Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still li...Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the anti-fungal mechanism of Neosartorya fischeri antifungal protein （NFAP）, a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting- effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP acti- vates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any in- fluence on the cell wall integrity pathway, but an un- known cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken to- gether, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related pro-teins from Aspergillus giganteus and Penicillium chrysogenum.展开更多
Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating succ...Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.展开更多
目的:探讨脾气虚证大鼠与鱼藤酮损伤大鼠之间下丘脑c AMP/PKA信号通路变化。方法:30只SPF级雄性大鼠,随机分为正常组,脾气虚组,鱼藤酮高剂量组(2.0 m L/kg),中剂量组(1.5 m L/kg),低剂量组(1.0 m L/kg),每组6只。采用ELISA法检测各组大...目的:探讨脾气虚证大鼠与鱼藤酮损伤大鼠之间下丘脑c AMP/PKA信号通路变化。方法:30只SPF级雄性大鼠,随机分为正常组,脾气虚组,鱼藤酮高剂量组(2.0 m L/kg),中剂量组(1.5 m L/kg),低剂量组(1.0 m L/kg),每组6只。采用ELISA法检测各组大鼠下丘脑组织c AMP活性,Real Time PCR法和Westernblot法检测蛋白激酶(cyclic-AMP-dependent protein kinase,PKA)、糖原磷酸化酶(glyeogenphospholase,Gp)、磷酸化酶激酶(phosphorylase kinase,PHK)的mRNA及其表达量。结果:与正常组大鼠比较,脾气虚证组及鱼藤酮3组大鼠的下丘脑组织c AMP活性及PKA、Gp、PHK mRNA和蛋白的表达均有不同程度的减弱,鱼藤酮中剂量组的结果与脾气虚证组最为相似。结论:脾气虚证大鼠与鱼藤酮损伤大鼠之间下丘脑c AMP/PKA信号通路的变化存在相关性,其中c AMP-PKA信号通路可能参与调控。展开更多
Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection.However,the regulatory mechanism of lipid droplets formation and the utilization during fungal developmen...Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection.However,the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown.Here,in Magnaporthe oryzae,we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization.Nem1 was highly expressed in conidia,but lowly expressed in appressoria,and its encoded protein was localized to lipid droplets.Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol(DAG)or triacylglycerol(TAG).NEM1 was required for asexual development especially conidia production.TheΔnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth.Remarkably,Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process.The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1.Together,our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus.展开更多
Docosahexaenoic acid(DHA)is a biologically active fatty acid that reduces the accumulation of lipids.However,the molecular mechanism underlying this process,particularly in fish,is not well understood.Recent studies s...Docosahexaenoic acid(DHA)is a biologically active fatty acid that reduces the accumulation of lipids.However,the molecular mechanism underlying this process,particularly in fish,is not well understood.Recent studies show that endoplasmic reticulum(ER)stress triggers the activation of the unfolded protein response,which has been revealed to play an essential role in lipid metabolism.In this study,we explored the effect of DHA on ER stress and investigated the potential molecular mechanisms underlying DHA-induced adipocyte lipolysis in grass carp(Ctenopharyngodon idella)both in vivo and in vitro.We found that DHA remarkably reduced the triglyceride content,increased the secretion of glycerol,pro-moted lipolysis in adipocytes and evoked ER stress,whereas inhibiting ER stress using 4-phenyl butyric acid(4-PBA)inhibited the effects of DHA(P<0.05).These results implied that ER stress potentially participates in DHA-induced adipocyte lipolysis.Additionally,STF-083010,a specific inositol-requiring enzyme 1a(IRE1a)-inhibitor,attenuated the effects of DHA on lipolysis,demonstrating that IRE1a and X-box binding protein 1 potentially participate in DHA-induced lipolysis.DHA also activated the cyclic adenosine monophosphate(cAMP)-dependent protein kinase A(PKA)pathway by increasing the level of cAMP and activating the PKA enzyme(P<0.05).Nevertheless,H89,a PKA inhibitor,weakened DHA-induced lipolysis by inhibiting the cAMP/PKA signaling pathway.Furthermore,inhibiting ER stress us-ing 4-PBA also inhibited lipolysis and alleviated DHA-induced activation of the cAMP/PKA signaling pathway,suggesting that ER stress may participate in DHA-induced lipolysis through the activation of the cAMP/PKA signaling pathway.Our data illustrate that DHA supplementation can be a promising nutritional strategy for ameliorating lipid accumulation in grass carp.The present study elucidated the molecular mechanism for DHA-induced lipolysis in grass carp adipocytes and emphasized the impor-tance of ER stress and the cAMP/PKA pathway in DHA-induced lip展开更多
Objective:To investigate the mechanism of c AMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction(芍药甘草汤,SGD)on the regulation of aquaporin 5(AQP5)and muscarinic receptor 3(M3 R)l...Objective:To investigate the mechanism of c AMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction(芍药甘草汤,SGD)on the regulation of aquaporin 5(AQP5)and muscarinic receptor 3(M3 R)levels in Sjogren’s syndrome(SS).Methods:Of the 30 mice,5 were randomly selected as control,and others were used for creating SS model.After successful modeling,mice were randomly divided into 5 groups(n=5 per group)and intragastrical y administered with saline(8 m L/kg),pilocarpine(1.4 mg/kg),or low,medium and high doses SGD(0.14,0.21,0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae,respectively)for 6 weeks.Human labial gland acinar cel s were treated with pilocarpine or varying doses of SGD with saline as the placebo.Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice.The serum levels of anti-SS antigen A(SS-A),anti-SS antigen B(SS-B),M3 R,andα-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay.Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3 R in acinar cells.Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA,c AMP,Epac1,AQP5,M3 R,nuclear factor kappa-B(NF-κB),and tumor necrosis factor(TNF)-αin submandibular gland tissues and cel s of each group.Results:Compared to normal mice,body weight,5-min salivary secretion,30-min secretion of tears and breakup time of tear film of model mice decreased at 1–6 weeks after immunization(al P<0.05),whereas water intake increased(al P<0.05).In the model group,glands of the submandibular glands showed atrophy,accompanied by acini of different sizes,decreased numbers and loose arrangement,with catheter dilatation and different degrees of lymphocyte infiltration.Conditions of mice in SGD groups were improved.The positive expression of AQP5 and M3 R were higher in the acinar cel s treated with al doses SGD compared to the normal group;serum levels of展开更多
文摘Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the anti-fungal mechanism of Neosartorya fischeri antifungal protein （NFAP）, a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting- effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP acti- vates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any in- fluence on the cell wall integrity pathway, but an un- known cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken to- gether, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related pro-teins from Aspergillus giganteus and Penicillium chrysogenum.
基金supported by the National Natural Science Foundation of China(31970472,32272547)the National Science Fund of Henan Province for Distinguished Young Scholars,China(202300410191)+3 种基金the Basic Research Project of the Key Scientific Research Projects of Universities in Henan Province,China(21zx013)the Henan Agricultural Research System,China(HARS-2209-G3)the Henan Special Support for High-Level Talents Central Plains Science and Technology Innovation Leading Talents,China(224200510018)the earmarked fund for China Agricultural Research System(CARS-27)。
文摘Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.
文摘目的:探讨脾气虚证大鼠与鱼藤酮损伤大鼠之间下丘脑c AMP/PKA信号通路变化。方法:30只SPF级雄性大鼠,随机分为正常组,脾气虚组,鱼藤酮高剂量组(2.0 m L/kg),中剂量组(1.5 m L/kg),低剂量组(1.0 m L/kg),每组6只。采用ELISA法检测各组大鼠下丘脑组织c AMP活性,Real Time PCR法和Westernblot法检测蛋白激酶(cyclic-AMP-dependent protein kinase,PKA)、糖原磷酸化酶(glyeogenphospholase,Gp)、磷酸化酶激酶(phosphorylase kinase,PHK)的mRNA及其表达量。结果:与正常组大鼠比较,脾气虚证组及鱼藤酮3组大鼠的下丘脑组织c AMP活性及PKA、Gp、PHK mRNA和蛋白的表达均有不同程度的减弱,鱼藤酮中剂量组的结果与脾气虚证组最为相似。结论:脾气虚证大鼠与鱼藤酮损伤大鼠之间下丘脑c AMP/PKA信号通路的变化存在相关性,其中c AMP-PKA信号通路可能参与调控。
基金supported by the National Natural Science Foundation of China(Grants 32072365 and 32272476)the Open Research Fund of the State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China(SKL-KF202216).
文摘Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection.However,the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown.Here,in Magnaporthe oryzae,we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization.Nem1 was highly expressed in conidia,but lowly expressed in appressoria,and its encoded protein was localized to lipid droplets.Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol(DAG)or triacylglycerol(TAG).NEM1 was required for asexual development especially conidia production.TheΔnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth.Remarkably,Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process.The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1.Together,our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus.
基金supported by the National Nature Science Foundation of China(NSFC,Grant Number:31772863,32072989).
文摘Docosahexaenoic acid(DHA)is a biologically active fatty acid that reduces the accumulation of lipids.However,the molecular mechanism underlying this process,particularly in fish,is not well understood.Recent studies show that endoplasmic reticulum(ER)stress triggers the activation of the unfolded protein response,which has been revealed to play an essential role in lipid metabolism.In this study,we explored the effect of DHA on ER stress and investigated the potential molecular mechanisms underlying DHA-induced adipocyte lipolysis in grass carp(Ctenopharyngodon idella)both in vivo and in vitro.We found that DHA remarkably reduced the triglyceride content,increased the secretion of glycerol,pro-moted lipolysis in adipocytes and evoked ER stress,whereas inhibiting ER stress using 4-phenyl butyric acid(4-PBA)inhibited the effects of DHA(P<0.05).These results implied that ER stress potentially participates in DHA-induced adipocyte lipolysis.Additionally,STF-083010,a specific inositol-requiring enzyme 1a(IRE1a)-inhibitor,attenuated the effects of DHA on lipolysis,demonstrating that IRE1a and X-box binding protein 1 potentially participate in DHA-induced lipolysis.DHA also activated the cyclic adenosine monophosphate(cAMP)-dependent protein kinase A(PKA)pathway by increasing the level of cAMP and activating the PKA enzyme(P<0.05).Nevertheless,H89,a PKA inhibitor,weakened DHA-induced lipolysis by inhibiting the cAMP/PKA signaling pathway.Furthermore,inhibiting ER stress us-ing 4-PBA also inhibited lipolysis and alleviated DHA-induced activation of the cAMP/PKA signaling pathway,suggesting that ER stress may participate in DHA-induced lipolysis through the activation of the cAMP/PKA signaling pathway.Our data illustrate that DHA supplementation can be a promising nutritional strategy for ameliorating lipid accumulation in grass carp.The present study elucidated the molecular mechanism for DHA-induced lipolysis in grass carp adipocytes and emphasized the impor-tance of ER stress and the cAMP/PKA pathway in DHA-induced lip
基金Supported by the National Nature Science Foundation of China(No.81503380)。
文摘Objective:To investigate the mechanism of c AMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction(芍药甘草汤,SGD)on the regulation of aquaporin 5(AQP5)and muscarinic receptor 3(M3 R)levels in Sjogren’s syndrome(SS).Methods:Of the 30 mice,5 were randomly selected as control,and others were used for creating SS model.After successful modeling,mice were randomly divided into 5 groups(n=5 per group)and intragastrical y administered with saline(8 m L/kg),pilocarpine(1.4 mg/kg),or low,medium and high doses SGD(0.14,0.21,0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae,respectively)for 6 weeks.Human labial gland acinar cel s were treated with pilocarpine or varying doses of SGD with saline as the placebo.Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice.The serum levels of anti-SS antigen A(SS-A),anti-SS antigen B(SS-B),M3 R,andα-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay.Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3 R in acinar cells.Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA,c AMP,Epac1,AQP5,M3 R,nuclear factor kappa-B(NF-κB),and tumor necrosis factor(TNF)-αin submandibular gland tissues and cel s of each group.Results:Compared to normal mice,body weight,5-min salivary secretion,30-min secretion of tears and breakup time of tear film of model mice decreased at 1–6 weeks after immunization(al P<0.05),whereas water intake increased(al P<0.05).In the model group,glands of the submandibular glands showed atrophy,accompanied by acini of different sizes,decreased numbers and loose arrangement,with catheter dilatation and different degrees of lymphocyte infiltration.Conditions of mice in SGD groups were improved.The positive expression of AQP5 and M3 R were higher in the acinar cel s treated with al doses SGD compared to the normal group;serum levels of
