الفهرس 
P53 tumor suppressor proteinOnly 14 pages are availabe for public view
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Abstract
The objectives of the present study are to evaluate azurin production among local Gram negative bacterial isolates from clinical sputum samples through screening the presence of azurin among six bacterial species (P. aeruginosa, K. pneumoniae, Citrobacter sp, E. cloacae, P. stuartii and S. rubidaea) via SDSPAGE, western blotting and PCR techniques. Moreover, azurin was purified from selected bacterial isolates through column chromatography and then subjected to study its antitumor activity against breast carcinoma cell line (MCF-7). Consequently, bioinformatics analysis was carried out to evaluate the activity of azurin through multiple sequence alignments and 3D-modeling of it. In addition, the antimicrobial activity of azurin was investigated against different types of pathogenic bacteria and fungi. The obtained results revealed that the most common bacteria isolated from the infected lungs was from Enterobacteriaceae family followed by P. aeruginosa isolates (53.3 - 46.6%, respectively). Also, the most studied virulence factors (protease, lipase, gelatinase, hemolysin and biofilm production) were secreted by P. aeruginosa isolates more than other species. Furthermore, the present data illustrated the significant association of biofilm production in Pseudomonas isolates, strong and moderate producing form, in comparison to Enterobacteriaceae (p<0.04). The different antibiotic resistance patterns were carried out and it was found that all Pseudomonas isolates were XDR; on the other hand, Enterobacteriaceae isolates were MDR. Statistical analysis showed that there was a direct correlation between biofilm production and antibiotic resistance. Furthermore, SDS-PAGE analysis revealed differences between the tested bacteria. Forexample, the induction of 54, 142 and 118 KDa proteins in some of the tested P. aeruginosa isolates. In addition, the absence of 37 and 39 kDa proteins in Enterococcus isolates besides 35 and 36 kDa proteins in Klebsiella isolates. Additionally, the results showed that most Pseudomonas and Enterobacteriaceae isolates expressed a 14 kDa protein. Also, western blotting analysis indicated the presence of azurin or similar proteins among P. aeruginosa, K. pneumoniae, S. rubidaea and P. stuartii while absence in E. cloacaea and Citrobacter sp. Meanwhile, PCR technique revealed that azu gene was detected in P. aeruginosa isolates only. Antitumor activities of CFS from the different bacterial species and purified azurin from 8 selected Pseudomonas isolates were estimated; the CFS from P. aeruginosa, K. pneumoniae, S. rubidaea and P. stuartii inhibited the proliferation of MCF-7 carcinoma cells. Additionally, variations in antitumor activity of azurin from the selected Pseudomonas isolates were observed; highest cytotoxicity was recorded from isolate no. 4(PA4) with IC50 at 17.6 μg/ml followed by isolates no. 12, 1, 2 and 7. On the other hand, IC50 values couldn’t be recorded for isolates no. 9, 11 and 20. Bioinformatics analysis of azurin was carried out through multiple alignment sequences for the obtained azu gene sequences and their deduced amino acids. Then, phylogenetic trees were constructed from both DNA and protein MSA of azurin; phylogenetic analysis showed that azu gene sequences from PA11, PA9 and PA20 are most closely related to the reference. In contrast, they are less related to reference according to amino acid sequence. Additionally, azu gene sequences from PA1, PA12 and PA4 which were less related to the reference are clustered according to protein sequences into the same group that was closely related to the reference. Consequently, validation of the azurin function was studied through predicting its 3-D structure among the 8 Pseudomonas isolates besides the ST strain and reference sequence; it was found that the secondary structure of azurin from isolates PA2, PA4 and PA12 were compatible with the reference secondary structure and had highest antitumor activities as well. On the other hand, lower confidence percentages were observed in azurin secondary structures of isolates PA9 followed by PA11 and PA20 and also showed lowest antitumor activities. Finally, antimicrobial activity of azurin was studied against some pathogenic bacteria and fungi. It was found that the antifungal effect of azurin was significantly higher than its antibacterial activity (p<0.001). B. cereus and A. flavus were the most sensitive microorganisms to azurin with MIC values at 12.5 and 6.25 μg/ml, respectively.