Investigation of structural and functional properties of the betaine transporter BetP from Corynebacterium glutamicum by using infrared spectroscopy

In this study, the structural and functional properties of the Na+/Betaine symporter BetP were investigated upon K+-induced activation. BetP regulates transport activity dependent on the amount of associated anionic lipids and the cytoplasmic K+-concentration. For this purpose, FTIR spectroscopy was implemented as a non-perturbing biophysical method which shed light on how the membrane lipids contribute to the molecular mechanisms of activation and regulatory response of BetP.Im Rahmen dieser Arbeit wurden die strukturellen und funktionellen Eigenschaften des Na+/Betain Symporters BetP auf die K+-induzierte Aktivierung hin untersucht. BetP reguliert die Transportaktivität in Abhängigkeit von der Menge an assoziierten anionischen Lipiden und der zytoplasmatischen K+-Konzentration. Für diesen Zweck wurde die FTIR Spektroskopie als eine zerstörungsfreie biophysikalische Methode verwendet um herauszufinden, wie die Membranlipide zum molekularen Mechanismus der Aktivierung und Regulation beitragen

Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells

Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated

Determination of Cellular Differences of Cd133+/Cd44+ Prostate Cancer Stem Cells in Two-Dimensional and Three-Dimensional Media By Fourier Transformation Infrared Spectroscopy

Amaç: Sferoid kültürleri, hücrelerin kendi iç dinamikleri ve diğer hücrelerle olan etkileşimleri açısından tek tabakalı kültürlere kıyasla tümör dokusunun özelliklerini daha iyi yansıtmaktadır. Bu çalışmanın amacı, üç boyutlu (3D) ve iki boyutlu (2D) kültür ortamlarında üretilen CD133+/CD44+ prostat kanser kök hücrelerinin (KKH) makromoleküllerindeki benzerlik ve farklılıklarının araştırılmasıdır. Gereç ve Yöntem: DU-145 prostat kanser hücre hattı içerisindeki CD133+/CD44+ yüzey belirteç özelliklerine sahip KKH’leri akış sitometrisi (FACS) kullanılarak izole edilmiştir. Agarla kaplı kültür kapları ile sferoid yapıları oluşturulmuştur. 2D ve 3D kültür ortamlarındaki KHK hücreleri Fourier dönüşümü kızılötesi (FTIR) spektroskopisi ile karşılaştırılmıştır. Bulgular: CD133+/CD44+ hücrelerin birinci haftada agarlı kültür ortamlarında mikro-agregatlar oluşturduğu gözlenmiştir. İkinci haftada ise, olgun sferoid yapıların oluştuğu saptanmıştır. 2D ve 3D (multisellüler tümör sferoidleri) kültür ortamlarında üretilen hücreler ile yapılan FTIR analizleri KHK hücre yapısındaki proteinler, lipitler, karbonhidratlar ve nükleik asitlerde (DNA, RNA) önemli derecede farklanmalar olduğunu göstermiştir. Membran lipit açil zincir uzunluğu ve hücre zarı kalınlığı, proteinlerin sekonder yapıları ve DNA oligonükleotitlerin baz sekanslarında veya fonksiyonel gruplarında önemli farklanmaların olduğu tespit edilmiştir. Elde edilen sonuçlar, 3D kültür ortamında üretilen sfreoid yapılarının in vivo tümör dokusu ile benzer özellikler sergilediğini göstermiştir. Sonuç: Hücre ortam koşulları ile yaratılmış olan fiziksel, kimyasal ve biyolojik özellikler hücrelerin kendi iç dinamiğini ve mikroçevresi içerisindeki etkileşimlerini önemli derecede etkilemektedir. 2D kültür ortamları ile hücreleri tek boyutta indirgemek hücrelerin gerçek özelliklerini yansıtmamaktadır. Bu nedenle, 3D kültür ortamları ile hücre dinamiklerinin incelenmesi gerekmektedir. Bu çalışmadan elde edilen sonuçlar, kanserde önemli bir hücre popülasyonunu oluşturan KKH’lerin membran yapısı, lipitler, proteinlerin sekonder yapıları ve DNA oligonükleotit yapılarının terapötik hedef olabileceğini göstermektedir.Objective: Spheroid cultures reflect properties of tumor tissue better than monolayer cultures in terms of internal dynamics and interaction with other cells. the aim of this study is to investigate the similarities and differences in CD133+/CD44+ prostate cancer stem cells (CSCs) produced in threedimensional (3D) and two-dimensional (2D) culture media. Material and Method: CSCs with CD133+/CD44+ surface marker properties in the DU-145 prostate cancer cell lines were isolated using flow cytometry (FACS). Spheroid structures were formed with agar-coated culture vessels. CSCs in 2D and 3D cultures were compared with Fourier transform infrared (FTIR) spectroscopy. Results: CD133+/CD44+ cells were observed to form micro-aggregates in cultured media in the first week. in the second week, mature spheroid structures were formed. FTIR analysis revealed that the 2D and 3D (multicellular tumor spheroids) models of CSCs exhibit significant differences in proteins, lipids and nucleic acids. Significant differences were detected in membrane lipid acyl chain length, membrane thickness, protein secondary structures and DNA oligonucleotides. the results showed that spheroids in 3D culture medium exhibit similar properties to in vivo tumor tissue. Conclusion: Physical, chemical and biological properties generated by environmental conditions significantly affect internal dynamics of cells and their interactions within the microenvironment. Reducing the cells in one dimension through the 2D culture medium does not reflect actual properties of cells. Therefore, cell dynamics in 3D culture media should be investigated. This study demonstrates that cellular lipids, membrane structure, protein secondary structures and nucleic acids of CSCs constituting an important cell population in cancer may be therapeutic targets

Meme Kanseri Kök Hücrelerinde Flavopiridolün Hücre Döngüsü, Apoptozis ve Biyomolekül Yapı Değişimleri Üzerine Etkisi

Objective: Cancer stem cells (CSCs) are a small population in cancer, which are responsible for therapeutic resistance, relapse and metastasis. Flavopiridol has antitumor activity against various types of cancer cells. the mechanism of action of flavopiridol on CD44+/ CD24- breast CSCs has not yet been fully elucidated. the aim of this study was to evaluate the mechanism of action of flavopiridol on breast CSCs (BCSC) in terms of apoptosis, cell cycle and biomolecular changes. Methods: in human breast cancer, cells with CD44+/CD24? markers were isolated from MCF-7 cell line using flow cytometry. the induction of apoptosis was investigated by Annexin-V. the effect of flavopiridol on cell cycle arrest was determined and the percent of cell populations at G0/G1, S and G2/M cycles were identified. the effect of the drug on three-dimensional cell cultures was investigated using a multicellular tumor spheroid model. in addition, the effect of flavopiridol on biomolecules has been evaluated using Fourier transform infrared (FTIR) spectroscopy, which has recently been used effectively in various scientific fields. Results: Flavopiridol especially induced early apoptosis. Cell cycle analyses revealed that flavopiridol induced cell cycle arrest in G0/G1 phase. Decreased number and diameter of spheroids was observed following flavopiridol treatment. ATR-FTIR data showed that treatment with flavopiridol led to significant changes in nucleic acids. Conclusion: According to the data obtained in this study, flavopiridol exhibits anticancer effects by altering the structure/ expression level of nucleic acids and changing cell cycle progression and inducing apoptosis. These finding reveals that flavopiridol can be an effective antitumor agent for the treatment of breast cancer after in vivo and phase studies are completed.Amaç: Kanser kök hücreleri (KKH) terapötik direnç, relaps ve metastazdan sorumlu olan oldukça küçük bir hücre popülasyondan oluşmaktadır. Flavopiridol (Alvocidib), çeşitli KKH’lerine karşı anti-tümör aktiviteye sahiptir. Flavopiridolün CD44+/CD24- meme KKH (MKKH) üzerindeki etki mekanizması henüz tam olarak aydınlatılamamıştır. Bu çalışmada, apoptozis, hücre döngüsü ve biyomoleküler değişiklikler dahil olmak üzere flavopiridolün MKKH üzerindeki etki mekanizmasının çeşitli şekillerde değerlendirilmesi amaçlanmıştır. Yöntemler: MCF-7 KKH hattı içerisindeki CD44+/CD24- yüzey belirteç özelliklerine sahip MKKH, akış sitometrisi kullanılarak izole edilmiştir. Apoptozis indüksiyonu Annexin-V yöntemi ile incelenmiştir. Flavopiridolün hücre döngüsü tutulumu üzerine etkisi Muse Cell Analyzer ile belirlenmiş ve G0/G1, S ve G2/M döngüsündeki hücre populasyonları yüzde olarak ifade edilmiştir. İlacın üç boyutlu hücre kültürlerindeki etkisi multiselüler tümör sferoid modeli kullanılarak incelenmiştir. Buna ek olarak, flavopiridolün biyomoleküller üzerindeki etkisi, son zamanlarda çeşitli alanlarda oldukça etkin bir şekilde kullanılan Fourier dönüşüm kızılötesi (FTIR) spektroskopisi kullanılarak değerlendirilmiştir. Bulgular: Flavopiridol spesifik olarak erken apoptozu indüklemiştir. Hücre döngü analizleri, flavopiridolün G0/G1 arrestine yol açtığını ortaya koymuştur. Flavopiridol uygulamasından sonra sferoid sayısı ve çapında azalma tespit edilmiştir. ATR-FTIR verileri, flavopiridol uygulamasının hücre içindeki nükleik asitlerde önemli değişimlere yol açtığını göstermiştir. Sonuç: Elde edilen bulgular, flavopiridolün nükleik asitlerin yapısını/expresyon seviyesini ve hücre döngüsünü değiştirdiğini ve apoptozu indükleyerek anti-kanser etkiler sergilediğini göstermektedir. Bu veriler, in vivo ve faz çalışmaları tamamlandıktan sonra flavopiridolün meme kanser tedavisi için etkili bir terapötik molekül olabileceğini ortaya koymaktadır

IR spektroskopi kullanılarak in vitro meme kanser kök hücrelerinin araştırılması

Aim: Cancer stem cells (CSCs) lead to tumor initiation, progression, relapse, metastasis and therapeutic resistance due to the ability of tumor to self-renewal and differentiate into other cell types. Therefore, the characteristic features of breast CSCs need to be determined in detail. The aim of this study was to investigate the differences in cell biochemistry at the molecular level by using Fourier transform infrared (FTIR) spectroscopy after breast CSCs were isolated with flow cytometry. Materials and Methods: Breast CSCs with CD44+/CD24- surface marker properties in the MCF-7 cancer cell lines were isolated by using flow cytometry (FACS). MCF10A, MCF-7 breast cancer cell line (cancer non-stem cells or non-CSCs) and breast CSCs were re-suspended into 0.9% NaCl, and each cell type was measured with the FTIR spectrometer. Results: The portion of breast CSCs with CD44+/CD24- surface marker properties in MCF-7 was 2.0- 2.3%. In the FTIR spectra, spectral similarities and differences among breast CSCs, non-CSCs and healthy cells were determined. In breast CSCs, the lipid and protein signals are quite strong accompanied with an increased cell membrane fluidity and dynamics. When non-CSCs are compared with healthy cells, a less amount of both ?-helical proteins and DNA is detected while an increase in the signals of negatively charged carboxyl groups is noticed. These data clearly show that breast CSCs exhibit a very different profile in terms of structure, content and dynamics of cellular macromolecules compared to both non-CSCs and healthy cells. Conclusion: Drug studies (targeted therapy, drug-action mechanism) can be performed by examining small changes in the molecular structure and content of breast CSCs. This study shows that FTIR spectroscopy can be used in advanced cell studies as well as in the analysis of biological samples in medical field due to rapid, label-free and accurate measurement without complex sample preparation procedures.Amaç: Kanser kök hücreleri (KKH), tümör içinde kendi kendilerini yenileme ve diğer hücre tiplerine farklılaşabilme kapasitesi sebebiyle tümörün başlaması, ilerlemesi, nüksetmesi, metastaz ve terapötik dirence yol açmaktadır. Bu nedenle, meme kanser kök hücrelerinin (MKKH) karakteristik özelliklerinin belirlenmesi gerekmektedir. Bu çalışmanın amacı, MKKH?lerin akış sitometrisi ile izole edildikten sonra Fourier dönüşümlü kızılötesi (FTIR) spektroskopisi kullanarak hücre biyokimyasındaki farklılaşmalarının moleküler seviyede araştırılmasıdır. Gereç ve Yöntem: MCF-7 meme kanser hücre hattındaki CD44+/CD24- yüzey belirteç özelliği gösteren MKKH?ler akış sitometrisi ile izole edilmiştir. MCF10A, MCF-7 kanser hücre (KH) hattı ve bu hattan izole edilen CD44+/CD24- yüzey belirteç özelliklerine sahip MKKH?'ler %0,9 NaCI içerisine resuspanse edildikten sonra FTIR spektrometre ile ölçülmüştür. Bulgular: MCF-7 içerisindeki CD44+/CD24- yüzey belirteç özelliğine sahip KKH?lerinin sort oranı %2,0-2,3 olarak belirlenmiştir. Elde edilen FTIR spektrumlarında, MKKH, meme kanser hücreleri (KH, non-KKH, bulk populasyon) ve sağlıklı hücreler arasında spektral benzerlikler ve farklılıklar tespit edilmiştir. MKKH?lerde lipit ve protein sinyalleri daha güçlü olup hücre zarı akışkanlığı ve dinamiği fazladır. Sağlıklı hücreler ile kıyaslandığında, KH?lerde ?-helikal proteinler ve DNA sinyallerinde azalmaya karşın negatif yüklü karboksil gruplarından kaynaklanan sinyallerde artış gözlenmektedir. Bu veriler, MKKH?lerin, sağlıklı ve KH?lere kıyasla yapı, içerik ve dinamiği bakımından oldukça farklı bir profil sergilediğini göstermektedir. Sonuç: Bu çalışma, MKKH?lerinin moleküler yapısı ve içeriğindeki değişikliklerin incelemesi vasıtasıyla terapötik hedefli ilaç çalışmaları yapılabileceğini ortaya koymaktadır. FTIR spektroskopisi boyar madde gerektirmeden, hassas ve hızlı ölçüm alınması, örnek hazırlamada kolaylık ve az miktarda örnek gerektirmesi sebebiyle ileri hücre çalışmalarında ve medikal alanda biyolojik örneklerin analizlerinde kullanılabileceği de gösterilmiştir

Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy

The Na+-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K+ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K+ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions

Specific binding of D-Amino neuraminic acid to ganglioside studied in prostate cancer cells

Aim: The aim of this study was to investigate the cellular binding site of human D-Amino Neuraminic Acid (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). The KDN molecule is a member of the sialic acid family, and its expression increases in cancer cells. KDN has been shown to bind to Monosialodihexosyl Ganglioside (GM3) in trout sperm. Materials and Methods: In this study, a prostate cancer cell line (DU145) was used. Each experimental group was divided into 3 groups: Control, Glucosylceramide synthase (GCS) enzyme inhibitor Genz-123346-treated, and GM3 synthesis inhibitor Triptolide-treated. Each group was stained using the immunocytochemical method for GM3, Disialosyllactosylceramide (GD3) and KDN. Fourier Transform Infrared (FTIR) Spectroscopy analysis was performed to elucidate the cellular changes after treatment. Results: The group of non-treated number 1 cells stained positive with GM3, GD3 and KDN, and the GCS enzyme was blocked with the Genz-123346 group of number 2 cells stained positive only with KDN. Furthermore, the group of GD3 synthase inhibitor Triptolide treated number 3 cells stained positive with GM3 and KDN. FTIR measurements showed apoptotic characteristics with Triptolide, while Genz-123346 did not have a negative effect on cell viability. There was a reduction in sugar structures and the results obtained with immunocytochemical staining were reinforced with FTIR. Conclusions: Determining the location of the bound KDN is important for the selection of new targets for cancer treatment research. KDN has been shown to be not inhibited by GM3 inhibition and GD3 inhibition. KDN can be on GM3 as well as connected to different places or can be free. In this study, it was demonstrated that it would not bind to any of the gangliosides in the pure GM or GD series

Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy

Cellular macromolecules play important roles in cellular behaviors and biological processes. In the current work, cancer (KLN205), normal (MSFs) and mouse embryonic stem cells (mESCs) are compared using ATR-FTIR spectroscopy. Modifications in the composition, concentration, structure and function-related changes in the cellular components were deciphered using the infrared spectra. Our results revealed that cancer and embryonic stem cells are very similar but highly different from the normal cells based on the spectral variations in the protein, lipid, carbohydrate and nucleic acid components. The longest lipid acyl chains exist in mESCs, while cancer cells harbor the lowest lipid amount, short lipid acyl chains, a high content of branched fatty acids and thin cell membranes. The highest cellular growth rate and accelerated cell divisions were observed in the cancer cells. However, the normal cells harbor low nucleic acid and glycogen amounts but have a higher lipid composition. Any defect in the signaling pathways and/or biosynthesis of these cellular parameters during the embryonic-to-somatic cell transition may lead to physiological and molecular events that promote cancer initiation, progression and drug resistance. We conclude that an improved understanding of both similarities and differences in the cellular mechanisms among the cancer, normal and mESCs is crucial to develop a potential clinical relevance, and ATR-FITR can be successfully used as a novel approach to gain new insights into the stem cell and cancer research. We suggest that targeting the cellular metabolisms (glycogen and lipid) can provide new strategies for cancer treatment.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Geleneksel Olarak Yara Tedavisinde Kullanılan Kudret Narı (Momordica charantia L.) Zeytinyağı Maseratı Kullanılarak Krem Formunun Geliştirilmesi ve İn Vitro Yara İyi Edici Etkisinin Araştırılması

Bu çalışma, halk arasında geleneksel olarak dahilen ve haricen yara iyileştirici olarak kullanılan kudret narı (Momordica charantia)-zeytinyağı karışımından yara iyi edici prototip krem formunun hazırlanması, hazırlanan krem formunun standardizasyonu ve kalite kontrol çalışmaları ile yara iyileştirici etkisinin belirlenmesi için in vitro hücre kültürü yöntemi kullanılarak araştırılması amacıyla dizayn edilmiştir. Çalışma kapsamında öncelikle kudret narı zeytinyağı karışımı hazırlanmıştır. Hazırlanan krem formunun total flavonoid içerikleri spektrofotometrik ve yüksek basınçlı sıvı kromatografi yöntemleri kullanılarak belirlenmiştir. Hazırlanan Kudret narı krem formunun invitro kalite kontrol çalışmalarında total flavonoid miktarı, pH, vizkozite ve stabilite tayinleri ile biyolojik aktivitelerinin belirlenmesi kapsamında, BJ ve HaCaT hücreleri ile gerçekleştirilen in vitro hücre migrasyonu çalışmaları ile yara iyileştirici etkileri belirlenmiştir. Kudret narı – zeytinyağı maseratı ve farmasötik kalitede yardımcı maddeler kullanılarak hazırlanan laboratuvar ölçekli krem formu total flavonoid içeriği üzerinden standardize edilmiş, yapılan 40 °C % 60 nemde yürütülen çalışmalarda ürünün stabil olduğu ve in vitro yara iyileştirme modelinde BJ ve HaCaT hücrelerinde artan dozlarla (1,10,30) birlikte kontrol grubuna karşılık hücre migrasyonunun arttığı gözlenmiştirThis study was designed for preparation of wound-healing cream form from the mixture of olive oil and Momordica charantia, used as wound healing agent. We also aimed to employ the standardization and quality control studies of prepared cream form and to investigate its wound healing effect using in vitro cell culture method. Within the scope of this study, first a mixture of olive oil and Momordica charantia was prepared. Total flavonoid contents of prepared cream form were determined by using spectrophotometry and high pressure liquid chromatography. Quality control studies of this cream were studied in vitro to determine the total amount of flavonoid, pH, viscosity and stability. Biological activities of prepared cream form were studied to determine the cell migration and wound healing effect on BJ and HaCaT cells by in vitro cell culture. The laboratory scale cream form, prepared using the mixture of olive oil and Momordica charantia together with pharmaceutical quality excipients, was standardized on the total flavonoid content. The product was found to be stable at 40°C with 60% humidity. In in vitro wound healing models of BJ and HaCaT cells with increasing doses of cream form, cell migration was observed to increase, compared to control group