulin G as the first antibody and a phosphatase-labeled anti-rabbit immunoglobulin G as the second antibody. For each drug, regression models were used to quantify the relationship between optical density values and antimicrobial agent concentrations in the cultures. RESULTS The 50% inhibitory concentrations (IC50) of ivermectin and sulphadiazine were found to be 0.2 μg/mL and 7.3 μg/mL after 48 h of exposure, respectively. None of the concentrations tested for each drugs demonstrated toxicity to Hep-2 cells after 72 h of incubation. CONCLUSION These results indicate that ivermectin significantly inhibited replication of the tachyzoites of T. gondii RH strain.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus in Foods of Animal Origin, Turkey

In the present study, 175 coagulase-positive Staphylococcus (CPS) isolates recovered from samples of beef (n = 110), raw milk n = 56), and fish (n = 9) were analyzed for methicillin resistance using MIC and PCR assays. Methicillin-resistant (MR) Staphylococcus aureus (SA) isolates were then characterized using pulsed-field gel electrophoresis (PFGE). According to findings, 62 (35.4%) of the isolates (44 from beef, 9 from milk, and 9 from fish) were identified as S. aureus based on the presence of the nuc gene. MRCPS was detected in 18 (10.3%) of 175 CPS isolates based on the presence of the mecA gene. Among these isolates, 15 (24.2%) were MRSA: 4 (26.7%) from beef, 2 (13.3%) from milk, and 9 (60%) from fish. However, based on the MIC assay, 21 (12.0%) of the CPS isolates (1 from beef, 15 from milk, and 5 from fish) were MRCPS, indicating a discrepancy between the results of these two methods. The PFGE results indicated genetic heterogeneity of the isolates; six PFGE clusters were found. These results confirm that MRSA is present in foods of animal origin, which is a concern to human health, and indicate the importance of method selection for determination of methicillin resistance. The identity of MR isolates should be verified by PCR to obtain more reliable results.

Determination of PER-1 and OXA-10-like β-lactamases in Ceftazidime-Resistant Pseudomonas aeruginosa Isolates by Molecular Methods

ABS TRACT Ob jec ti ve: Pse u do mo nas ae ru gi no sa is one of the im por tant no so co mi al pat ho gens and re sis tant to many an ti bi o tics inc lu ding β-lac tams. PER-1 and OXA-10 type ex ten ded-spec trum βlac ta ma ses (ESBLs), the ma jor β-lac ta ma ses, we re iden ti fi ed in P. ae ru gi no sa. The aim of this study was to iden tify PER-1 (bla PER-1) and OXA-10 (bla O XA-10)-li ke β-lac ta ma ses in cef ta zi di meresis tant no so co mi al P. ae ru gi no sa stra ins. Ma te ri al and Met hods: The pre sen ce of PER-1 and OXA10 li ke β-lac ta ma ses was in ves ti ga ted by polymerase chain reaction in 50 cef ta zi di me-re sis tant P. ae ru gi no sa stra ins iso la ted from pa ti ents hos pi ta li zed in va ri o us cli nics of Ondokuz Mayıs Uni ver sity, Scho ol of Me di ci ne bet we en 2007 and 2008. The PER and OXA-10-li ke β-lac ta ma ses we re analy zed by res tric ti on frag ment length poly morp hism (RFLP) and fol lo wed by pul sed-fi eld gel elec trop ho re sis (PFGE) for the de ter mi na ti on of clo nal re la ti ons hip of the stra ins. Re sults: The bla PER-1 ge ne and bla O XA-10 li ke ge ne we re de tec ted in 23 (46%) and 39 (78%) of the 50 of cef ta zi di me-re sis tant P. ae ru gi no sa iso la tes, res pec ti vely. In ad di ti on, both of the two ß-lac ta ma se ge nes we re al so de tec ted in 12 (23%) of the iso la tes. PER-1 and OXA-10, -11, -14, -16 types we re iden ti fi ed by RFLP analy sis. Alt ho ugh the PFGE typing re sults yi el ded 11 dif fe rent ban ding pat terns, 74% (n= 37) of the all P. ae ru gi no sa stra ins we re inc lu ded in four ma in pat terns. Conc lu si on: It was conc lu ded that the pre va len ce of PER-1 and OXA-10 enz ymes was com mon among cef ta zi di me re sis tant P. ae ru gi no sa iso la tes. PER-1 and OXA-10 enz ymes pro du ced the iso la tes we re be ing transmit ted ho ri zon tally as the most of the iso la tes we re clo nally re la ted.

Investigation Of Panton-valentine Leukocidine Presence In The Clinical Strains Of Staphylococcus Aureus

Amac: Bu calismada Staphylococcus aureus klinik izolatlarinda PantonValentin Lokosidin (PVL) varligi PZR yontemiyle arastirilmistir.Gerec ve Yontemler: Bu calismada, Şubat 2006-Haziran 2008 tarihleri arasinda cesitli klinik orneklerden izole edilen 130 MRSA ve 20 MSSA izolati test edildi. S. aureus izolatlari ile bu izolatlardaki metisilin direncinin dogrulanmasi multipleks polimeraz zincir reaksiyonu (PZR) ile yapildi. PVL gen varligi hem multipleks PZR hem de single-target PZR ile de test edildi. Bulgular: Calisma sonucunda, 3 izolatta PVL pozitif bulundu. Bunlardan ikisi MRSA, digeri ise MSSA izolati idi. PVL pozitif MRSA izolatinin biri yara orneginden digeri kan kultur orneginden tanimlandi. PVL pozitif MSSA izolati ise idrar orneginden tanimlandi. PVL pozitif izolatlarin ucu de tetrasikline direncliydi. Kan kulturunden izole edilen PVL pozitif MRSA izolati gentamisin ve siprofl oksasine de direncli bulunurken, diger PVL pozitif MRSA izolati duyarli bulundu. Sonuc: Toplumda ve hastanede PVL pozitif izolatlarin yayilimini engellemek ve PVL pozitif izolatlarin prevalansi ve genetik ozellikleri hakkinda bilgi edinebilmek icin ileri calismalara ihtiyac vardir