TUE-454
Metabolomic analysis of fission yeast at the
onset of nitrogen starvation
K. Sajiki, T. Pluskal, M. Yanagida
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University (OIST), 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
Microorganisms naturally respond to changes in nutritional condi-tions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively examined by genetic, transcriptomic and proteo-mic studies. However, since nitrogen starvation is ultimately a met-abolic condition, complete understanding of its effect cannot be achieved without studying the intracellular metabolome. In addi-tion, the most immediate response to nitrogen starvation might not be mediated on the transcriptional level, as the transcription/trans-lation machinery responds with an inevitable delay. In this study, we conducted time course metabolomic analysis immediately after nitrogen starvation, prior to any visible changes in cell morphol-ogy. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min after nitrogen source removal, are the rapid increase of trehalose, 2-oxoglutarate, and succinate, and the sharp decline of purine biosynthesis intermedi-ates. At 30–60 min, free amino acids decreased, although several modified amino acids, including trimethylated amino acids, increased. Most energy metabolites such as ATP, S-adenosyl-methionine or NAD+remained stable during the first 1 h. The fast
shut-off of purine biosynthesis and the sharp rise of 2-oxoglutarate and succinate may be caused by the depletion of NH4Cl from the
culture medium. Glutamate dehydrogenase catalyzes the reaction of 2-oxoglutarate and NH4Cl to synthesize glutamate and
subse-quently succinate. The gene gdh1 + encodeing glutamate dehy-drogenase is essential for S. pombe enter the G0 phase under nitrogen starvation. The level change of key metabolites such as 2-oxoglutarate, succinate and glutamate, may represent an important mechanistic step to trigger subsequent cellular regulations under nitrogen starvation. We extened our metabolome experiments for longer time after nitrogen starvation (6, 12 and 24 h), and will present results that suggest the importance of 2-oxoglutarte udner long-term nitrogen starvation.
Reference
Sajiki, K., Pluskal, T., Shimanuki, M., Yanagida, M. (2013): Metabolomic Analysis of Fission Yeast at the Onset of Nitro-gen Starvation. Metabolites 3: 1118–1129.
Keywords: 2-oxoglutarate, Nitrogen starvation, Yeast metabolism.
TUE-455
Metformin reduces palmitate induced
endoplasmic reticulum stress and apoptosis in
rat insulinoma cell line
M. Kokas1,2, L. Simon-Szabo1, J. Mandl1, G. Keri2,3, M. Csala1 1Department of Medical Chemistry, Molecular Biology and
Pathobiochemistry, Semmelweis University,2MTA-SE
Pathobiochemistry Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, MTA,
3Vichem Chemie Research Ltd, Budapest, Hungary
Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pan-creatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic
reticu-lum (ER) stress response can lead to evenb-cell death (lipoapop-tosis). Since improvement ofb-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. We mea-sured palmitate induced cell death using Tryphan Blue exclusion method. Assessment of palmitate-induced lipoapoptosis by detec-tion of caspase-3 showed a significant decrease in metformin trea-ted cells. Attenuation ofb-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2a (eIF2a), c-Jun N-terminal kinase (JNK), splicing of X box binding protein 1 mRNA and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that theb-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.
Keywords: apoptosis, Endoplasmic reticulum stress.
TUE-456
Microbial removal of remazol blue by different
bacteria
S. Ertugrul Karatay, N. Kocßberber Kilic, G. D€onmez Biology, Ankara €Universitesi, Ankara, Turkey
Textile, pulp, paper, tanning, and dyeing wastewaters include toxic many substances such as highly colored chemicals. These wastewaters affect the environment negatively and need to be treated. In the current study, 3 different bacteria (isolate A, iso-late B, and isoiso-late C) were isoiso-lated from dye containing textile wastewaters. These bacteria were tested with their possible usage in treating wastewaters including dyestuff. For this purpose, molasses media was prepared and Remazol Blue (RB) was used as the pollutant. Experiments were carried out in a rotary shaker (100 rpm) with an incubation period of 7 days at 30°C. Isolate A, isolate B, and isolate C were investigated with regards to dif-ferent pH levels (6, 7, 8, and, 9) and dye concentrations (approxi-mately 25, 50, 75, and 100 mg/l). All the tested bacteria were cultivated in molasses media with approximately 25 mg/l dye at different pH levels. The residual dye concentration and optical densities of the bacteria were determined spectrophotometrically. According to the data from the experiments, all the three bacteria removed this dye with the maximum levels at pH 7. In the exper-iments investigating the effect of initial dye concentration onto dye removal, with an increase in pollutant concentration removal of dye decreased. Within the three bacteria, isolate C, removed dye with higher efficiencies than other bacteria tested.
Keywords: Bioremoval, Remazol Blue, Wastewater.
TUE-457
Modulation of global gene expression profile
with resveratrol in rat liver tissues
M. C. Baloglu1, G. Sadi2 1
Department of Genetic and Bioengineering, Kastamonu University, Kastamonu,2Department of Biology, Karamanoglu
Mehmetbey University, Karaman, Turkey
Analyzing gene expression profile by using microarray technology is one of the fastest-growing new technologies in genetic researches. Resveratrol, a strong antioxidant in plants, is a natu-ral phytoalexin and polyphenolic compound and regulates the expression of various genes. In this study, microarray analysis was performed to indicate effects of resveratrol treatment on glo-bal gene expression profiles of rat liver tissues. RMA algorithm (Robust Multiarray Analysis) was used for microarray raw data normalization. Significantly expressed probe sets with p-values lower than 0.05 were determined by One way ANOVA. Among
FEBS Journal 281 (Suppl. 1) (2014) 65–783
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significantly expressed probe sets, fold change of at least two was considered as differentially expressed probe sets. Principal com-ponents analysis (PCA) was used to simplify the analysis and visualization of multidimensional data sets. PCA revealed that three biological replicates within a one group clustered together and separated from control group. The Venn diagram was con-structed to indicate the number of separated and overlapping probe sets between resveratrol and control groups. After the res-veratrol treatment, 186 and 494 transcripts were up and down regulated, respectively. Gene Ontology Enrichment Analysis Soft-ware Toolkit (GOEAST) was used for determination of annota-tion and biological processes for significantly different probe sets. According to the GO database, after resveratrol treatment, up-regulated differentially expressed probes were functionally catego-rized into 10 groups. These included not only functionally well-defined categories, such as cell part, cytoplasm, positive regula-tion of biological process but also response to stress, organic sub-stance, immune system, and biotic stimulus. Resveratrol treatment led to an increase in the expression of genes including Lcn2 and Usp2 which are responsible for regulation of apoptosis. Additionally, expression level of cellular mobility element genes (Akta1-Mylpf), and acute inflammation of the calcium binding protein gene (S100a9) increased. So, it can be concluded that res-veratrol reduced apoptosis and increased cell motility, and immune response. Expression levels of genes involved in the syn-thesis of the proteins found in the nucleus and nucleolus reduced after resveratrol treatment. Expression level of Pdcd4 gene acting as an inhibitor of apoptosis and Tp53 bp2 gene encoding tumor protein p53 binding protein decreased by 8.8 and 2.8 folds, respectively. It can be suggested that resveratrol has ability to reduce cell death. Finally, microarray results were validated with qRT-PCR using selected genes such as Cat, Usp2, Igfbp2, Cyp1a1, and Cyp8b1.
Keywords: Microarray, Rat Liver Tissue, Resveratrol.
TUE-458
Modulation of metallothionein and apoptotic
activities in zinc nanooxide exposed mussel by
heat stress and nifedipine
O. Stoliar1
, H. Falfushynska1, I. Sokolova2, L. Gnatyshyna1, O. Fedoruk1, A. Ivanina2
1
Ternopil National Pedagogical University, Ternopil, Ukraine,
2
University of North Carolina at Charlotte, Charlotte, USA Nanooxide ZnO (n-ZnO) is one of the most common types of used nanoparticles. N-ZnO can be a source of Zn for the aquatic organ-isms. Zn is an essential co-factor of many metalloezymes and tran-scription factors and can also modulate function of ionic channels; however, it is toxic at high concentrations. This study investigated the effects of n-ZnO and Zn2+on metal binding and cellular stress response, and possible modulation of these effects by heat stress (25°C) or Ca-channel blocker nifedipine (NFD) in a model organ-ism, a mussel Unio pictorum. Male U. pictorum were exposed for 14 days to Zn2+(3.1lM), NFD (10 lM), n-ZnO (3.1 lM), com-bination of n-ZnO and NFD at 18°C (n-ZnO + NFD), and n-ZnO at 25°C (n-ZnO + T). Metal binding capacity was determined by measuring levels of metallothioneins (MT) in the digestive gland tissue. Cellular stress response was assessed in the digestive gland by measuring levels of antioxidants (reduced and oxidized glutathi-one, GSH & GSSG, respectively) and activity of superoxide dismu-tase (SOD), levels of oxiradicals and oxidative lesions of proteins (carbonyls, PC) and lipids (LPO), DNA fragmentation, and activ-ity of the main effector enzymes in the apoptotic cascades, caspase-3 and cathepsin D (total and free). Exposure to Zn, ZnO and n-ZnO+ NFD induced significant upregulation of MT levels by ~ 30%, while NFD alone depleted the MT level. Notably, the
upreg-ulation of MT in response to n-ZnO exposure was abolished at the elevated temperature (25°C). All exposures except n-ZnO + T led to upregulation of the activity of an antioxidant enzyme, SOD, accompanied by a 2–3-fold decrease in the levels of PC, while the concentrations of LPO products did not change. In contrast, the combined exposure to n-ZnO+ T abolished upregulation of SOD activity and induced oxidative stress as indicated by elevated levels of protein carbonyls (by~40%), LPO products (by over 100%) and a~2-fold increase in the levels of oxidized glutathione (GSSG). Exposures to the Zn, n-ZnO+ T and NFD led to a significant increase in DNA fragmentation. Cathepsin D-related apoptotic activity was induced by all exposures except n-ZnO+ T and NFD, while the caspase-3 mediated cascade was induced prominently by n-ZnO+ T and decreased by n-ZnO, NFD and n-ZnO + NFD. NFD exposure alone caused elevation of oxyradical formation and release of cathepsin D. Overall, our data show that in the complex exposures, the heat stress drastically impacts MT-dependent metal binding and oxidative stress responses on the exposure to n-ZnO, whereas NFD leads to a change in the apoptotic activity and doesn’t affect MT functions.
Keywords: apoptosis, heat stress, metallothionein.
TUE-459
Molecular characterization of nicotinate
phosphoribosyltransferase from
Mycobacterium tuberculosis H37Rv, and
inhibition of its activity by pyrazinoic acid
S. Mori1
, H. Kim1, E. Rimbara1, Y. Arakawa2, K. Shibayama1
1
Bacteriology II, National Institute of Infectious Diseases, Tokyo,
2
Bacteriology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Nicotinate phosphoribosyltransferase (NAPRTase) catalyzes the reaction of nicotinic acid (NA) with 5-phosphoribosyl-1-pyro-phosphate (PRPP) to produce nicotinic acid mononucleotide and diphosphate. It plays an important role in nicotinamide adenine dinucleotide biosynthesis. Recently, two enzymes in Mycobacte-rium tuberculosis H37Rv—PncB1 (Rv1330c) and PncB2 (Rv0573c)—were reported to exhibit NAPRTase activity. In this study, we investigated the properties and kinetics of PncB1. PncB1 requires bivalent metal ions for its catalytic activity, and its optimal temperature is 40°C. In addition, PncB1 exhibits ATP hydrolysis activity. PncB1 can catalyze the NAPRTase reaction in the presence and absence of ATP. However, by comparing the PncB1 kinetic values in the presence and absence of ATP, it was found that ATP affects the reaction by altering the apparent Km
values, whereas, the Kcat values were only slightly affected. The
Km values for NA and PRPP in the presence of ATP were
0.16 mM and 0.22 mM, respectively; and 3.22 mM and 2.71 mM, respectively, in the absence of ATP. On the other hand, the Kcatvalues for NA and PRPP in the presence of ATP
were 0.14 and 0.15 s 1, respectively; and 0.13 and 0.09 s 1, respectively, in the absence of ATP. The Kcat/Kmvalues for both
NA and PRPP were approximately 25-fold greater in the pres-ence of ATP than in the abspres-ence of ATP. Hpres-ence, it is suggested that PncB1 utilizes the energy obtained through its facultative ATPase activity for effective catalysis. Pyrazinamide (PZA) is an anti-tuberculosis drug, which is converted to its active form, pyr-azinoic acid (POA), by pyrazinamidase/nicotinamidase. PZA and POA are nicotinamide and nicotinic acid analogs, respectively. Although PZA and POA cannot be used as substrates for PncB1 instead of NA, the enzymatic activity of PncB1 is strongly inhib-ited by POA at pH 5.4. The results of molecular modeling and molecular simulation of PncB1 revealed that the binding site of POA is the same as that of NA. POA requires an acidic
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