Green Synthesis of Silver Nanoparticles with Antimicrobial Properties Using Phyla dulcis Plant Extract.

Foodborne illnesses caused by the consumption of food contaminated with foodborne pathogens at infectious doses are becoming a common health issue throughout the world. Metal nanoparticles with potential antimicrobial properties are an area that can be explored to discover novel antimicrobial agents. The traditional synthesis methods of metal nanoparticles involve the use of toxic chemicals and the generation of harmful byproducts. In this study, a greener method to synthesize silver nanoparticles (AgNPs) with potential antimicrobial properties was investigated. The aqueous extract of the medicinal plant Phyla dulcis Trev. (verbenaceae) was used as the reducing and stabilizing agent to synthesize AgNPs using microwave irradiation. The formation of AgNPs was confirmed using ultraviolet-visible spectroscopy by the appearance of characteristic surface plasmon resonance peaks in the 430-440 nm wavelength range. The size and stability of the AgNPs were studied using Zetasizer nano-series for 5 weeks after synthesis. The average particle size remained between 63 and 76 nm during the first 4 weeks and increased to 114 nm in the fifth week showing possible aggregation after the fourth week. The zeta potential remained between -20 and -24 mV throughout the 5 weeks showing relatively good stability. Scanning electron microscopy/energy dispersive X-ray spectroscopy showed the association of phytoconstituents with the AgNPs. X-ray photoelectron spectroscopy analysis confirmed the formation of metallic nanoparticles starting from silver nitrate. Finally, the AgNPs were tested to be effective against Escherichia coli O157:H7 (ATCC 43888), Salmonella Typhimurium (novobiocin and nalidixic acid resistant strain), Listeria monocytogenes (4b; ATCC 19115), and Staphylococcus aureus (ATCC 6538) strains, which are known to be common foodborne pathogens.

Molecular modeling of metabolism for allergen-free low linoleic acid peanuts.

It is necessary to eliminate linoleic acid and allergenic arachins from peanuts for good health reasons. Virginia-type peanuts, harvested from plots treated with mineral salts combinations that mimic the subunit compositions of glutamate dehydrogenase (GDH) were analyzed for fatty acid and arachin compositions by HPLC and polyacrylamide gel electrophoresis, respectively. Fatty acid desaturase and arachin encoding mRNAs were analyzed by Northern hybridization using the homologous RNAs synthesized by peanut GDH as probes. There were 70-80 % sequence similarities between the GDH-synthesized RNAs and the mRNAs encoding arachins, fatty acid desaturases, glutamate synthase, and nitrate reductase, which similarities induced permutation of the metabolic pathways at the mRNA level. Modeling of mRNAs showed there were 210, 3,150, 1,260, 2,520, and 4,200 metabolic permutations in the control, NPKS-, NS-, Pi-, NH(4)Cl-, and PK-treated peanuts, respectively. The mRNA cross-talks decreased the arachin to almost zero percent in the NPKS- and PK-treated peanuts, and linoleate to ~18 % in the PK-treated peanut. The mRNA cross-talks may account for the vastly reported environmentally induced variability in the linoleate contents of peanut genotypes. These results have quantitatively unified molecular biology and metabolic pathways into one simple biotechnology for optimizing peanut quality and may encourage small-scale industry to produce arachin-free low linoleate peanuts.

RNA synthetic activity of glutamate dehydrogenase: determination of enzyme purity, RNA characteristics, and deamination/amination ratio.

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.

Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.