RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to l-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways

Association between active pulmonary tuberculosis and circulating microRNAs: a preliminary study from Turkey

Background/aim: Tuberculosis is a public health problem that still remains significant. For prevention, diagnosis, and treatment of tuberculosis more effective novel biomarkers are needed. MicroRNAs can regulate innate and adaptive immune responses, alter host-pathogen interactions, and affect progression of diseases. The relationship between microRNA expression and active pulmonary tuberculosis (APT) has not yet been investigated in the Turkish population. We aimed to test the potential diagnostic value of some microRNAs whose levels were previously reported to be altered in APT patients. Materials and methods: Using two different references (U6 and miR-93), we compared the expression levels of potentially important microRNAs in serum of APT patients with healthy individuals using quantitative polymerase chain reaction (qPCR). Results: miR-144 expression level was down-regulated in APT patients when either U6 or miR-93 was used for normalization. When data was normalized with miR-93, a statistically significant decrease in miR-125b (0.8 fold) and miR-146a (0.7 fold) expression levels were observed, while no differences were detected for U6. The receiver operating characteristic suggested that miR-144 may be a candidate biomarker for discriminating APT patients and controls (p < 0.05) both for U6 and miR-93. Conclusion: These findings suggest that miR-144 can have potential as a biomarker for APT. Using a single reference may be misleading in evaluation of microRNA expression. U6 and miR-93 can be used in combination as references for normalization of serum microRNA expression data

Hydrogen peroxide-induced oxidative damages in Schizosaccharomyces pombe

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H2O2-treated and non-treated control cells. It was observed that increased H2O2 concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells

IRAP-PCR As A Tool For Screening HERV Polymorphisms In Nasal Mucosal Swabs

Objective: Inter-retrotransposon polymorphism Polymerase Chain Reaction (IRAP-PCR) technique allows for detecting insertional polymorphisms via amplification of the DNA fragment between two retrotransposons in plant genomes. However, this method has not been reported to be used for analyzing human samples to date. Recently, Human Endogenous Retrovirus (HERV) polymorphisms gained interest due to their potential effect on pathophysiology of certain diseases. Nevertheless, the association between HERV polymorphisms and the risk for developing nasal polyposis (NP) has not been studied. In this study, we aimed to investigate whether or not IRAP-PCR could be performed in nasal swab samples for comparing HERV polymorphisms in different nasal mucosal samples

Inter-retrotransposon polymorphism polymerase chain reaction as a tool for screening HERV polymorphisms in nasal mucosal swabs

Objective: Inter-retrotransposon polymorphism Polymerase Chain Reaction (IRAP-PCR) technique allows for detecting insertional polymorphisms via amplification of the DNA fragment between two retrotransposons in plant genomes. However, this method has not been reported to be used for analyzing human samples to date. Recently, Human Endogenous Retrovirus (HERV) polymorphisms gained interest due to their potential effect on pathophysiology of certain diseases. Nevertheless, the association between HERV polymorphisms and the risk for developing nasal polyposis (NP) has not been studied. In this study, we aimed to investigate whether or not IRAP-PCR could be performed in nasal swab samples for comparing HERV polymorphisms in different nasal mucosal samples

Investigation of the Relationship Between Oxidative Stress and Glucose Signaling in Schizosaccharomyces pombe

The invertase mutant defective in the glucose signaling pathway of () is resistant to glucose repression. This mutant is able to consume sucrose alongside glucose and grows in glucose-containing media with a generation time close to that of the wild type. Intracellular oxidation, protein carbonyl, and reduced glutathione levels and catalase, superoxide dismutase, and glutathione peroxidase activity were investigated in , to determine the relationship between oxidative stress response and glucose signaling. The expression profiles of some genes involved in regulation of glucose repression ( fructose-1,6-bis-phosphatase; hexokinase) and stress response ( and transcription factors; catalase; Cu,Zn superoxide dismutase) were analyzed using the quantitative real-time PCR technique. Oxidative stress response in seems to be affected by glucose signaling in a manner different from that caused by glucose deprivation

Autophagy as a molecular target for cancer treatment

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed

Selective and oxidative stress-mediated cell death of MCF-7 cell line induced by terpinolene

In this study, we compared the effects of terpinolene against the anti-cancer agent cisplatin on proliferation, apoptotic activity and intracellular ROS (reactive oxygen species) production in cancerous (MCF-7) and non-cancerous (HEK-293) cell lines. Cisplatin presented a strong growth-inhibitory effect on both MCF-7 (IC25: 18.77 mu M) and HEK-293 (IC25: 15.03 mu M) cell lines. The growth-inhibitory effect of terpinolene was found to be weaker than cisplatin (IC25: 291.18 mu M for MCF-7 and 345.35 mu M for HEK 293 cells). The expression levels of BAX (by 1.86-fold), cleaved-PARP (by 2.23-fold) and pro-caspase-8 (by 1.74-fold) proteins increased in response to terpinolene treatment in MCF-7 cells. Curiously, the increase in the expression levels of these apoptotic markers was less pronounced in terpinolene-treated non-cancerous HEK 293 cells. Terpinolene, promoted a higher rate of apoptotic and necrotic cell death on MCF-7 cells (apoptotic cells: 25.28 %; necrotic cells: 17.70 %) when compared with HEK-293 cell line (apoptotic cells: 15.47 %; necrotic cells: 9.42 %). Interestingly, terpinolene caused a marked increase (2.05-fold) in intracellular ROS production in MCF-7 cells while HEK-293 cells appeared to be resistant to terpinolene or cisplatin treatments. In conclusion, although terpinolene showed a weaker growth-inhibitory effect on MCF-7 cancer cells, it exhibited a better selectivity than cisplatin in inducing cell death and intracellular oxidative stress in MCF7 breast cancer cells