Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
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Article Citation - WoS: 3Citation - Scopus: 3Endogenous Heat Shock Protein Groel of A. Actinomycetemcomitans Preferentially Targets Primary Human Cd8+t Cells(TÜBİTAK, 2015) Kant, Melis; Akgül, Bünyamin; Nalbant Aldanmaz, AytenApoptosis can be used to manipulate host cells by bacterial products such as bacterial heat shock proteins (Hsp). One of the virulence factors of periodontal pathogen Aggregatibacter actinomycetemcomitans is heat shock protein GroEL (AaGroEL), which has been shown to interact with host cells. AaGroEL (Hsp64) also has the potential to modulate immune system cells. In this study we used endogenous AaGroEL protein as an antigen to study bacterial Hsp-induced apoptosis in different immune system cells. Human peripheral blood mononuclear cells and cell lines were cultured with different doses (50-1000 ng/mL) of endogenous AaGroEL at various time points. Apoptosis of the cells was measured by Annexin V and 7AAD labeling. Apoptotic cells were analyzed by flow cytometry. Our data suggested that AaGroEL-responding primary CD8+ T cells were more susceptible to apoptosis than CD4+ T cells. Furthermore, the magnitude of apoptosis in the Jurkat T cell line was higher than that in primary CD8+ T cells. There was no statistically significant level of apoptosis in the chronic myeloid leukemia (K562) cell line, which belongs to myeloid lineages. Thus, A. actinomycetemcomitans GroEL protein has more potent apoptotic effect on cells that are derived from a lymphoid progenitor.Article Citation - WoS: 15Citation - Scopus: 16The Usability of Juniperus Virginiana L. as a Biomonitor of Heavy Metal Pollution in Bishkek City, Kyrgyzstan(Taylor & Francis, 2015) Kurmanbekova, Gülbübü; Severoğlu, Zeki; Özyiğit, İbrahim İlker; Doğan, İlhan; Demir, Göksel; Yalçın, İbrahim Ertuğrul; Kaşoğlu, GültenUncontrolled and unplanned urbanization and industrialization due to increase of population and rapid industrial development have created severe environmental problems in Kyrgyzstan during the last few decades. In this study, Juniperus virginiana, a dioecious species, was employed in order to make assessment of the heavy metal pollution rate in the area and of the heavy metal pollution impact on the mineral nutrient status of the plant. For this study, leaf (washed and unwashed) and bark samples of J. virginiana, and its co-located soil samples were collected from eight different stations, all in the capital of Kyrgyzstan, Bishkek, in 2012 vegetation period. The standard procedures were used and the determinations of heavy metal and nutrient element contents (Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Pb and Zn) in all samples were done using inductively coupled plasma-optical emission spectroscopy. According to our measurements, J. virginiana was found to be capable of accumulating a considerable amount of metals and the mineral nutrient uptake pattern was altered because of metal deposition in the plant, which showed a contamination risk in the area.Article Characterization of the Human Sialidase Neu4 Gene Promoter(TÜBİTAK - Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, 2014) Seyrantepe, Volkan; Delman, MuratThere are 4 different sialidases that have been described in humans: lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), and lysosomal/mitochondrial (Neu4). Previously, we have shown that Neu4 has a broad substrate specificity and is active against glyco-conjugates, including GM2 ganglioside, at the acidic pH of 3.2. An overexpression of Neu4 in transfected neuroglia cells from a Tay-Sachs patient shows a clearance of accumulated GM2, indicating the biological importance of Neu4. In this paper, we aimed to characterize a minimal promoter region of the human Neu4 gene in order to understand the molecular mechanism regulating its expression. We cloned 7 different DNA fragments from the human Neu4 promoter region into luciferase expression vectors for a reporter assay and also performed an electrophoretic mobility shift assay to demonstrate the binding of transcription factors. We demonstrated that -187 bp upstream of the Neu4 gene is a minimal promoter region for controlling transcription from the human Neu4 gene. The electrophoretic mobility shift assay showed that the minimal promoter region recruits a c-myc transcription factor, which might be responsible for regulation of Neu4 gene transcription. The data we obtained might be useful to discover small molecules, which control selective high expression of the human Neu4 gene, resulting in the normal morphological phenotype in the lysosomes of Tay-Sachs patients.Article Citation - WoS: 19Citation - Scopus: 21Altered Conductance and Permeability of Cx40 Mutations Associated With Atrial Fibrillation(Rockefeller University Press, 2015) Cruz, Ana Santa; Meşe, Gülistan; Valiuniene, Laima; Brink, Peter R; White, Thomas W.; Valiunas, VirginijusGap junctions ensure the rapid propagation of the action potential throughout the myocardium. Three mutant forms of connexin40 (Cx40; A96S, M163V, and G38D), the primary component of the atrial gap junction channel, are associated with atrial fibrillation and retain the ability to form functional channels. We determined the biophysical properties of these mutant gap junctions in transiently transfected HeLa and N2A cells. All three mutants showed macroscopic junctional conductances over the range of 0.5 to 40 nS, and voltage dependences comparable to those of wild-type (WT) Cx40. However, the unitary conductance of G38D channels was ~1.6-fold higher than that of WT Cx40 channels (~220 vs. ~135 pS), whereas the unitary conductances of the A96S and M163V mutants were similar to that of WT Cx40. Furthermore, the M163V and G38D channels exhibited approximately two- and approximately fivefold higher permeability to the anionic dye Lucifer yellow (LY) relative to K+ (LY/K+) compared with that of WT Cx40, whereas A96S LY transfer was similar to that of WT (G38D > M163V > A96S ? Cx40WT). In contrast, G38D channels were almost impermeable to cationic ethidium bromide (EtBr), suggesting that G38D alters channel selectivity. Conversely, A96S and M163V channels showed enhanced EtBr permeability relative to WT Cx40, with the following permeability order: M163V > A96S > Cx40WT > G38D. Altered conductive and permeability properties of mutant channels suggest an essential role for Cx40-mediated biochemical and electrical coupling in cardiac tissues. The altered properties of the three single-base substitution mutants may play a role in mechanisms of reentry arrhythmias. © 2015 Santa Cruz et al.Letter Citation - Scopus: 9A Call for Benchmark Data in Mass Spectrometry-Based Proteomics(Proteomass Scientific Society, 2012) Allmer, JensProteomics is a quickly developing field. New and better mass spectrometers, the platform of choice in proteomics, are being introduced frequently. New algorithms for the analysis of mass spectrometric data and assignment of amino acid sequence to tandem mass spectra are also presented on a frequent basis. Unfortunately, the best application area for these algorithms cannot be established at the moment. Furthermore, even the accuracy of the algorithms and their relative performance cannot be established. This is due to the lack of proper benchmark data. This letter first introduces the field of mass spectrometry-based proteomics and then defines the expectations of a well-designed benchmark dataset. Thereafter, the current situation is compared to this ideal. A call for the creation of a proper benchmark dataset is then placed and it is explained how measurement should be performed. Finally, the benefits for the research community are highlighted. © 2012, Proteomass Scientific Society. All rights reserved.Article Citation - WoS: 13Citation - Scopus: 15Cytotoxic Tolerance of Healthy and Cancerous Bone Cells To Anti-Microbial Phenolic Compounds Depend on Culture Conditions(Humana Press, 2019) Karadaş, Özge; Meşe, Gülistan; Özçivici, EnginCarnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.Article Citation - WoS: 24Citation - Scopus: 23Polyethers Isolated From the Marine Actinobacterium Streptomyces Cacaoi Inhibit Autophagy and Induce Apoptosis in Cancer Cells(Elsevier, 2019) Khan, Nasar; Yılmaz, Sinem; Aksoy, Semiha; Uzel, Ataç; Tosun, Çiğdem; Ballar Kırmızıbayrak, Petek; Bedir, ErdalPolyether compounds, a large group of biologically active metabolites produced by Streptomyces species have been reported to show a variety of bioactivity such as antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Since some of these compounds target cancer stem cells and multi-drug resistant cancer cells, this family of compounds have become of high interest. In this study, three polyether-type metabolites (1-3), one of which was a new natural product (3), were isolated from the marine derived Streptomyces cacaoi via antimicrobial activity-guided fractionation studies. As several polyether compounds with structural similarity such as monensin have been linked with autophagy and cell death, we first assessed the cytotoxicity of these three compounds. Compounds 2 and 3, but not 1, were found to be cytotoxic in several cell lines with a higher potency towards cancer cells. Furthermore, 2 and 3 caused accumulation of both autophagy flux markers LC3-II and p62 along with cleavage of caspase-3, caspase-9 and poly (ADP-ribose) polymerase 1 (PARP-1). Interestingly, prolonged treatment of the compounds caused a dramatic downregulation of the proteins related to autophagasome formation in a dose dependent manner. Our findings provide insights on the molecular mechanisms of the polyether-type polyketides, and signify their potency as chemotherapeutic agents through inhibiting autophagy and inducing apoptosis.Article Citation - WoS: 9Citation - Scopus: 10Characterization of Long Living Yeast Deletion Mutants That Lack Mitochondrial Metabolism Genes Dss1, Ppa2 and Afg3(Elsevier, 2019) Muid, Khandaker Ashfaqul; Kimyon, Önder; Reza, Shahadat Hasan; Karakaya, Hüseyin Çağlar; Koç, AhmetMolecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2 Delta, dss1 Delta, and afg3 Delta) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondria] DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.Article Citation - WoS: 2Citation - Scopus: 2Identification of Cytoplasmic Sialidase Neu2-Associated Proteins by Lc-ms/Ms(Türk Biyokimya Derneği, 2019) Akyıldız Demir, Seçil; Seyrantepe, VolkanBackground: Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, gly-copeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective: The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods: In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results: Here, mass spectrometry analysis showed four proteins; a-actin, beta-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions: Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.Article Citation - WoS: 7Citation - Scopus: 9Genomewide Elucidation of Drug Resistance Mechanisms for Systemically Used Antifungal Drugs Amphotericin B, Caspofungin, and Voriconazole in the Budding Yeast(American Society for Microbiology, 2019) Balkan, Çiğdem; Ercan, İlkcan; Işık, Esin; Akdeniz, Esra Şahin; Balcıoğlu, Orhan; Kodedova, Marie; Koç, AhmetThere are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae. We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up-or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae. Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.