فهرست مطالب farangis ataei

SH-SY5Y is a neuroblastoma cell line which used as a cancer and neurodegenerative disorders model and its neuro-experimental studies. The different diseases cause by a defect in apoptosis pathway. Disruption of apoptotic proteins has an effect on the treatment process and response to drugs. In nerve cells, due to the high expression of apoptosis inhibitory proteins, the efficacy of drugs is low. Combination therapy is one of the developing treatment methods. The aim of this research is to evaluate the effectiveness of doxorubicin drug on apoptosis in SH-SY5Y cells under the conditions of high expression of caspase9. Caspase9 is a key enzyme in intrinsic apoptosis. First, cell viability was obtained through MTT assay under the different drug concentrations. Then, caspase9 gene was transfected in cells and affected by the concentration lower than IC50 of drug, and cell energy level and cell death were checked by different methods. ATP assay showed that the expression of caspase9 with drug lead to ATP decreases. Caspase3/7 activity indicated an increase in cell death by drug and caspase. Propidium staining to hoechst showed that the expression of caspase9 in combination with doxorubicin induce more death. To ensure the expression levels of protein that induces cell death, the amount of caspase3 protein was checked by western blotting, which showed a significant increase in combination of caspase9 and drug. Our findings showed that the induction of caspase9 expression intensifies the effect of drug and the combined treatment may be effective on the responsiveness of neuronal diseases.

One family of anti-apoptotic proteins named the inhibitor of apoptosis proteins (IAPs) prevents cell death by blocking the downstream region of the caspase activation pathways. Survivin is a small member of the family of proteins that suppress apoptosis. Survivin performs various tasks that help cancer cells survival, including cytoprotection, preventing cell death, and controlling the cell cycle, particularly during the mitotic process. Cancer cells may survive with the help of survivin, as it is consistently up-regulated in human tumors, connected to poor prognosis, resistance to chemotherapy or radiation therapy, and associated with these treatments. Survivin is often regulated at two levels: the transcriptional level and the post-translational levels. In this review, the different proteins influence the progression of survivin degradation in post-translation adjustment were discussed such as FAT10, Usp22, Csn5; and LNC473. Finding and developing a therapy strategy that can adequately address the range of the aforementioned issues might be aided by understanding regulators and their mechanisms of action.

Programmed cell death is a vital cellular process that is highly conserved in evolution. Apoptosis, as a common mode of programmed cell death, is disturbed in the most human malignancies and leads the resistance of cancers to current treatment strategies. Caspase 9 is a key protein in mitochondrial apoptosis. Activated Caspase 9 leads to activation of Caspase 3/7, initiating a caspase cascade and killing cell. In this study, Caspase 9 gene was cloned into pcDNA3.1(+) and its expression and function evaluated in cell.

PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.

Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.

The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.

Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.

Luciferase from firefly Photinus pyralis (P .py) is a peroxisomal enzyme that converts a heterocyclic substrate luciferin to an excited state oxyluciferin in the presence of Mg+2-ATP and O2. Excited oxyluciferin with the emission of visible light is changed to its ground state. The combination of rapidity, sensitivity, and convenience has led to the development of a broad range of luminescence applications. In spite of wide ranges applications, firefly luciferase is unstable against changes in chemical and physical conditions, thereby reduce its precision and sensitivity. The most undesirable instability of the luciferase is low thermostability and high susceptibility to proteolytic degradation. According to previous studies, limited proteolysis by trypsin of P .py luciferase indicated six cleavage sites on two accessible regions: 206-220 (Including K206, R213, and R218) and 329-341 (Including K329, R330, and R337) on N-terminal domain. In this study, we used site-directed mutagenesis to introduce one point mutation on the 329-341 accessible regions of P. py luciferase, in order to investigate the role of R330 on the enzyme structure and function which R330 changed to Q. Based on limited proteolysis data, R330Q mutant didn’t significantly change compared to wild type, but this mutation caused several alterations in enzymatic properties including shifting the pH optimum from 7.5 to 8 and increasing the thermal inactivation. Based on the results, it can be concluded that whilst Arg330 is a conserved residue but not effects on trypsinolysis stability.

The enzyme, Phenylalanine dehydrogenase (L-phe DH; NAD oxidoreductase, deaminating; EC 1.4.1.20) belongs to the amino acid dehydrogenase family of enzymes which catalyzes the reversible oxidative deamination reaction of L-phenylalanine to their respective α- ketoacids. An assay technique with a high sensitivity for blood L-phenylalanine level, an important marker for the screening of Phenylketonuria (PKU), has been established by means of PheDH. This enzyme is being used as a commercial and valuable biocatalyst in medical and pharmaceutical industries. The enzymes of this family are closely related in structure and function. Swiss-Pdb Viewer used for analysis and Rhodococcus sp. M4 was chosen because of the availability of several crystal structures with bound substrates and its high specific activity. Rhodococcus sp. M4 and B. badius PheDHs are very different from each other on a sequence level, sharing only %32 identity and %50 similarity. Coming from the same structural sub-family, they share a much stronger correlation in their folding motifs. Since there currently is no crystal structure available for the B. badius PheDH, the sequence was folded over the 1BW9 crystal structure and superimposedover the original scaffold using SuperPose.

The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic or mitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from the intermembrane space of mitochondria and binding to Apaf-1 molecules, a heptameric apoptosome complex is formed and triggers the downstream cascade of caspases. Here, for the first time we present spontaneous mutations and recombinations of the Apaf-1 gene and its neighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector with Nluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesis method, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37ºC. In 21 of these cases, we found 38 different mutations. Our data suggest that there is a direct relationship between bacterial incubation temperatures and the number of unwanted spontaneous mutations. During our experiment we found that the Apaf-1 gene is much less susceptible to spontaneous mutations when it is transformed into XL10-Gold at 18 ºC. In contrast, a large number of spontaneous mutations were found when the gene of interest transformed into DH5α at 37ºC.