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.
The unprejudiced of this education is to gauge the ability of the retinoic acid to induce apoptotic cell death in hematological tumors through caspase dependent or independent apoptotic pathway, The cytotoxicity effects of retinoic acid of different concentrations (400,350,300,250,200,150,100,50,25,12.5 μg\ml) and exposure for all hematological malignancy cell lines (Human non-Hodgkin lymphoma SR and human multiple myeloma (COLO 677) and Human Monocytic Leukemia THP1 and Acute promyelocytic leukemia NB4) have been determined using a microtetrazolium (MTT) assay. Propodeum iodide and alcidine orange (AO/PI) paired discoloration was used to study the ability of retinoic acid to induce apoptosis in the infected cells and examined under fluore
... Show MoreIn this work, enhancement to the fluorescence characteristics of laser dye solutions hosting highly-pure titanium dioxide nanoparticles as random gain media. This was achieved by coating two opposite sides of the cells containing these media with nanostructured thin films of highly-pure titanium dioxide. Two laser dyes; Rhodamine B and Coumarin 102, were used to prepare solutions in hexanol and methanol, respectively, as hosts for the nanoparticles. The nanoparticles and thin films were prepared by dc reactive magnetron sputtering technique. The enhancement was observed by the narrowing of fluorescence linewidth as well as by increasing the fluorescence intensity. These parameters were compared to those of the dye only and the dye solution
... Show MoreA synthesis series of new heterocyclic derivatives (A2-A7) (pyrrole, pyridazine, oxazine and imidazol) derived from 4-acetyl-2,5-dichloro-1-(3,5-dinitrophenyl)-1H-pyrrole-3-carboxylate(A1) have been synthesised. Synthesis of compound (A2) by the reaction of starting material (A1) with hydroxyl amine hydrochloride in the presence of pyridine. Compound (A2) was reacted with hydrazine hydrate in dry benzene to give (A3) derivative. The compound )A3( deals with sodium nitrite to give diazonium salt, and the reaction diazonium salt with ethyl acetoacetate to produce compound (A4). To a mixture of compound (A4) and hydroxyl amine with sttired to yield (A5).Compound (A6) was prepared by reaction compound (A4) with thiosemicarbazide in presence
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(3)
Background: The aim of this study was to evaluate the expression of ?broblast growth factor-2 and Heparanase in oral squamous cell carcinoma, and to correlate the two studied marker with each other and with clinicopathologicalfinding including grade, stage. Methods: Sections of 30 formalin-fixed paraffin embedded blocks specimens of oral squamous cell carcinoma were immunostained to assess the expression of ?broblast growth factor-2 and Heparanse in oral squamous cell carcinoma cases. Results: The expression of fibroblast growth factor-2 and Heparanase were positive in all oral squamous cell carcinoma cases (100%). The positive expression of fibroblast growth factor-2 was significantly correlated with tumor site (p=0.016),and clinical pres
... Show MoreKlebsiella pneumoniae has been found in the urinary tract of some bladder cancer patients. Bacterial presence within tumor tissue may affect the tumor-microenvironment and consequently influence cancer behavior, development, and treatment response. This study investigated mesenchymal and stemness transdifferentiation of bladder cancer cell line due to environmental stress of K. pneumoniae. Cultures of urothelial bladder cancer cell line (T24) were infected with K. pneumoniae with different multiplicity of infection (MOI) for two and four days. Transdifferentiation-associated features were morphologically assessed.
Moreover, transdifferentiation markers were estimated using Q-PCR and immunohistoc
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Background: The study was designed to evaluate the effect of local application of exogenous VEGF/collagen I separately and as a combination in socket healing. Sixty male Albino Wistar rats were subjected for a surgical tooth extraction of upper 1st molar of both sides (right side was considered as experimental site, while left be the control one, treated with 1µL of normal saline). The rats were scarified at 3, 7, 14, 28 days post extraction. Socket healing was histologically examined with immunohistochemistochemical localization of ALP&FGF2. Materials and Method: Sixty male Albino Wistar rats were subjected for a surgical tooth extraction of upper 1stmolar of both sides (right side was considered as experimental site, while left be the
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The interleukin-1 family has multifaceted roles in men٫s reproductive syste. Out of these is interleukin-1 receptor antagonist (IL-1RN) which exists in men gonads, and in case of infection and inflammatory process, its activity is increased. The current study aims to verify a possible linkage of Variable Number Tandem Repeat (VNTR) polymorphism of the IL-1RN gene with human men infertility. The study groups enrolled included 100 infertile men and 100 fertile and healthy men. Their seminal fluids were subjected to analysis. Also peripheral blood samples were collected for the assessment or detection of polymorphic Variable Number Tandem Repeats (VNTR) polymorphism of interleukin-1 receptor antagonist gene (IL-1RN). Two a
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