The aim of study To purify GPCR from a local strain of S. cerevisiae using Ion exchange and gel filtration chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to the already used in published researches, which depend on the costly affinity chromatography and other expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of G-protein coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University, Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30 C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar (YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological, microscopic characterization and biochemical test . The GPCR that extract from whole cell of S.cerevisiae was purified by ion exchange chromatography using DEAE-Sepharose ,the bound proteins (negatively charged) were then eluted using gradient concentration of NaCl ranged between( 0.1 -0.5M). Gel filtration chromatography using Sepharose 6B was applied as a second step of purification. The optical density for each fraction was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution. Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by using SDS-PAGE electrophoresis . In ion exchange chromatography the fraction were collected with 5 ml tube at a flow rate 0.5 ml/ min and eluted with gradient (0.1-0.5M) of sodium chloride solution. Two proteins peaks appeared after eluted by the gradient concentration of sodium chloride, while no protein peaks appeared in the washing fractions. The GPCR concentration was measured in the fractions of these two protein peaks, data indicated that GPCR located in the first protein peak (eluted at 0.1M of NaCl) at fraction numbers between 3 and 9, the maximum concentration of GPCR was 9.281 with specific activity 71.58(ng/mg)protein , 3.125 purification folds and72.9(%) yield while the second peaks (eluted at 0.4 M of NaCl) don't give any concentration for GPCR, thus its neglected. Gel filtration chromatography was used as second step of purification which applied by using sepharose 6B. Results show single active protein peaks appeared that identical with the peak of GPCR at fractions numbers(29-35). The maximum concentration of GPCR was 9.082 (ng/ml)was observed in these fractions. The specific activity for these fractions was 151.37 (ng/mg) protein with 6.608 purification folds and 39.64 (%) yield. The present study a chive a relatively high purification of GPCR from whole cell of a local strain S. cerevisiae with fold purification 6.608 and a yield of 39.64 % and molecular weight about~33KD.
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Objective: Zerumbone (ZER) is a well-known natural compound that has been reported to have anti-cancer effect. Thus, this study investigated the ZER potential to inhibit Thymidine Phosphorylase (TP) and the ability to trigger Reactive oxygen species (ROS)-mediated cytotoxicity in non-small cell lung cancer, NCI-H460, cell line. Material and Method: The antiangiogenic activity for ZER was evaluated by using the thymidine phosphorylase inhibitory test. Reactive oxygen species (ROS) production was determined via DCFDA dye by using flow cytometry. Result and Discussion: ZER was found to be potent TP inhibitory with the IC50 value of 50.3± 0.31 μg/ml or 230±1.42 µM. NCI-H460 cells upon treatment with ZER produced sign
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Increasing the power conversion efficiency (PCE) of silicon solar cells by improving their junction properties or minimizing light reflection losses remains a major challenge. Extensive studies were carried out in order to develop an effective antireflection coating for monocrystalline solar cells. Here we report on the preparation of a nanostructured cerium oxide thin film by pulsed laser deposition (PLD) as an antireflection coating for silicon solar cell. The structural, optical, and electrical properties of a cerium oxide nanostructure film are investigated as a function of the number of laser pulses. The X-ray diffraction results reveal that the deposited cerium oxide films are crystalline in nature and have a cubic fluorite. The field
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Objective: Zerumbone (ZER) is a well-known natural compound that has been reported to have anti-cancer effect. Thus, this study investigated the ZER potential to inhibit Thymidine Phosphorylase (TP) and the ability to trigger Reactive oxygen species (ROS)-mediated cytotoxicity in non-small cell lung cancer, NCI-H460, cell line. Material and Method: The antiangiogenic activity for ZER was evaluated by using the thymidine phosphorylase inhibitory test. Reactive oxygen species (ROS) production was determined via DCFDA dye by using flow cytometry. Result and Discussion: ZER was found to be potent TP inhibitory with the IC50 value of 50.3± 0.31 μg/ml or 230±1.42 µM. NCI-H460 cells upon treatment with ZER produced sign
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This work presents a completely new develop an analyzer, named NAG-5SX1-1D-SSP, that is simple, accurate, reproducible, and affordable for the determination of cefotaxime sodium (CFS) in both pure and pharmaceutical drugs. The analyzer was designed according to flow injection analysis, and conducted to turbidimetric measurements. Ammonium cerium nitrate was utilized as a precipitating agent. After optimizing the conditions, the analysis system exhibited a linear range of 0.008-27 mmol. L-1 (n=29), with a limit of detection of 439.3 ng/sample, a limit of quantification of 0.4805 mg/sample, and a correlation coefficient of 0.9988. The repeatability of the responses was assessed by performing six successive injections of CFS at concentra
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Background: Oral SCC is a complex malignancy where environmental factors, viral infections and genetic alterations most likely interact, and thus give rise to the malignant condition. The HSP70 play a direct role in apoptosis inhibition by aligning the improved integrity of a cell’s proteins with the improved chances of that particular cell’s survival.P21 gene produces p21 protein which is a potent cyclin-dependent kinase inhibitor that plays a significant role in carcinogenesis. The aims of the study were to evaluate and compare the immun-histochemical expression of the HSP70 and cell cycle protein p21in NOM, OED, and OSCC. Correlate both marker expressions with each other. Materials and methods: Forty six formalin-fixed, par
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Mammalian cell culture refers to culturing mammalian cells in a medium that provide nutrients for cells to be able to grow in vitro under environment that closely mimic the in vivo conditions. By enabling culturing these cells outside living biological entities, investigation on intra- and intercellular activities and flux; genetic and phenotyping analysis; proteomics, study of toxicology, drug discovery and development can be carried out without manipulation of living animals. In this chapter, detail protocol of media preparation, cell culture maintenance and preservation are elaborated for both types of mammalian cell culture, monolayer or suspension cultures. Determination of number of cells is discussed as well.
This study was conducted at the poultry farm of the Department of Animal Production/College of Agriculture/University of Baghdad/Abu Ghraib, on 252 birds (180 females and 72 males). This study aims to observe the effect of melatonin implantation and exposure to different light colors and their interaction on characteristics of fertility and hatching of local Iraqi chickens. The birds were divided into three sections (white, red and green) each section contains two lines, one of which has been planted melatonin under the skin of the neck of birds and the other has not been planted hormones. The results showed that melatonin implantation and exposure to different light colors did not significantly affect the hatching rate of fertilized eggs a
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