Stable Silver Nanoparticles Synthesis by Citrus Sinensis (Orange) and Assessing Activity Against Food Poisoning Microbes / 生物医学与环境科学（英文）

Silver nanoparticles are considered as good antimicrobial agent. AgNPs were synthesized by mixing silver nitrate solution with citrus sinesis extract for 2 h at 37 °C and analyzed by UV-visible spectra, SEM, XRD, and FTIR. AgNPs were tested against B. subtilis, Shigella, S. aureus, and E. coli. Minimum inhibitory concentration of AgNPs was 20 µg/mL for B. subtilis and Shigella and 30 µg/mL for S. aureus and E. coli. Antibiofilm activity (80% to 90%) was observed at 25 µg/mL. AgNPs were stable for five months with sustained antimicrobial activity. Biosynthesized AgNPs can be used to inhibit food poisoning microbial growth.

Anticancer medicines (Doxorubicin and methotrexate) conjugated with magnetic nanoparticles for targeting drug delivery through iron.

The uptake of iron is increased by cancer cells. Iron magnetic nanoparticles (MNP) can be used as a nanovehicle for immobilization of anticancer medicines and to integrate them at a target site. The anticancer medicines doxorubicin (DOX) and methotrexate (MTX) were immobilized separately and in combination onto MNP by a glutaraldehyde activation method and confirmed by magnetic nanoparticles linked immunosorbent assay (MagLISA) and Fourier-transform infrared (FTIR) spectroscopy. The phenol peaks of DOX and MTX at 2896.6 cm⁻¹ to 2912.5 cm⁻¹ in FTIR spectra of immobilized medicines indicated the conjugation. Affinity-purified anti-DOX and anti-MTX antibodies were used to evaluate the coupling of DOX and MTX onto MNP, and the binding was found 34.6% to 37.2% and 51.8% to 54.3% separately, respectively. The immobilization of DOX and MTX in combination onto MNP was 18% and 27%, respectively. HeLa and B cells were cultured with DOX-MNP, MTX-MNP, and DOX-MNP-MTX separately, and MagLISA indicated that the binding of DOX-MNP/MTX-MNP was 41.5% to 45% with HeLa cells and 20% to 26% with B cells. No significant difference was observed in binding of DOX-MNP-MTX with HeLa and B cells. Results also indicated that the release of medicines at pH 5.0 is more (39% to 44%) than at pH 7.4 (3.7% to 10.2%). Sixteen to 22% more killing effect was observed on HeLa cells than on B cells. In immunohistochemical staining, more deposition of brown color on HeLa cells than on B cells may be due to more expression of iron-binding sites on cancer cells. The dual property of MNP can be used for binding of medicines and for targeting drug delivery.

Biocatalytic activity of recombinant human ß-mannosidase immobilized onto magnetic nanoparticles for bioprocess.

Recombinant human ß-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.

Determination of oxygen derived free radicals producer (xanthine oxidase) and scavenger (paraoxonase1) enzymes and lipid parameters in different cancer patients.

BACKGROUND: Cancer is a major cause of death throughout the world. Xanthine oxidase (XO) is considered an oxygen derived free radicals producer and paraoxonasel (PON1) is known as a free radicals scavenger. This study was undertaken to determine the activity of PON1 and XO and their concentration along with the lipid profile, lipid peroxide, and thiol level in cancer patients and healthy persons. METHODS: Antigen competitive ELISA was used to determine the level of paraoxonasel and xanthine oxidase in plasma. Paraoxonase and arylesterase activity of the PON1 enzyme and the activity of the XO enzyme were also determined. PON1 and XO activity, cholesterol, LDL-C, HDL-C, lipid peroxides, and thiol level in patients with breast cancer (n = 50), prostate cancer (n = 20), lung cancer (n = 12,), cervix cancer (n = 15), Non-Hodgkin lymphoma (n = 10), and acute lymphoblastic lymphoma (n = 8) and total age matched healthy persons (n = 115) were studied. RESULTS: Low plasma paraoxonase/arylesterase activities (p < 0.05) and the concentration of the PON1 enzyme were observed in all cancer patients. An elevated level of XO activity was observed in cancer patients. Among different cancers, comparatively high level of XO activity was noted in acute lymphoblastic lymphoma patients (0.18 +/- 0.03, p = 0.001) except cervix cancer patients (0.053 +/- 0.03, p = 0.0029) where a low level was observed. Low HDL-C (p < 0.01), cholesterol (p < 0.01), triglycerides (p < 0.01), thiol levels (p < 0.01) and high lipid peroxide levels (p > 0.05) were observed in cancer patients. CONCLUSIONS: Lower PON1 and high XO enzyme activities cause an imbalance of the free radical system which enhances the lipid peroxidation and other pathological conditions. Lower HDL-C level is also indicative of the low level of PON1 enzyme activity.

PCR assay targeting virulence genes of Helicobacter pylori isolated from drinking water and clinical samples in Lahore metropolitan, Pakistan.

Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and adenocarcinoma and its high infection rate is observed in overcrowded and lower socioeconomic groups in developing countries. This study was designed to identify the role of drinking water in the transmission and prevalence of H. pylori (HP). Selective HP medium was developed for enrichment and presumptive identification of H. pylori by urease, catalase and species specific 16S rRNA tests. The virulence genes (vacA 's' and 'm' regions and cagA) of H. pylori in 90 out of 225 H. pylori positive drinking water samples were present (40%). Ten out of 18 biopsies (55.55%) and 15 out of 50 vomiting fluids of gastric disease patients (30%) were also positive for virulence genes. Anti-H. pylori antibodies were also detected in 31 out of 50 patients' sera. The presence of virulence genes was also directly confirmed by hybridization studies using non-radioactive DNA probes of 16S rRNA, vacA and cagA genes. The presence of H. pylori in water is due to poor sanitary conditions, improper waste disposal and lack of public health education. PCR-based analysis and colony hybridization can be used for detection of H. pylori in clinical and environmental samples.

Immunobiochemical analysis of Paraoxonase1 (anti-oxidant), xanthine oxidase (oxidant) enzymes and lipid profile of cardiac disease patients in Lahore Metropolitan, Pakistan.

Cardiac diseases are the major cause of death. Paraoxonase1 (PON1) is known as free radicals scavenger/anti-atherosclerosis, whereas xanthine oxidase (XO) is a free radicals generator. This study was undertaken to determine and compare the Paraoxonase and arylesterase activities of PON1 enzyme and activity of XO enzyme. The concentration of XO and PON1 enzymes along with lipid profile, lipid peroxides, and thiol level in plasma of cardiac patients (n=200) and healthy persons (n=200) of Lahore metropolitan, Pakistan was also determined. Anti-PON1 and anti-XO antibodies were developed, purified, and used to measure the concentration of PON1 and XO by competitive ELISA. It is observed that low paraoxonase (P=0.0073)/arylesterase activity (P=0.0038) of PON1 enzyme and its low concentration (P=0.0049) were observed in cardiac patients, whereas elevated level of XO activity (P=0.0129) and its concentration (P=0.0097) was observed in cardiac patients as compared with healthy persons. Low levels of HDL (P=0.0013), thiol (P=0.0014) and high level of cholesterol (P=0.0025), triglycerides (P=0.0018), LPO (P=0.0014), and LDL level (P=0.05) were observed in cardiac patients admitted in intensive care unit as compared with hypertensive patients and control subjects. It is concluded that overall low PON1 and high XO activities do cause imbalance of free radical system which ultimately leads to or enhance the cardiac pathological conditions.

Synthesis of cholesterol-conjugated magnetic nanoparticles for purification of human paraoxonase 1.

Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe(3)O(4) nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm(-1), and at 2,334.9 cm(-1) confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg(-1) protein, and the purification fold was 515 with 73% yield. The K (m) values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg(+2), Mn(+2), glycerol, (NH(4))(2)SO(4), PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb(+2), Co(+2), Zn(2+), ethanol, beta-mercaptoethanol, and acetone reduced the activity while Ni(2+), Cd(2+), Cu(2+), iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml(-1), respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.

PCR targeting of antibiotic resistant bacteria in public drinking water of Lahore metropolitan, Pakistan.

OBJECTIVE: To investigate the prevalence of kanamycin (kan) and ampicillin (amp) resistant bacteria in public drinking water. METHODS: Bacteria containing kan and amp resistant genes were amplified by PCR and further characterized by colony hybridization and transformation studies. The genus of kan and amp resistant bacteria was determined with standard methods. RESULTS: Among the 625 drinking water samples, 400 contained kan and amp resistant bacteria and the percentage was 42.5% and 57.5%, respectively, which was further confirmed by the amplification of a 810 bp kan resistant gene and a 850 bp amp resistant gene. Of the 170 kan resistant bacteria, 90 were Gram negative and 80 were Gram positive. Of the 230 amp resistant bacteria, 160 were Gram negative while 70 were Gram positive. Salmonella, Shigella, Staphylococcus, Streptococcus, and E.coli were detected as 13%, 11%, 17%, 30%, and 29%, respectively. Bacterial strain DH5alpha transformed with plasmids isolated from kan and amp resistant bacteria confirmed that the antibiotic resistant genes were mediated by plasmids. CONCLUSION: Drinking water is contaminated with kan and amp resistant bacteria due to poor sanitary conditions.