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Öğe Conserved amino acid residues that affect structural stability of candida boidinii formate dehydrogenase(Springer, 2021) Bulut, Huri; Yüksel, Büşra; Gül, Mehmet; Eren, Meryem; Karataş, Ersin; Kara, Nazlı; Yılmazer, Berin; Koçyiğit, Abdurrahim; Labrou, Nikolaos E.; Bi?nay, BarışThe NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme’s thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.Öğe Cuscuta campestris tedavisi ile mide kanseri hücrelerinde apoptozun indüklenmesi ve reaktif oksijen türlerinin oluşumu yoluyla proliferasyonun engellenmesi(DergiPark, 2021) Bulut, Huri; Durmuş, Ezgi; Hacıosmanoğlu, Ebru; Bozalı, Kübra; Şentürk, Hilal; Koçyiğit, AbdurrahimAmaç: Kanser tahrip edici, ölümcül bir hastalıktır ve dünya çapındaki ölüm nedenleri arasında ikinci sırada yer almaktadır. Mide kanseri gibi kanser türleri için farklı tedavi yöntemleri mevcut olsa da çoğu tedavi yöntemleri birçok yan etkiye sahiptir. Çeşitli iyileştirici özelliklere sahip Cuscuta campestris' in kanser hücreleri üzerindeki etkilerinin araştırılması yeni bir alandır ve etkileri tam olarak çalışılmamış ve aydınlatılmamıştır. Bu çalışmada, C. campestris özütünün farklı konsantrasyonlarının mide kanseri hücreleri üzerindeki sitotoksik, genotoksik, apoptotik ve reaktif oksijen türlerinin üretimindeki etkilerini araştırdık. Gereç ve yöntem: Araştırmamızda, C. campestris için antioksidan, pro-oksidan ve radikal temizleyici aktiviteleri değerlendirildi ve miktar tayini LC-MS / MS yöntemi ile analiz edildi. C. Campestris’ in normal hücrelere kıyasla mide kanseri (AGC) hücreleri üzerindeki seçiciliğini göstermek için insan normal deri fibroblastik (CCD-1079Sk) hücre hattı kullanıldı. Apoptoz belirteçlerinin tayininde akridin oranj / etidyum bromür çift boyama, akış sitometrisi ve western blot metotlarından faydalanıldı. Genotoksik aktivite tayini, Comet analizi ile gerçekleştirildi. Bulgular: Sonuçlarımız, C. Campestris etanolik özütünün doza bağlı bir şekilde, mide kanseri hücre hattı üzerinde normal hücrelere kıyasla önemli ölçüde daha yüksek sitotoksik etkilere sahip olduğunu göstermiştir. Ayrıca, elde ettiğimiz veriler C. campestris'in mide kanseri hücre hatlarında reaktif oksijen türlerinin oluşumunu arttırdığını ve DNA hasarına neden olduğunu açıkça göstermiştir. Yapılan apoptoz tayin ölçümleri C. campestris' in bölünmüş kaspaz-3, bölünmüş kaspaz-9, bölünmüş PARP ve P-53'ü aktive etmesiyle kanser hücreleri üzerinde apoptotik bir etkiye sahip olduğunu da doğrulamıştır. Sonuç: Bu çalışmanın sonucu olarak, C. Campestris’in mide karsinomu tedavisinde umut vadeden bir antikanser ajanı olabileceği düşünülmektedir.Öğe Induction of Apoptosis through Oxidative Stress Caused by Rubus tereticaulis Leaves Extracts in A549 Cells(Istanbul University Press, 2024) Öter, Gamze Nur; Durmuş, Ezgi; Şen, Ali; Koçyiğit, AbdurrahimObjective:Plants have been used for medicinal purposes since the beginning of human history and form the basis of modern medicine, and they are also the source of most chemotherapeutic drugs for cancer treatment. This study aims to investigate for the first time the cytotoxic and apoptotic effects of the active ethanol (RTE) and chloroform (RTC) extracts of Rubus tereticaulis leaves in the A549 non-small-cell lung cancer cell line. Materials and Methods:A549 cells were treated with RTE and RTC individually. The MTT assay was used to quantitatively detect RTE and RTC's cytotoxic effects. The fluorescent signal indicator H2DCF-DA was used to detect cellular reactive oxygen species (ROS) production. Apoptosis was evaluated by fluorescence microscope after acridine orange/ethidium bromide fluorescent staining, annexin V-FITC and immunoblotting analyses, immunofluorescence, and imaging. Results:Both RTE and RTC induced cytotoxicity in A549 cells in a dose-dependently, which was accompanied with induced ROS accumulation. Both early and late apoptotic cells detected by flow cytometry were increased in the RTE- and RTC-treated cells. In addition, the results show RTC to have higher cytotoxic and apoptotic effects and increased ROS-generation capacity than RTE. Therefore, the polarity of the solvent used to exert the anticancer effect of R. tereticaulis leaves is crucial. Conclusion:This is the first anti-cancer activity study on R. tereticaulis. The results suggest R. tereticaulis leaves to have an anti-cancer effect on lung cancer cells through ROS-mediated apoptosis and RTC to be an effective therapeutic/adjuvant strategy in cancer treatment. © 2024 Istanbul University Press. All rights reserved.Öğe Investigation of diagnostic proteins by 2D electrophoresis in major de-pression model induced by forced swim test in rats(2021) Duman, Tuğçe; Akyüz, Enes; Bulut, Huri; Koçyiğit, Abdurrahim; Tülek, Ahmet; Karataş, Ersin; Kırpınar, İsmetBackground: Aside from its pervasiveness, whereby it affects as much as 20% of the world's population, depression continues to be one of the most crucial psychiatric problems due to the loss of power it causes by disrupting daily life functioning, containing economic consequences, and having a high suicidal tendency. Major depression (MD) is a systemic and multifactorial disorder involving complex interactions between genetic predisposition and disturbances of various molecular pathways. Objectives: In our current study, we aimed to identify the proteins obtained from serum samples that change during depression with the MD model. Methods: The MD model was applied through the forced swim test in rats. 14 Winstar Albino male rats were divided into two equal groupsas follows: depression and control groups. Serum samples were separated by chromatographic methods and then compared with two-dimensional (2D) electrophoresis. Results: A total of 9 potential diagnostic protein sequences were identified, which were distinguished with computer soft-ware. During the last phase of the study,the Matrix-Assisted Laser Desorption/Ionization -Time of Flight (MALDI-TOF) analysis, the previous expression sequences identified among the groups were determined and classified. By comparing protein expressions, it was concluded that 9 different points could be used together as a potential biomarker. Conclusion: Results can help us identify a new diagnostic system that can be used to diagnose MD.Öğe Preparation and evaluation of inflammation targeted nano-micellar formulation of celecoxib(Bezmialem Vakif Univ, 2019) Bahadori, Fatemeh; Büyük, Ayşe Şeyma; Kozanoğlu, Ahmed Serdar; Eskandari, Zahra; Ankaralı, Handan; Kepekçi Tekkeli, Şerife Evrim; Koçyiğit, AbdurrahimObjective: Celecoxib (CLX), brand named Celebrex, which belongs to non-steroidal anti-inflammatory drugs family, selectively inhibits the cytokine related cyclooxygenase-2 isoenzyme and thus, possesses less gastrointestinal side effects, have shown to cause stroke, myocard infarction and even death in some cases. In this study we aimed to target inflammation site using CLX uploaded nano-micelles (nano-CLX) made of poly (lactic-co-glycolic) acid (PLGA) to protect other tissues from its side effects. Methods: CLX was physically entrapped in PLGA micelles using w/o/w emulsion method, resulted in obtaining mono-dispersed particles with 112 nm size. 50 mg PLGA was able to carry 50 mg CLX in 20 mL (2.5 mg/mL) with encapsulation efficiency of 85%. Rheumatoid arthritis model was achieved by injection of complete Freund's Adjuvant to the hint paw of Wistar rats. Infected groups received oral Celebrex, intravenous (i.v.) Celebrex and nano-CLX. Each group was compared with a healthy control group receiving the drug via the same routes. The obtained serums and the hint paw sizes were studied for 6 hours in 3 time periods. Results: Prostaglandin E2 and tumor necrosis factor-a levels were found to be decreased for longer time period by application of nano-CLX compared to oral and i.v. Celebrex. Interleukin-1 (IL-1) and IL-6 levels showed a dramatic decrease at orally administered Celebrex groups, showing the accumulation of these pro-inflammatory Factors at inflammation area. Conclusion: Based on the hypothesis that the ratio of blood parameters is inversely proportional to accumulation at inflammation site, thus, our nano-formulation is targeted to the tissues in the systemic blood flow and have a better selective inhibition.
