The increasing number of multi-drug-resistant bacteria and cancer cases, that are a real threat to humankind, forces research world to develop new weapons to deal with it. Biogenic silver nanoparticles (AgNPs) are considered as a solution to this problem. Biosynthesis of AgNPs is regarded as a green, eco-friendly, low-priced process that provides small and biocompatible nanostructures with antimicrobial and anticancer activities and potential application in medicine. The biocompatibility of these nanoparticles is related to the coating with biomolecules of natural origin. The synthesis of AgNPs from actinobacterial strain was confirmed using UV-Vis spectroscopy while their morphology, crystalline structure, stability, and coating were characterized using, transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta potential and Fourier transform infrared spectroscopy (FTIR). Antibacterial activity of biogenic AgNPs was evaluated by determination of minimum inhibitory and minimum biocidal concentrations (MIC and MBC) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The potential mechanism of antibacterial action of AgNPs was determined by measurement of ATP level. Since the use of AgNPs in biomedical applications depend on their safety, the in vitro cytotoxicity of biosynthesized AgNPs on MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell lactate dehydrogenase (LDH) release and measurement of reactive oxygen species (ROS) level were assessed. The nanoparticle protein capping agent that can be involved in reduction of silver ions to AgNPs and their stabilization was identified using LC-MS/MS. Nanoparticles were spherical in shape, small in size (mean 13.2 nm), showed crystalline nature, good stability (−18.7 mV) and presence of capping agents. They exhibited antibacterial activity (MIC of 8–128 μg ml−1, MBC of 64–256 μg ml−1) and significantly decreased ATP levels in bacterial cells after treatment with different concentrations of AgNPs. The in vitro analysis showed that the AgNPs demonstrated dose-dependent cytotoxicity against RAW 264.7 macrophages and MCF-7 breast cancer cells but higher against the latter than the former. Cell viability decrease was found to be 42.2–14.2 and 38.0–15.5% while LDH leakage 14.6–42.7% and 19.0–45.0%, respectively. IC50 values calculated for MTT assay was found to be 16.3 and 12.0 μg ml−1 and for LDH assay 102.3 and 76.2 μg ml−1, respectively. Moreover, MCF-7 cells released a greater amount of ROS than RAW 264.7 macrophages during stimulation with all tested concentrations of AgNPs (1.47–3.13 and 1.02–2.58 fold increase, respectively). The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis revealed the presence of five protein bands at a molecular weight between 31.7 and 280.9 kDa. These proteins showed the highest homology to hypothetical proteins and porins from E. coli, Delftia sp. and Pseudomonas rhodesiae. Based on obtained results it can be concluded that biogenic AgNPs were capped with proteins and demonstrated potential as antimicrobial and anticancer agent.
Abscission is a natural process that occurs to facilitate shedding of no longer needed organs, but on the other hand, can be triggered by certain environmental conditions, e.g. biotic or abiotic stresses. Regardless of the stimuli, organ shedding takes place specifically at the abscission zone (AZ). A signaling pathway that controls this process in Arabidopsis thaliana from ligand to receptors has been proposed. However, knowledge concerning the influence of plant hormones on these molecular elements still remains enigmatic. Excessive and premature flower abscission in the crop species Lupinus luteus L. is a process of substantial interest to the agricultural industry, as it can affect yield. Our strategy combined molecular studies, comprehensive ultrastructural and histological analysis, as well as exogenous hormone treatment to describe the contribution of the Lupinus IDA-like gene in flower abscission. In the AZ of the naturally abscised flowers, the differentiation of morphologically distinct cells characterized by progressive degradation processes was accompanied by LlIDL mRNA accumulation. A similar effect was observed following early steps of AZ activation and after abscisic acid or ethylene treatments. These phytohormones, previously pointed out as key stimulators of flower separation, altered the temporal expression pattern of LlIDL. Exogenous EPIP peptide synthesized on the basis of LlIDL sequence, significantly increased flower abortion rate, which indicates that this motif governs protein activity. In conclusion, our data provide new evidence for LlIDA involvement in both the early and late events of flower abscission supported by detailed spatiotemporal characterization of AZ cell structure and ultrastructure.
Density functional theory (DFT) quantum chemical calculations of the structure and NMR parameters for highly strained hydrocarbon [2.2]paracyclophane 1 and its three derivatives are presented. The calculated NMR parameters are compared with the experimental ones. By least-squares fitting of the (1)H spectra, almost all J(HH) coupling constants could be obtained with high accuracy. Theoretical vicinal J(HH) couplings in the aliphatic bridges, calculated using different basis sets (6-311G(d,p), and Huz-IV) reproduce the experimental values with essentially the same root-mean-square (rms) error of about 1.3 Hz, regardless of the basis set used. These discrepancies could be in part due to a considerable impact of rovibrational effects on the observed J(HH) couplings, since the latter show a measurable dependence on temperature. Because of the lasting literature controversies concerning the symmetry of parent compound 1, D(2h) versus D(2), a critical analysis of the relevant literature data is carried out. The symmetry issue is prone to confusion because, according to some literature claims, the two hypothetical enantiomeric D(2) structures of 1 could be separated by a very low energy barrier that would explain the occurrence of rovibrational effects on the observed vicinal J(HH) couplings. However, the D(2h) symmetry of 1 with a flat energy minimum could also account for these effects.
Summary UDP-glycosyltransferases (GTases, UGT) catalyze the transfer of the sugar moiety from the uridine-diphosphate-activated monosaccharide (e.g. uridine-diphosphate-5’-glucose, UDPG) molecule to the specific acceptor. Glycosides contain aglycons attached by a β-glycosidic bond to C1 of the saccharide moiety. Glycosylation is one of the mechanisms maintaining cellular homeostasis through the regulation of the level, biological activity, and subcellular distribution of the glycosylated compounds. The glycosides play various functions in plant cells, such as high-energy donors, or signalling molecules, and are involved in biosynthesis of cell walls. Plant cells exhibit structural and functional diversity of UGT proteins. The Arabidopsis thaliana genome contains more than 100 genes encoding GTases, which belong to 91 families, and are deposited in the CAZY (Carbohydrate Active enzyme) database (www. cazy.org/GlycosylTransferases.html). The largest UGT1 class is divided into 14 subfamilies (A-N), and includes proteins containing highly conserved 44-amino acid PSPG (Plant Secondary Product Glycosyltransferase) motif at the C-terminus. The PSPG motif is involved in the binding of UDP-sugar donors to the enzyme. UGT1’s catalyze the biosynthesis of both ester-type and ether-type conjugates of plant hormones (phytohormones). Conjugation of the phytohormones is an important mechanism that regulates the concentration of physiological active hormone levels during growth and development of plants. Glycoconjugation of phytohormones is widespread in the plant kingdom and all known phytohormones are able to form these conjugates. Most plant hormone conjugates do not indicate physiological activity, but rather are involved in transport, storage and degradation of the phytohormones. UDPG-dependent glycosyltransferases possess high substrate specificity, even within a given class of phytohormones. In many cases, the phenotype of plants is strongly affected by loss-of-function mutations in UGT genes. In this paper, advances in the isolation and characterization of glycosyltransferases of all plant hormones: auxin, brassinosteroids, cytokinin, gibberellin, abscisic acid, jasmonates, and salicylate is described
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