Antimicrobial activity and mode of action of terpene linalyl anthranilate against carbapenemase-producing Klebsiella pneumoniae / 药物分析学报

Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance.This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate(LNA)against carbapenemase-producing Klebsiella pneumoniae(KPC-KP).LNA alone exhibited bacteri-cidal activity at 2.5％(V/V),and in combination with meropenem(MPM)at 1.25％(V/V).Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins,indicating membrane damage in LNA-treated KPC-KP cells.Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress.Zeta po-tential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability.Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells,inferring membrane damage.Furthermore,intracel-lular leakage of nucleic acid and proteins was detected upon LNA treatment.Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular ma-terials.LNA was found to induce oxidative stress by generating reactive oxygen species(ROS)that initiate lipid peroxidation and damage the bacterial membrane.In conclusion,LNA generates ROS,initiates lipid peroxidation,and damages the bacterial membrane,resulting in intracellular leakage and eventually killing the KPC-KP cells.

A Review of Formation, Toxicity of Reactive Oxygen Species by Heavy Metals and Tolerance in Plants

Aims: This whole review tells about origin of ROS, Cell death, role of ROS, defense system in numerous developmental pathways.Methodology: In plant reactive oxygen species are the main factor of cell mechanism deterioration. The stability of the reduction and regeneration is disturbed under stress environment. ROS continuously damages the main organelles of cell in plants as well as inactivate several enzymes. Break down of lipids, nucleic acid, proteins, pigments, damages in membrane which may lead to cell death.Conclusion: ROS is diffusible measures in pathways of signal transduction in numerous developmental pathways in plants. ROS acts as a messenger. Plants protect cell from misbalancing and also damages ROS production. ROS produced in excess amount rather than required for numerous metabolic reaction. In aerobic respiration ROS is produced

Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A2

Background Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 (Lys49-PLA2) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation. Methods Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2 myotoxicity. In addition, K115A and K116A mutants - which contribute to decrease cytotoxicity - and the K122A mutant - which decreases both myotoxicity and cytotoxicity - were also used. The H48Q mutant - which does not interfere with membrane damage or myotoxic activity - was used to evaluate if the PLA2 catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls. Results Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response. Conclusions The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain.(AU)

Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A2

Abstract Background Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 (Lys49-PLA2) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation. Methods Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2 myotoxicity. In addition, K115A and K116A mutants which contribute to decrease cytotoxicity and the K122A mutant which decreases both myotoxicity and cytotoxicity were also used. The H48Q mutant which does not interfere with membrane damage or myotoxic activity was used to evaluate if the PLA2 catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls. Results Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response. Conclusions The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain.

Do seed VLCFAs trigger spongy tissue formation in Alphonso mango by inducing germination.

Spongy tissue is a physiological disorder in Alphonso mango caused by the inception of germination-associated events during fruit maturation on the tree, rendering the fruit inedible. Inter-fruit competition during active fruit growth is a major contributing factor for the disorder which leads to reduced fat content in spongy tissue affected fruits. This study was, therefore, carried out to determine the possible association between seed fats and ST formation. The study of the fat content during fruit growth showed that it increased gradually from 40% fruit maturity. At 70% maturity, however, there was a sudden increase of fat content of whole fruit, leading to acute competition and resulting in differential allocation of resources among developing fruits. As a result, the seed in spongy-tissue-affected mature ripe fruit showed a marked drop in the levels of fats and the two very long chain fatty acids (VLCFAs), tetracosanoic acid and hexacosanoic acid together with an increase of linolenic acid and a fall in oleic acid contents, which are known to be key determinants for the initiation of pre-germination events in seed. Subsequently, a rise in the level of cytokinin and gibberellins in ST seed associated with a fall in abscisic acid level clearly signalled the onset of germination. Concurrently, a significant reduction in the ratio of linolenic acid/linoleic acid in pulp led to the loss of membrane integrity, cell death and the eventual formation of spongy tissue. Based on the above, it is concluded that a significant reduction in the biosynthesis of VLCFAs in seeds during fruit growth might trigger pre-germination events followed by a cascade of biochemical changes in the pulp, leading to lipid peroxidation and membrane injury in pulp culminating in ST development. Thus, this study presents crucial experimental evidence to highlight the critical role played by VLCFAs in inducing ST formation in Alphonso mango during the pre-harvest phase of fruit growth.