| Since1944, clinical application of aminoglycoside antibiotics has come a long way. Bacteria also have more resistance mechanisms. Aminoglycoside antibiotic resistance mechanisms include:antibiotics acetylation, phosphorylation and adenylylation; outer membrane permeability changed, active efflux and drug trapping;30S ribosomal subunit target mutation; antibiotic binding site methylation. The expression of induced resistance genes has become the major problem of antibiotic resistance.AAC (6’)-IIb located in the chromosome of Pseudomonas fluorescens, which showed resistance for gentamycin, tobramycin, netilmycin, sisomicin and dibekacin. The5’-UTR sequence of AAC (6’)-IIb (aac) has the same with ermC and fexA, which can be translated into short peptides. It showed that aac displayed different structures at different temperatures by Vienna RNA structure prediction, which indicated that the different structures may affect the AAC (6’)-IIb expression. We constructed pGEX-aac-lacZa expression vector to study the function of aac. LacZ activity increased by adding kanamycin B, which confirmed that AAC (6’)-IIb expression could be induced with aminoglycosides. LacZα transcriptional level increased slightly in the antibiotic group by real-time PCR, which suggested that kanamycin B may stabilize the structure of aac. The different concentrations of kanamycin B had no significant changes of lacZa in transcripts, which suggested that kanamycin B changed the secondary structures of aac transcript. Neomycin and gentamycin further verified the activity of lacZa, which suggested that aminoglycoside antibiotics may bind to aac and influence lacZa expression.Disc-assay displayed that13kinds of aminoglycosides except spectinomycin could induce the expression of lacZa, which suggested that there was no binding between aac and spectinomycin for amino group. So there was unchanged with lacZa expression. SPR experiments showed the binding between aac and aminoglycosides except spectinomycin, and accessed to the binding constants. We labeled the aac RNA at the3’end with biotin, which validated no significant degradation of RNA in this process, and high labeling efficiency and production. In molecular biology, a riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene’s activity. The post-translational regulation mechanism of ermC and fexA is still unknown and the leader peptides could not bind to corresponding antibiotics. Therefore ermC, fexA and aac may be riboswithes.Construction of pGEX-RmtB-aac-lacZα reporter vector, which caused16S rRNA methylation, Disc-assay still displayed induction with aminoglycosides except spectinomycin. So it proved that aminoglycosides could direct binding to aac. aac is a riboswitch while aminoglycosides as ligands. The blue strings were not obvious with RmtB strain, which suggested that the modified ribosomes had effect on lacZα translation. The induction happened with higher concentrations of aminoglycosides, which suggested that the binding concentration of aminoglycosides reduced after16S rRNA methylation in vivo. The mechanisms need to be further study.SHAPE experiments confirmed that the complex of aminoglycosides and aac-RNA has effect with aac-RNA structure. The binding open the translation initiation of SD-2and AUG-2, but aac-RNA did not bind spectinomycin. RNA structure prediction by Vienna online and homology analysis revealed that a conserved sequence of aac (AGUC) presented in16S rRNA. It suggested that binding sites may occur in the relevant location. The SHAPE data analysis found that the binding sites-"CCC"near "AGUC". It’s because the "CCC" signal decreased with aminoglycosides. But more precise binding sites have yet to be studied further.In summary, the project found a new switch "aac", with the ligands of aminoglycosides. And it was the first report of antibiotics as ligands of riboswitch. The resistance mechanism of induction gene expression opened up a new field. Hydrogen sulfide is the third largest gas signal molecules, involved in vascular tone, myocardial contraction, nerve transmission and regulation of insulin secretion. When the body lack of hydrogen sulfide, there will be arterial and pulmonary hypertension, Alzheimer disease, gastric mucosal damage and a variety of liver cirrhosis in animal models. Exogenous hydrogen sulfide may improve myocardial dysfunction associated with ischemia/reperfusion injury and reduce gastric mucosal injury. On the other hand, its excessive production may contribute to inflammatory diseases, septic shock, stroke, mental disorders in patients with Down syndrome incidence. So reducing its production in these diseases may be a potential therapeutic value.The study of hydrogen sulfide is still in the preliminary stage. The molecular mechanism of hydrogen sulfide and its target molecules is still unknown. We used fission yeast microarray to elucidate the cellular effects of hydrogen sulfide, and find new targets for major diseases. Compared the gene expression in S.pombe treated with and without hydrogen sulfide, we found out the differential expression genes. It’s help with analyzing signaling pathways of cell cycle, cell proliferation and apoptosis. And it paved the way for studying important protein factors.After verification and analysis of experiments, we used50μM sodium hydrosulfide (hydrogen sulfide donor) to treat the fission yeast. After the genome-wide RNA extraction, then reverse transcription and hybrid, we standardized of the signal analysis system using SAS, t-test analysis. There were83differentially expressed genes of2-fold and280genes of1.5-fold. The cluster showed three biological repeat experiments with repeatability and reliability.1.2-fold genes taken to real-time quantitative PCR, it concluded that the data of gene chip is reliability. So we used1.5-fold genes as the main study.By DAVID online software with the default setting (counts>2, EASE score<0.1) for functional classification of differentially expressed genes, GO biological process showed20down-regulated genes,63genes up-regulated of2-fold genes, and156up-regulated genes,124genes down-regulated of1.5-fold genes. It’s interesting that31genes of2-fold associated with stress response,18genes encoding predicted or known transport proteins,11genes encoding cell cycle/meiosis-related proteins and10genes encoding proteins involved in oxidative reduction. Stress-related genes comparison with up-regulated genes of hydrogen sulfide, there were46%identical genes for hydrogen peroxide,43%for cadmium and49%for heat. However, only12%and24%genes were same with methyl sorbitol and methane sulfonate salt. The genes associated with hydrogen peroxide may indicate that hydrogen sulfide could protect cells from oxidative stress. MAPK signaling pathway involved in a number of changes in gene expression, which may be target genes of hydrogen sulfide.Genome-wide localization study,480genes of4954genes located in the mitochondria (9.6%of genes located in the mitochondria). There were124down-regulated genes with23genes (18.5%) located in the mitochondria. So hydrogen sulfide repressed a number of mitochondrial gene expression, which may have effects on the mitochondrial function.Oxygen consumption and mitochondrial membrane potential of the fission yeast verified the impact of hydrogen sulfide on mitochondrial function. The experimental results showed that the fission yeast with hydrogen sulfide treatment decreased oxygen consumption and mitochondrial membrane potential, which proved mitochondrial dysfunction and fully confirmed the results of gene-chip data that hydrogen sulfide caused decreased mitochondrial gene expression.Some differentially expression genes caused by hydrogen sulfide provided clues for human gene expression studies and molecular target for homologous proteins. It’s paved the way for further exploration of the mechanism associated with hydrogen sulfide and treatment of human disease. |
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