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Öğe Amplification of GC-rich ADAMTS-2 and URG4/URGCP promoter regions with optimized combination of PCR enhancers(Turkish Journal of Biology, 2016) Alper, Meltem; Tokay, Esra; Köçkar, FerayPCR is an effective and widely used method for the amplification of target DNA fragments in vitro. The templates that have high guanine-cytosine (GC) content, complex secondary structures, or short tandem repeats are difficult to amplify by the conventional PCR methods. Human genome regulatory regions such as promoters are rich in terms of GC base compositions. To amplify these regions some special modifications or additives are required. Using different PCR strategies such as hot-start/touchdown PCR or the utilization of various additives into the reaction mixture such as organic molecules can improve PCR yield. In the present study we optimized the PCR conditions for the amplification of the human ADAMTS-2 gene promoter region featuring an extremely high GC content and a secondary structure. We show that a combination of three additives, betaine, dimethyl sulfoxide, and 7-deaza GTP, is essential to obtain a correct PCR amplicon. To demonstrate whether the optimized PCR condition was applicable to other GC-enriched regions, a similar procedure was repeated for the amplification of the human URG4/URGCP gene promoter.Öğe Effect of angiogenesis related growth factors VEGF-a and FGF-1 on osteosarcoma cell proliferation(Adıyaman Üniversitesi, 2020) Alper, MeltemOsteosarcoma is the most common bone tumor in children and adolescents. Alterations in the expression of some genes, cytokines and growth factors are responsible for the development of the malignant phenotype in osteosarcoma. Some members of the VEGF and FGF families have been associated with poor prognosis and the metastasis in various tumor types including osteosarcoma. Among the members of the family, information about effects of VEGF-a and FGF- 1 on osteosarcoma cell proliferation is limited. In the present study, it was aimed to elucidate the effects of VEGF-a and FGF-1 on osteosarcoma cell proliferation. Saos-2 cells, having osteoblastic features, were used for osteosarcoma model. Cells were treated 20 ng/mL VEGF-a and FGF-1 after 24 hours of serum starvation. MTT assay was applied to measure cell viability after incubation for 1-72 hours. Results indicated that VEGF-a promoted cell proliferation for all incubation times. Maximum increase was observed after 48 hours of incubation (1.7 fold) with a statistically important manner. FGF-1 led to very slight increase in Saos-2 cell proliferation. Consequently, these findings can contribute development of new therapy strategies for osteosarcoma.Öğe IL-6 upregulates a disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2) in human osteosarcoma cells mediated by JNK pathway(Springer, 2014) Alper, Meltem; Köçkar, Feray TuraADAMTS-2 and ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin type 1 motif 2) belong to the procollagen aminoproteinase subfamily of ADAMTS proteases. They play crucial roles in the collagen metabolism. To understand the regulation of ADAMTS-2 gene expression in osteoblastic cells, we have cloned a functional 760 bp of human ADAMTS-2 promoter. Sequence analysis of the ADAMTS-2 promoter region showed the absence of a TATA box, but identified a GC box, a CpG island, several GAGA boxes and several transcriptional factor binding sites, which may be valuable in the regulation of ADAMTS-2 transcription. We also elucidated that Interleukin 6 (IL-6) increases ADAMTS-2 and ADAMTS-3 mRNA and protein levels in different osteosarcoma cell lines namely, MG-63 and Saos-2. IL-6 also increases the transcriptional activation of the ADAMTS-2 gene promoter. Pathway inhibition studies revealed that ADAMTS-2 upregulation by IL-6 was mediated by JNK pathway.Öğe Induction of human ADAMTS-2 gene expression by IL-1 alpha is mediated by a multiple crosstalk of MEK/JNK and PI3K pathways in osteoblast like cells(Elsevier, 2015) Alper, Meltem; Aydemir, A. Tugsen; Koçkar, FerayUp-regulation of ADAMTS genes with proinflammatory cytokines is important for some pathological conditions such as osteoarthritis (OA) that is a disease based on ECM degradation in cartilage. IL-1 alpha is a proinflammatory cytokine and important both to normal and pathophysiologic conditions in cartilage and bone. Effects of some proinflammatory cytokines such as TNF-alpha and IL-1 beta on the some members of ADAMTS family have been investigated in some chondrocyte tissues or cell lines. However the effect of the IL-1 alpha on the expression of ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cell lines, remains unclear. Therefore, the aim of this study is to investigate the effect of IL-1 alpha on ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cells, Saos-2 and MG-63. The present study, for the first time, demonstrated that IL-1 alpha increases ADAMTS-2 and ADAMTS-3 gene expressions in both Saos-2 and MG-63 cells. Having correlation to mRNA induction, the upregulation of ADAMTS-2,-3 protein levels by IL-1 alpha stimulation is also observed. The inhibition studies showed that this upregulation occurred at the level of transcription, and there was no effect of IL-1 alpha on ADAMTS-2 mRNA half-life in Saos-2 cells. Transactivation potential of IL-1 alpha on ADAMTS-2 promoter was investigated by transient transfection assay. Specifically, IL-1 alpha strongly increased -658/+112 and -530/+112 ADAMTS-2 promoter constructs. Further, we analyzed signaling pathways involved in ADAMTS-2 induction. Pathway inhibition studies revealed that this upregulation depends on the activation of MEK, JNK and PI3K pathways. These findings suggested that IL-1 alpha is a strong positive regulator of ADAMTS-2 and ADAMTS-3 expression. These findings would provide novel insight into the pathophysiology of OA. (C) 2015 Published by Elsevier B.V.Öğe İnsan ADAMTS-3 gen promotorunun fonksiyonel analizi(TÜBİTAK, 2016) Alper, Meltem; Köçkar, FerayADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) ailesi memelilerde ve omurgasızlarda bulunan ekstraselüler proteinazlardır. ADAMTS’ler prokollajenin amino ucunun işlenmesi, matriks proteoglikanlarından agrekan, versikan ve brevikanın degredasyonu, anjiyogenezin inhibisyonu, kan pıhtılaşma homoeostazisinde önemli rollere sahiptir. ADAMTS-3 temel olarak eklem kıkırdağının spesifik bileşeni olan tip II kollajenin işlenmesini sağlar. Ayrıca osteoartirit, miyokard enfarktüs ve göğüs kanseri gibi bazı patolojik durumlarda da bağlantılı olduğu düşünülmektedir. ADAMTS-3’ün fonksiyonel özellikleri detaylı bir şekilde çalışılmış olmasına rağmen genin transkripsiyonel regülasyonunun nasıl olduğu bilinmemektedir. Bu proje ADAMTS-3 geninin transkripsiyonel regülasyonunu ve bu regülasyonda SP1, USF ve C/EBP? transkripsiyon faktörlerinin rolünü aydınlatmayı hedeflemektedir. Bu kapsamda, transkripsiyonel olarak en aktif bölgenin belirlenmesi amacıyla dört farklı uzunlukta ADAMTS-3 promotor parçası [-1340/+40] [-899/+40], [-576/+40], [-131/+40] lusiferaz vektörüne klonlandı. Saos-2 ve MG-63 hücrelerinde yapılan geçici transfeksiyon analizleri ile bütün parçaların aktif olduğu ve transkripsiyonel aktivite için en küçük promotor parçasının yeterli olduğu belirlendi. Her iki hücre hattında da 616 bç’lik promotor parçasının [-576/+40] en kuvvetli bazal aktiviteye sahip olduğu belirlendi. 1380 [1340/+40] ve 916 bç’lik [-899/+40] promotor parçalarının Saos-2 hücrelerindeki aktivitesinin MG-63 hücre hattına göre daha düşük olduğu tespit edildi. Biyoinformatik analizlere göre ADAMTS3 promotoru 40bç’lik 5’UTR bölgesi içermektedir ve TATA kutusu bulundurmamaktadır. Saos-2 ve MG-63 hücrelerinde yapılan ko-transfeksiyon deneyleriyle ADAMTS-3 promotor bölgesinde potansiyel bağlanma motifleri bulunan SP1, USF ve C/EBP? gibi bazı transkripsiyon faktörlerinin ADAMTS-3 promotor aktivitesini azaltıcı yönde etki gösterdiği belirlendi. Çalışmanın diğer bir kısmında SP1, USF ve C/EBP?’nın Saos-2 ve MG-63 hücrelerinde geçici aşırı ifadesi sağlanarak ADAMTS-3 ve kollajenlerin (tip I,II III) mRNA seviyelerine olan etkisi qRT-PCR, protein seviyesine olan etkisi de westernblot yöntemiyle belirlendi. Bahsedilen transkripsiyon faktörlerinin ADAMTS-3 mRNA ve protein düzeyini azalttığı belirlendi.Kollajen tiplerinin ekspresyonlarına olan etkisinin hücre tipine göre farklılık gösterdiği belirlendi. DNA-protein etkileşim çalışmalarında ise, SP1,C/EBP?,USF,AP1,E2F,NF?? ve STAT transkripsiyon faktörlerinin ADAMTS-3 promotor bölgesine fonksiyonel olarak bağlandığı EMSA deneyleri ile belirlendi. Ayrıca Saos, MG-63, Panc, HUVEC, PC-3 ve DU-145 hücre hatlarında ADAMTS-3 mRNA ekspresyon seviyeleri de RT-PCR ile belirlendi. Sonuç olarak bu çalışma ile insan ADAMTS-3 promotoru ilk kez klonlandı ve fonksiyonel olarak analizi yapıldı.SP1, USF ve C/EBP? transkripsiyon faktörlerinin ADAMTS3’ün ve kollajenlerin (tip I, II, III) transkripsiyonel regülasyonuna olan etkisi belirlenerek literatüre önemli katkılar sağlanmış oldu.Öğe Molecular characterization of zeta class glutathione S-transferases from Pinus brutia Ten.(Indian Academy Sciences, 2015) Öztetik, Elif; Köçkar, Feray Tura; Alper, Meltem; İscan, MesudeGlutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants. In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1.Öğe Molecular cloning and biochemical characterization of a Tau class glutathione S-transferase from Pinus brutia Ten(Springer, 2020) Alper, Meltem; Öztetik, Elif; Yaşar Kaya, Mihrap; Tura Köçkar, FerayKey message: A new Tau class GST gene was cloned from Pinus brutia Ten. cDNA sequence was analysed for conserved sequences. Substrate specificity, optimum pH, and temperature values of the recombinant PbGST Tau enzyme were determined. Abstract: Tau class glutathione S-transferases (GSTs) are essential enzymes for detoxification in plants. To date, a lot of the members of this family have been characterized from different plants but the studies on the conifers are very scarce. This study investigates for the first time molecular cloning and biochemical characterization of a Tau class GST gene (PbGST Tau) from Pinus brutia Ten. The full length PbGST Tau ORF was 687 bp having a molecular mass of 27.37 kDa. Catalytic and ligand binding sites of PbGST Tau are well conserved and shared maximum identity with Pinus tabulaeformis GST Tau. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ECA) as substrates exhibited a Km of 3.66 mM and 0.3 mM, respectively. PbGST Tau enzyme had an optimum activity at pH 6.0 and 8.0 when CDNB and ECA were used as substrate, respectively. The highest activity was measured at 25 °C. Through enzyme assays, phylogenetic analysis and structural modelling, we provide a detailed characterization of the PbGST Tau gene and the enzyme. This study is going to provide new insights into the phylogenetic and biochemical analysis of GST family in conifers.Öğe Molecular cloning and in silico analysis of human ADAMTS-3 (a disintegrin and metalloprotease with thrombospondin motifs) gene promoter(WILEY-BLACKWELL, 2014) Alper, Meltem; Aydemir, A.; Koçkar, Feray[Abstract Not Available]Öğe SP1-mediated downregulation of ADAMTS3 gene expression in osteosarcoma models(Elsevier Science Bv, 2018) Aydemir, A. Tuğşen; Alper, Meltem; Koçkar, FerayADAMTS3 is a member of procollagen N-proteinase subfamily of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family. It has an important function in the procollagen maturation process. The removal of N-peptidases is required for the accurate processing of fibrillar collagens. Otherwise, several disorders can occur that is related with the collagenous tissues. ADAMTS3 mainly maturates type II collagen molecule which is the main component of the bone and cartilage. There are several expression studies about ADAMTS3 gene however its transcriptional regulation has not been lightened up, yet. Here we first time cloned and functionally analyzed the promoter region of ADAMTS3 gene, approximately 1380 bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171 bp fragment is sufficient to activate gene expression in both Saos-2 and MG63 cells. In silico analysis showed that ADAMTS3 has a TATA-less promoter and contains several SP1/GC box binding motifs and a CpG island. Therefore we mainly investigated the SP1 dependent regulation of ADAMTS3 promoter. SP1 downregulated ADAMTS3 transcriptional activity. As consistent with the transcriptional activity, mRNA, and protein expression levels were also decreased by SP1. On the other hand, functional binding of the SP1 on multiple regions of ADAMTS3 promoter was confirmed by EMSA studies. As ADAMTS3 is responsible for the collagen maturation and biosynthesis, further we investigated the effect of SP1 on type I II and III collagen gene expressions. We point out that SP1 increased type II and III collagen expression and in contrast decreased type I collagen expression levels in Saos-2 cells. mRNA expression level was decreased for all collagen types in MG63 model. Decrease in the type II collagen expression was also demonstrated at the protein level by SP1. Collectively these results provide first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines.Öğe Transactivation of the human ADAMTS-2 gene promoter through proinflammatory cytokine TNF-alpha in osteoblast-like cells(Wiley-Blackwell, 2015) Alper, Meltem; Aydemir, T.; Koçkar, F....Öğe USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells(Pleiades Journals, 2021) Alper, Meltem; Aydemir, T.; Köçkar, F.Abstract: Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.
