Background: Nowadays, the environmentally friendly procedures must be developed to avoid using harmful compounds in synthesis methods. Their increase interest in creating and researching silver nanoparticles (AgNPs) because of their numerous applications in many fields especially medical fields such as burn, wound healing, dental and bone implants, antibacterial, viral, fungal, and arthropodal activities. Biosynthesis of nanoparticles mediated pigments have been widely used as antimicrobial agent against microorganisms. Silver nanoparticles had synthesized by using melanin from locally isolate Pseudomonas aeruginosa, and used as antimicrobial activity against pathogenic microorganisms. Aim of the study: Isolation of Pseudomonas aeruginosa that produce melanin and extraction of melanin. Synthesis and characterization silver nanoparticle and study of the antimicrobial activity of silver nanoparticles in the presence of melanin against UTI pathogens. Materials and methods: The samples swab inoculated on cetrimide agar as selective media and incubated aerobically for 24 hours at 37 °C. Used nutrient agar with nutrient broth supplement with 1% tyrosine for screening for melanin production by P. aeruginosa isolates, silver nanoparticles synthesis from P. aeruginosa was done according to biological method and was characterized with AFM, UV-Visible, XRD, FTIR and FE-SEM. Agar well diffusion method was used to examine the effect of combination against UTI pathogens. Results: The synergistic effects of AgNPs and melanin were evaluated to compare between the two treatments (silver nanoparticles alone and combination of silver nanoparticles and melanin). The results revealed that the combination showed the highest antimicrobial activity in compare with silver nanoparticles alone.
The apoptotic activity of methionine γ- lyase from Pseudomonas putida on cancer cell lines was indicated by measuring the concentration of cytochrome c in the supernatants of cell lines. The result revealed high concentration of cytochrome c in the supernatants of cancer cell lines (RD, AMGM and AMN3) respectively while the concentration of anti-apoptotic protein (Bcl-2) was very low.
Many human malignant cell lines and primary tumours have absolute requirements for L-methionine (1). Under normal circumstances methionine comes from dietary proteins, most normal tissues can also synthesize methionine from either homocysteine or methylthioadenosine (2). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and became arrested in the late S/ G2 phase of the cell cycle (3).Methionine cleaving enzyme (methionine γ- lyase) has been found to be an effective antitumour agent in vitro as well as in vivo (4). Methionine γ- lyase is a pyridoxal 5-phosphate dependent enzyme that catalyzes the α, γ- elimination of L-methionine to α-ketobutyrate, methanethiol and ammonia (5). Rhabdomyosarcoma (RD) cell line at passage 65, AhmedMohammed-Nahi-2003(AMN3) cell line at passage 180, AMGM (Ahmed – Majeed-Glioblastoma-Multiform) cell line at passage 65 and normal Rat Embryo Fibroblast (REF) cell line at passage 56, kindly provided by Iraqi center for cancer and medical genetics research (ICCMGR) were cultured in PRMI 1640 medium supplement with 10% heat inactivated fetal calf serum and incubated at 37 ºC with 5% CO2. Examination of cytotoxicity of methionine γ- lyase Cytotoxicity was determined with the crystal violet stain as previously described (7). In briefly , 1x105 cell/ml was seeded onto 96-well culture plates in 200 µl RPMI 1640 medium and incubated until the cell reached confluent monolayer(vary according to the kind of cell-line). After incubation period the medium was removed and 200 μl of various concentrations (1000, 500,250 and 125 μg/ml) from methionine γ- lyase were added to the plate by using 3- well replicates for each concentration. The plates were incubated at 37ºC for 96 hr. The control (cancer cell lines without treatment with enzyme) (3-well replicates) was treated with 200 μl of serum free medium and incubated at 37ºC for 96 hr. At the end of incubation period, the enzyme and medium was removed from plate and washed with PBS to remove unattached (dead) cells. Two hundred μl of crystal violet was added to each well and left for 20 min at 37ºC.The stain was removed by washing with tap water several times, and the plates were left to dry. After drying, each plate was read by using ELISA microplate spectrophotometer at 492nm wave length. The percentages of Inhibitory Rate (IR %) were estimated (8).
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