Fungicidal activity of novel quinazolin-6-ylcarboxylates and mode of action on Botrytis cinerea.

BACKGROUND: Fungal diseases remain important causes of crop failure and economic losses. As the resistance toward current selective fungicides becomes increasingly problematic, it is necessary to develop efficient fungicides with novel chemotypes. RESULTS: A series of novel quinazolin-6-ylcarboxylates which combined the structures of pyridine or heterocyclic motif and the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety, a binding group of ATP-binding site of gefitinib, were evaluated for their fungicidal activity on different phytopathogenic fungi. Most of these compounds showed excellent fungicidal activities against Botrytis cinerea and Exserohilum rostratum, especially compound F17 displayed the highest activity with EC50 values as 3.79 µg mL-1 against B. cinerea and 2.90 µg mL-1 against E. rostratum, which was similar to or even better than those of the commercial fungicides, such as pyraclostrobin (EC50 , 3.68, 17.38 µg mL-1 ) and hymexazol (EC50 , 4.56, 2.13 µg mL-1 ). Moreover, compound F17 significantly arrested the lesion expansion of B. cinerea infection on tomato detached leaves and strongly suppressed grey mold disease on tomato seedlings in greenhouse. The abilities of compound F17 to induce cell apoptosis of the non-germinated spores, to limit oxalic acid production, to reduce malate dehydrogenase (MDH) expression, and to block the active pocket of MDH protein were demonstrated in B. cinerea. CONCLUSION: The novel quinazolin-6-ylcarboxylates containing ATP-binding site-directed moiety, especially compound F17, could be developed as a potential fungicidal candidate for further study. © 2023 Society of Chemical Industry.

Exposure to emamectin benzoate confers cytotoxic effects on human molt-4 T-cells and possible ameliorative role of vitamin E and dithiothreitol.

Emamectin benzoate (EMB) is an avermectin insecticide that is extensively used for pest control, but there are few reports concerning its cytotoxic effects on human lymphocytes. In the current study, the hematotoxicity of EMB was evaluated in Molt-4 T-cells, a human T-lymphoblastic cell line with high motility, and the role of vitamin E (VitE) and dithiothreitol (DTT) in attenuating EMB cytotoxicity was characterized. Exposure of Molt-4 cells to EMB decreased cell viability and proliferation, induced a loss of cell clusters, and significantly increased membrane collapse and chromatin condensation. Moreover, EMB significantly increased cell death and suppressed transglutaminase activity. EMB treatment modulated the NF-κB signaling pathway, decreased the expression of p105, p50, and p65/RelA in cytosolic and nuclear fractions, and increased nuclear IκBα expression. EMB increased oxidative stress, as demonstrated by a significant increase in the levels of reactive oxygen species (ROS). Treatment with non-cytotoxic concentrations of VitE or DTT ameliorated the hematotoxicity induced by pretreatment with EMB, increased Molt-4 cell viability, raised the IC50 values of EMB, limited intracellular ROS generation, and mitigated EMB-mediated effects on NF-κB signaling. The results indicate the potential cytotoxicity of EMB on human lymphocytes, and demonstrate that VitE and DTT treatment can reduce the cytotoxic effects of EMB.

The importance of selecting crystal form for triazole fungicide tebuconazole to enhance its botryticidal activity.

The growing evidences of resistant fungi stimulate fully understanding tebuconazole regarding its crystal structure on fungicidal activity. In this study, the crystal structures of six technical tebuconazoles (BX, HH, JP, QZ, SJ, and YT) were characterized by using high-resolution X-ray powder diffraction and three-dimensional crystal structure modeling. A structure-activity relationship of the tebuconazoles on the susceptible (HLS and YJS) or resistant (XHR) Botrytis cinerea isolates was analyzed, the differential tricarboxylic acid (TCA) cycle metabolism was determined, and molecular docking with sterol 14α-demethylase (CYP51) was performed. The results showed that tebuconazole existed in three types of crystal forms: an overlapping-pair conformation, a side-by-side-pair conformation, and a parallel-pair conformation. QZ with the parallel-pair conformation and the minimum crystal cell volume exhibited a higher activity and a lower resistant level. XHR possessed a higher content of TCA cycle metabolites and phosphate than YJS, but the exposure to QZ significantly reduced the contents of citrate, isocitrate, α-ketoglutarate and oxaloacetate in XHR, as did the exposure to other technical tebuconazoles. Moreover, the point mutations F487L, G464S, and G443S altered the binding properties of chiral stereoscopic R-QZ with CYP51 protein. Especially the G443S mutation promoted a weak linking of R-QZ with LEU380 and TYR126, and greatly slashed the binding action at lower docking score. In conclusion, our results evidenced an efficient crystal conformation of tebuconazole to improve botryticidal activity and a potential adaptability of B. cinerea to tebuconazole inhibition in TCA cycle metabolism and CYP51 protein mutation.

Novel Galactosyl Moiety-Conjugated Furylchalcones Synthesized Facilely Display Significant Regulatory Effect on Plant Growth.

The expansion of weed infestation has increased the demand on new herbicides. A series of novel galactosyl moiety-conjugated furylchalcones was facilely synthesized in which the furyl group (A ring) was combined with the substituted benzene group (B ring), and a galactosyl moiety was introduced. All these galactosyl furylchalcones were predicted to be phloem-mobile. Most of the galactosyl furylchalcones significantly promoted early seedling growth of sorghum and barnyardgrass under dark conditions, but all of them revealed considerable anti-growth ability on illuminated pot plants; especially, 1-(3'-(4″-O-ß-d-galactopyranosyl)furyl)-3-(4″-nitrophenyl)-2-en-1-one (B11) had a better herbicidal activity against rapeseed and Chinese amaranth than haloxyfop-R-methyl. The median efficient concentrations (EC50) of compound B11 against cucumber and wheat were 9.55 and 26.97 mg/L, respectively, also showing a stronger suppressing capacity than 2,4-D. Molecular docking with phosphoenolpyruvate carboxylase protein showed a stable binding conformation in which the galactosyl group interacted with LYS363 and GLU369, the furan ring and carbonyl bound with ARG184, and the crosslink of the nitro group with GLU240 formed a salt bridge. The results demonstrate that galactosyl furylchalcones possess the great potential as new herbicides for weed management, and further evaluations on more weeds are required for practical application.

The effect of α-tocopherol and dithiothreitol in ameliorating emamectin benzoate cytotoxicity in human K562 cells involving the modulation of ROS accumulation and NF-κB signaling.

Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (p < 0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.

Photosensitization of Chinese hamster V79 cells to photoactivated alpha-terthienyl involving membrane damage and oxidative stress.

Photosensitization of V79 Chinese hamster lung fibroblasts was tested to investigate if the cells can fit the photoactive effect of alpha-terthienyl for safety application. Using 15-min photoirradiation of a black light (320-400 nm, 40 W), alpha-terthienyl was significantly photoactivated and caused V79 cells to be shrinkage, detachment and necrosis. The photoactivated alpha-terthienyl played a concentration-dependent stress to decrease cell survival and to induce cell death with median inhibitory concentration (IC50) as 4.78 µg/ml. Cell viability in MTT assays also fell down to 10.58% of the control in the treatment of 10.0 µg/ml photoactivated alpha-terthienyl. As the irradiation time prolonged and the concentration of photoactivated alpha-terthienyl increased, cell death increased significantly, the intracellular level of reactive oxygen species (ROS) and the content of extracellular malondialdehyde were gradually increased. The changes of peroxidase, superoxide dismutase and catalase activities in V79 cells were positively responsive to the oxidative stress caused by photoactivated alpha-terthienyl. Moreover, using non-photosensitizing condition, the increased cell death and oxidative stress in the treatment of alpha-terthienyl at >7.0 µg/ml were also observed. The results showed the maladjustment response of V79 cells with membrane damage and cell death, clearly demonstrating the photosensitization of animal cells to the photoactivated cytotoxic effect of alpha-terthienyl.

Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae (Noctuidae, Lepidoptera).

A novel transglutaminase (MsTGase) from Mythimna separata larvae was separated and purified; its biochemical property and enzymatic catalytic activities were investigated. MsTGase was obtained chromatographically by the precipitation of Sephadex G-100 gel and DEAE-Cellulose-52 ion-exchange column with 48-fold purification and a reproducible yield of approximately 12%. Molecular weight of the MsTGase was 63.5 KDa and its N-terminal amino acid sequence was GKIEEG-LVI. Michaelis constant of the MsTGase for the substrate N-CBZ-Gln-Gly was 12.83mM with a Vmax of 7.99U/mL. Optimum conditions for MsTGase activity were at 42°C and pH7.5. The enzyme didn't possess metal ion at its catalytic active site; its activity could be significantly inhibited by Mg2+, but activated by Ca2+. Chlorpyrifos and spinosad showed a strong potential to increase MsTGase activity, supporting the view that MsTGase was a novel target. Moreover, the formation of intermolecular cross-links of casein and bovine serum albumin polymerized by MsTGase in the presence of DTT was observed. These findings pave the way for future studies on the physiological role of MsTGase and the potential impact of its regulation on MsTGase-associated pest management.

Emamectin benzoate induces ROS-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cells.

Emamectin benzoate (EMB), a novel macrocyclic lactone insecticide, possesses high efficacy and beneficial selective toxicity in agriculture, but so far the EMB-induced cytotoxic action in arthropod insect remains unclear. The present studies were carried out to characterize the property of EMB on the induction of reactive oxygen species (ROS)-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cell model. Following the exposure to EMB at 2.5, 5, 10 or 15 µM, the cells changed to be round, suspended and aggregated, and the decline of cell proliferating ability and cell viability was positively related with the exposure time. Median inhibitory concentration (IC50) of EMB on cell viability was 3.72 µM during 72 h exposure. Apoptosis was induced in 29.8% (24 h) and 39.5% (48 h) of the cells by EMB at 15 µM, showing chromatin condensation in nuclei. The content of ROS in the cells increased rapidly as the concentration of EMB increased, and the pre-incubation of the cells with vitamin E significantly reduced the ROS accumulation. In the treatment of 15 µM EMB, the migrated cell nucleus with DNA strand breaks appeared a teardrop, pear-shaped, or large fan-like tail, and 63.1% of γH2AX-positive cells contained more than four foci, accompanying with high expression level of caspase-3 in time-dependent manner, which consequently led to cell apoptotic death. These evidences in ROS-mediated DNA damage and cell apoptosis induced by EMB may be helpful for deep understanding the cytotoxic action of EMB based on cell model.

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10µM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10µM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca2+]i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia.

Antioxidative cellular response of lepidopteran ovarian cells to photoactivated alpha-terthienyl.

Photodynamic sensitizers as useful alternative agents have been used for population control against insect pests, and the response of insect ovarian cells towards the photosensitizers is gaining attention because of the next reproduction. In this paper, antioxidative responses of lepidopteran ovarian Tn5B1-4 and Sf-21 cells to photoactivated alpha-terthienyl (PAT) are investigated. PAT shows positive inhibitory cytotoxicity on the two ovarian cells, and its inhibition on cell viability is enhanced as the concentrations are increased and the irradiation time is extended. Median inhibitory concentrations (IC50) are 3.36µg/ml to Tn5B1-4 cells, and 3.15µg/ml to Sf-21 cells at 15min-UV-A irradiation 2h-dark incubation. Under 10.0µg/ml PAT exposure, 15min-UV-A irradiation excites higher ROS production than 5min-UV-A irradiation does in the ovarian cells, the maximum ROS content is about 7.1 times in Tn5B1-4 cells and 4.3 times in Sf-21 cells, and the maximum malondialdehyde levels in Tn5B1-4 and Sf-21 cells are about 1.47- and 1.36-fold higher than the control groups, respectively. Oxidative stress generated by PAT strongly decreases the activities of POD, SOD and CAT, and induces an accumulation of Tn5B1-4 cells in S phase and Sf-21 cells in G2/M phase in a concentration-dependent fashion. Apoptosis accumulation of Tn5B1-4 cells and the persistent post-irradiation cytotoxicity are further observed, indicating different antioxidative tolerance and arrest pattern of the two ovarian cells towards the cytotoxicity of PAT.

A comparative assessment of cytotoxicity of commonly used agricultural insecticides to human and insect cells.

The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (P<0.05). Methomyl, even 20µg/ml, showed little cytotoxicity at 24h exposure whereas emamectin benzoate possessed the strongest cytotoxic potential in a dose-dependent fashion. The results revealed comparable cytotoxic property of agricultural insecticides against intact cells.

Oxytetracycline biosynthesis improvement in Streptomyces rimosus following duplication of minimal PKS genes.

Oxytetracycline (OTC) is a widely used antibiotic, which is commercially produced by Streptomyces rimosus. The type II minimal polyketide synthases (minimal PKS) genes of the oxytetracycline biosynthesis cluster in S. rimosus, consisting of oxyA, oxyB and oxyC, are involved in catalyzing 19-C chain building by the condensation of eight malonyl-CoA groups to form the starting polyketide. This study aimed to investigate the effects of overexpression of the minimal PKS gene in a model S. rimosus strain (M4018) and in an industrial overproducer (SR16) by introduction of a second copy of the gene into the chromosome. Increased levels of oxyA, oxyB and oxyC gene transcription were monitored using reverse transcription quantitative real-time PCR. Overexpression of the minimal PKS gene elicited retardation of cell growth and a significant improvement in OTC production in corresponding mutants (approximately 51.2% and 32.9% in M4018 and SR16 mutants respectively). These data indicate that the minimal PKS plays an important role in carbon flux redirection from cell growth pathways to OTC biosynthesis pathways.

[Cloning and expression of the redox-sensing transcriptional repressor Rex and in vitro DNA-binding assay of the Rex and rex operator in Streptomyces rimosus M4018].

OBJECTIVE: The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018. METHODS: We cloned the rex of S. rimosus M4018 (Sr-rex) based on its homologoussequence in Streptomyces coelicolor A3(2) and its upstream rex operator (ROP) fragment using PCR and genome walking. An electrophoretic mobility shift assay (EMSA) was applied to analyze the regulation of rex to ROP in vitro. RESULTS: Sr-rex is 846 bp in length and has a 84% identity with the one in S. coelicolor A3(2) in amino acid sequence. It was deposited in Genbank under the accession number GQ849479. The expressed Sr-Rex by E. coli was mainly composed of alpha-helixes and beta-sheets, which was in compliance with the prediction. An Electrophoretic Mobility Shift Assay (EMSA) confirmed the specific binding activity of Sr-Rex with ROP. Meanwhile, we synthesized a 22 bp DNA fragment (ROP1) based on the minimal binding site of ROP. The maximal binding ratio of this fragment to Sr-Rex was 5:1 (molar). NADH negatively affected the binding activity, however, NAD+ had no impact on it. CONCLUSION: In S. rimosus M4018, the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD (H).

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway.

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.

[Biological characteristics of different forest soils in Nanjing-Zhenjiang mountan area].

Studies on the biological characteristics, including soil microbe, soil enzyme activity, soil nutritient content, and litter decomposition of different forest soils in Nanjing-Zhenjiang mountain area showed that the amounts of microbes and the activities of six enzymes in forest soils changed regularly in different forests during different months. The contents of nutritional elements varied regularly with forest growth bio-cycles, and were inerrelated prominently with the amounts of soil microbes and the activities of soil enzymes. There existed temporal-spatial differences in the decomposition rate of litters and the reverted velocity of nutrients among different forests. The comparisons of various biological characteristics among secondary Quercus variabilis forest, Phllostachys pubescens forest, and Cunninghamia lanceolata forest indicated that secondary Quercus variabilis forest had the most abundant nutrients in soil, and possessed the strongest ability of self-fertilization. Therefore, to construct coniferous forests with broadleaf trees in this area could avoid or abate the decline of soil fertility.