Letm1 Gen Ekspresyonu Ile Jnk Aktivasyonu Arasındaki Ilişkinin Araştırılması

LETM1 (Leucine zipper/EF hand-containing transmembrane-1) proteini hücre içi iyon homeostazisi ve oksidatif fosforilasyon zinciri biyogenezinde rol oynayan bir mitokondriyal iç membran proteinidir. Proteinin fonksiyonuna yönelik çalışmalar daha ziyade Ca2+ ve K+ taşınmasına yönelik olmakla birlikte maya homoloğunda protein sentezi ile olan ilişkisi ele alınmaktadır. Bir üçüncü çalışma alanı olarak hücre ölümündeki rolünü sorgulayan çalışmalar ise sınırlı sayıdadır. Proje çalışmamız kapsamında LETM1 ekspresyonundaki artışın JNK fosforilasyonu üzerine etkisinin olup olmadığı ele alınmıştır. Bu çerçevede Lipofektamin ile HeLa hücrelerinin transfeksiyonunu takiben LETM1 ve JNK düzeyleri western blot yöntemi ile test edilmiştir. Ayrıca LETM1 ekspresyonundaki artışın hücre canlılığına etkisi MTS ile belirlenmiştir. Çalışmamızda Lipofektin aracılı transfeksiyon sonrası LETM1 ekspresyonunun arttığı ve bu hücrelerde JNK fosforilasyonunun değiştiği belirlenmiştir. Ayrıca hücre canlılığının da LETM1 ekspresyonundaki artışa paralel olarak azaldığı görülmüştür. LETM1 aşırı ekspresyonunun JNK yolağındaki rolü laboratuvarımızda halen devam etmekte olan bir araştırma konusudur. Bu amaçla yeni projeler ile LETM1 fonksiyonu hakkında daha fazla veri alınması planlanmaktadır.LETM1 (Leucine zipper/EF hand-containin transmembrane-1) is a mitochondrial inner membrane protein which plays role in intracellular ion homeastasis and biogenesis of oxidative phosphorilation. In general, most of the current studies focusing on the Ca and K transport function and its role in protein synthesis in its yeast homolouge of LETM1. There are limited number of studies questioning its role in cell death. In this study, effect of increased expression of LETM1 on phosphorilation of JNK studied. JNK and LETM1 levels were tested by western blotting after Lipofectamine transfection of HeLa cells. Also effect of increased LETM1 levels on cell viability was tested by MTS. According to our data, LETM1 levels were significantly increased and phoshorilation status of JNK was changed in transfected HeLa cells. Also, cell viability was significantly decreased in regard to increase in LETM1 levels. Effect LETM1 overexpression on JNK cascade is still under investigation in our laboratory. We are planning to extend our research with new funds to gain more data on the LETM1 function

Letm1 Ekspresyonuna Bağlı Jnk Fosforilasyonundaki Değişimde Perk' In Rolünün Araştırılması

LETM1 (Leucine zipper/EF hand-containing transmembrane-1) proteini hücre içi iyon homeostazisi ve oksidatif fosforilasyon zinciri biyogenezinde rol oynayan bir mitokondriyal iç membran proteinidir. Proteinin fonksiyonuna yönelik çalışmalar daha ziyade Ca2+ ve K+ taşınmasına yönelik olmakla birlikte maya homoloğunda protein sentezi ile olan ilişkisi ele alınmaktadır. Bir üçüncü çalışma alanı olarak hücre ölümündeki rolünü sorgulayan çalışmalar ise sınırlı sayıdadır. Proje çalışmamız kapsamında geçmiş projemizde belirlemiş olduğumuz JNK aktivasyonun da endoplazmik retikulum stresinde rol oynayan PERK' in rolü ele alınmış ayrıca c-jun fosforilesyonu incelenmiştir. Bu çerçevede Lipofektamin ile HeLa hücrelerinin transfeksiyonunu takiben LETM1 ve c-jun ve PERK düzeyleri western blot yöntemi ile test edilmiştir. Çalışmamızda Lipofektin aracılı transfeksiyon sonrası LETM1 ekspresyonunun arttığı ve bu hücrelerde c-jun fosforilasyonu belirlenmiştir. Ancak PERK proteininin varlığı gösterildiği halde bu proteinin fosforilasyonu görülmemiştir. LETM1 ekspresyonundaki değişimlerin hücresel etkilerinin incelenmesine yönelik çalışmalarımız TÜBİTAK destekli projemiz kapsamında devam etmektedir. Bu çerçevede gerek hücre içi sinyal iletim yollarının belirlenmesi gerekse mitokondriyal fonksiyon üzerine etkilerin çalışılması planlanmaktadır.LETM1 (Leucine zipper/EF hand-containin transmembrane-1) is a mitochondrial inner membrane protein which plays role in intracellular ion homeastasis and biogenesis of oxidative phosphorilation. In general, most of the current studies focusing on the Ca and K transport function and its role in protein synthesis in its yeast homolouge of LETM1. There are limited number of studies questioning its role in cell death. In this study, effect of increased expression of LETM1 on phosphorilation of PERK and c-jun in the light of the data obtained from our previous study. LETM1, c-jun and PERK levels were tested by western blotting after Lipofectamine transfection of HeLa cells. According to our data, LETM1 levels were significantly increased and phosphorilated c-jun was detected in transfected HeLa cells. On the other hand PERK phosphorilation did not observed. Cellular effects of LETM1 expression are still under investigation in our laboratory with the project granted by TUBITAK. In this regard, we are planning to determine intracellular signal cascades as well as mitochondrial function in response to LETM1 expression changes

Geology of the Paleotetis units at the northern part of Edremit Bay

İnceleme alanı Kuzeybatı Anadolu’da Biga Yarımadası’nın güneyinde, Edremit Körfezi ve kuzeyinde yer alan Kazdağ ve çevresini kapsar. Kazdağ Grubu, amfbolit-granulit fasiyesinde metamorfik bir istiftir.  Kazdağ Grubu üzerinde, bir sıyrılma fayı dokanağı ile yeşil şist fasiyesinde metamorfik birimlerden oluşan Karakaya Karmaşığı bulunur. Kazdağ Grubu, okyanus kabuğu, üzerinde gelişen okyanus platosu çökel ve volkanikleri, Karakaya Karmaşığı, riftt çökelleri, denizaltı-dağı, denizaltı platosu, hendek çökelleri, dalma-batma gerisi havza çökelleri temsil eden bir eklenir prizmadır. Kazdağ Grubu ve Karakaya Karmaşığı Paleotetis Okyanusu’nun Permo-Karbonifer’de oluşumu ve Triyas’ta kapanmasının hemen hemen tüm aşamalarını temsil eder. Anahtar Kelimeler: Paleotetis, Kazdağ Grubu, Karakaya Karmaşığı, jeodinamik evrim.Study area is located to the south of the Biga Peninsula, NW Anatolia. It includes the Edremit Bay and the Kazdağ Group to the north. The rock groups represent a geological period starting from the Carboniferous to present. On the basement a metamorphic series in amphibolite-granulite facies take place. These series form the Kazdağ Group itself and are made up of the Babadağ Formation, Sarıkız Formation, Kavurmacılar Formation and Altınoluk Formation. A detachment fault and the metamorphic Karakaya complex (green schist facies) take place on the Kazdağ Group. To the east, the Karakaya Complex starts with the Fazlıca, Kınar and Kalabak units, which contain shale, schist, fillate, basalt and marble, on a Palaeozoic granodiorite basement. These units are overlain by the units of Nilüfer, which is made up of tectonically thrusted spilits, and Tepeoba, which is made up of felsic fillate and tuffs, respectively. The unit Hodul passes laterally into the unit Nilüfer and it is made up of arkozic sandstone, rare spilit and chert alternations. On top of these formations, the unit Çal is located with a tectonic contact and it contains Permian-Trias limestone blocks in a size of a mountain. The study area forms the pieces of Palaeotethys ocean dominated between Carboniferous and Triassic. The rocks of the Kazdağ Group form the Laurassia part of the ocean crust while the Karakaya Complex represents the southern environments of the south-dipping oceanic crust. These environments include the sea-mount (Nilüfer unit), accretional prism (Hodul unit), marginal basin (Tepeoba unit) and passive Cimmeria margin of this basin (Fazlıca+Kınar+Kalabak). The Laurassia and Sakarya continents collided during Middle-late Triassic and the units between these continents formed the Karakaya Complex in the form of tectonic slices.Keywords: Paleotethys, Kazdağ Group, Karakaya Complex, geodynamic evolution

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients. Genet Mol Biol

Abstract Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named "common deletion", has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named “common deletion”, has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer

A novel approach for rapid screening of mitochondrial D310 polymorphism

BACKGROUND: Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. METHODS: 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. RESULTS: Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. CONCLUSION: In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

DNA’nın aljinatla kuvvetlendirilmiş kitozan boncuklar içinde enkapsülasyon ve serbestleşme özelliklerinin incelenmesi

DNA' nın Aljinatla Kuvvetlendirilmiş Kitozan Boncuklar İçinde Enkapsülasyon ve Serbestleşme Özelliklerinin İncelenmesi Viral olmayan gen taşıyıcı sistemler son yıllarda çok yaygın olarak araştırılmaktadır. Diğer taraftan kitozan bir taşıyıcı olarak ilgi çekmektedir. Bu çalışmada gen taşıyıcı olarak kitozan boncuklar, iki farklı DNA tipi, pMK3 plazmid DNA ve salmon testis DNA kullanılarak hazırlanmıştır. pMK3 plazmid DNA ile, iyonotropik jelleşme yöntemi uygulanarak 680-760 mm boyutunda, sürekli DNA serbestleştiren boncuk hazırlanmıştır. Aljinatla kuvvetlendirilmiş kitozan boncuklarda, DNA' nın hazırlama sırasında ve serbestleşme deneyleri süresince stabil kaldığı ve yapısal özelliklerini muhafaza ettiği görülmüştür. Denenen formulasyon faktörlerinden; DNA konsantrasyonu ve formunun boncuk özelliklerinde önemli olduğu saptanmıştır. Boncuk büyüklüğü, DNA miktarına bağlı olarak artmıştır. Doğrusal DNA ile yapılan çalışmada, DNA' nın boncukta tutulma oranı, her üç formu içeren plazmid DNA' ya kıyasla daha düşüktür. Diğer taraftan, plazmid DNA formu ve konsantrasyonu, boncuklardan serbestleşme üzerine etkili değildir. Benzer çalışmalar salmon testis DNA ile yürütülmüştür. Yine iyonotropik jelleşme yöntemi ile, 673 mm büyüklüğünde muntazam şekilli boncuklar elde edilmiştir. Ancak serbestleşme çaışmasında; DNA' nın boncuk içerisinde immobilize halde kaldığı, serbestleşmediği görülmüştür. Salmon testis DNA ile kitozan mikroküre hazırlanması incelenmiş ve 1.34 mm boyutunda kitozan mikroküreler hazırlanmıştır. Ancak bu tip yapıda da DNA' nın immobilize halde kaldığı saptanmıştır. Burada, DNA ve kitozan arasında kompleks oluşabilirliği düşünülerek, bu konu üzerine yoğunlaşılmıştır. Uyguladığımız yöntemlerde DNA' nın yapısının değişmediği görülmüştür. DNA' nın kitozanla oluşturduğu komplekste 1:0.3 oranının önemli olduğu, bunun üzerindeki oranlarda, DNA' nın tümüyle kitozana bağlandığı ve serbest DNA kalmadığı görülmüştür. Boncuklara ilişkin çalışmaların ikinci bölümünde plazmid DNA yüklü kitozan boncukların in vivo transfeksiyon özelliği incelenmiştir. Fare bacak kasına enjekte edilen örneklerde, 180 gün sonra çıplak DNA'ya kıyasla yaklaşık 2 kat yüksek gen ekspresyonu saptanmıştır. Sonuç olarak yapılan bu ön çalışmada, DNA tipine bağlı olarak farklı amaçlara yönelik sistem geliştirilebileceği, plazmid DNA' nın kitozanda enkapsülasyonu ile in vivo gen ekspresyonu elde edilebileceği, salmon testis DNA enkapsülasyonunda ise immobilize bir sisten oluşturulabileceği konusunda olumlu sonuçlar elde edilmiştir. Investigation of Encapsulation and Release Properties of DNA Encapsulated in Alginate Reinforced Chitosan Beads. Non-viral gene delivery vectors is currently a very important area of research. On the other hand, chitosan have received considerable attentions as a carrier. In this study, chitosan beads were succesfully prepared as a gene delivery carrier by using two different type of DNA, namely pMK3 plasmid DNA and salmon testis DNA. pMK3 -loaded chitosan beads were prepared by ionotropic gelation method and the 680-760 mm bead size were obtained. In alginate treated chitosan beads, DNA remained stable and DNA integrity did not changed during the encapsulation and release processes. The form and the concentration of the DNA is considerably important on bead properties. Bead size incerased as the DNA amount increase. In linear DNA loaded chitosan beads, loading capacity was lower than supercoiled DNA. On the other hand, form and concentration of DNA did not affect the release from beads. Salmon testis DNA was studied as well as pMK3 plasmid DNA. Spherical and good shaped chitosan beads with the 673 mm bead size were obtained by using ionotropic gelation method. In release studies, DNA did not release from beads and immobilized. Salmon testis DNA loaded chitosan microspheres were investigated and the 1.34 mm microsphere size were obtained. Salmon testis DNA was also immobilized in this structure. From this point of research, we focused on chitosan-DNA complexes. Integrity of the DNA did not change from experimental procedures. In chitosan-DNA complexes, the ratio of DNA:chitosan was important and there was no free DNA when this ratio above 1:0.3 and the DNA was completely bind to chitosan. In the second part of the study, in vivo transfection properties of plasmid DNA loaded chitosan beads were investigated. Chitosan beads were injected into the leg muscle of mice and two fold higher expression was obtained with the beads than the naked DNA. As a result, in this preliminary study, we obtained two different promising systems by using two different DNAs, first, by encapsulating the plasmid DNA we had a controlled release system which have in vivo transfection activity, and the second, by encapsulating salmon testis DNA, we obtained an immobilized system