Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral ...Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.展开更多
The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecu...The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecular biology experiments to reveal that tomato phytoene synthase 1(SlPSY1)is responsible for the accumulation of an important flavor chemical,6-methyl-5-hepten-2-one(MHO).To dissect the function of SlPSY1 in regulating fruit quality,we generated and analyzed a dataset encompassing over 2000 compounds detected by GC-MS and LC-MS/MS along with transcriptomic data.The combined results illustrated that SlPSY1 deficiency imparts novel flavor to yellow tomatoes with 236 volatiles significantly changed and improves fruit firmness,possibly due to accumulation of seven cutins.Further analysis indicated SlPSY1 is essential for carotenoid-derived metabolite biosynthesis by catalyzing prephytoene-PP(PPPP)to 15-cis-phytoene.Notably,we showed that SlPSY1 can influence the metabolic flux between carotenoid and flavonoid pathways,and this metabolic flux was confirmed by silencing SlCHS1.Our study provided insights into the multiple effects of SlPSY1 on tomato fruit metabolome and highlights the potential to produce high-quality fruit by rational design of SlPSY1 expression.展开更多
AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negati...AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS： In situ hybridization of EBV-encoded small RNA-1 （EBER-1） and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS： 12% of the cases of GC （25/208） showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION： Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.展开更多
In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach...In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.展开更多
Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa T...Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa TPS1 generated a 5/8/6/5 tetracyclic sesteraltererol(1)and its absolute stereochemistry was determined by X-ray crystallographic analysis of its derivative 10,11-epoxysesteraltererol(2),which enabled revision of the absolute configuration of C7 of sesterfisherol produced by Nf SS and PTTS014 characterized previously and its derivative 10,11-epoxysesterfisherol.Aa TPS2 produced a 5/15 bicyclic preterpestacin I(3).Site-directed mutagenesis suggested that F192 in Aa TPS1 was likely involved in controlling of the hydroxylation of C12,and eight amino acids were important for the enzyme activity of Aa TPS1 and Aa TPS2.The engineered Escherichia coli and Saccharomyces cerevisiae strains were constructed for the productions of compounds 1 and 3,and the highest titer of compound 1 reached 62.3 mg/L in shake-flask culture.Both compounds 1 and 2 showed anti-adipogenic activity.展开更多
Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia...To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.展开更多
Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm sp...Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm species. In this study, enzymes and starch were extracted from P. edulis seedlings germinated in the dark at room temperature. Granule_bound proteins of 58 kD and 91 kD were recognized by a pea SS Ⅱ antiserum. The 58 kD granule_bound protein was purified and identified as granule_bound starch synthase Ⅰ by alignment of the N_terminal sequence with that of granule_bound starch synthase Ⅰ from several angiosperms. Elution of soluble starch synthase activity from a DEAE_Sepharose column showed two starch synthase activity peaks, indicating at least two isoforms of soluble starch synthases. Primer affinities of soluble starch synthases were investigated. Glycogen from rabbit was the best primer for soluble starch synthase. The enzymological properties of Pinus starch synthases appear to be similar to those reported for angiosperms.展开更多
Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β...Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.展开更多
Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles i...Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles in determining the size and shape of cells throughcontrolling the rate and direction of cell expansion·Wallsare also involved in ahsorption of nutrients,transductionof sigmals and initial resistance to pathogen attack oncells.Wall polysaccharides from different plants are alsofood sources,used as building and paper-making materi-als and have other industrial applications.展开更多
Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,w...Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.展开更多
Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted i...Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted in a few plant species.MTSs are responsible for the complex cyclization of monoterpene precursors,resulting in the production of diverse monoterpene products.These enzymatic reactions are considered exceptionally complex in nature.Therefore,it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products.In our study,we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products.By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic,monocyclic and bicyclic products in MTSs,we observed that even a single amino acid mutation can change the specificity and diversity of MTS products,which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing.Notably,residues N340,I448,and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations.Alterations in these residues,either directly or indirectly,can impact the synthesis of single monoterpenes or their mixtures.By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs,it will be possible to reasonably design and engineer different catalytic activities into existing MTSs,laying a foundation for the artificial design and industrial application of MTSs.展开更多
Objectives:Terpenes are important volatile organic compounds that impact fruit aroma and flavor quality.Terpene synthases(TPSs)are the key enzymes responsible for the biosynthesis of basic backbone structure of terpen...Objectives:Terpenes are important volatile organic compounds that impact fruit aroma and flavor quality.Terpene synthases(TPSs)are the key enzymes responsible for the biosynthesis of basic backbone structure of terpenes.The identification and characterization of TPSs are critical for comprehending the biosynthesis of terpenes in fruit.Materials and Methods:The present study utilized cultivated peach(Prunus persica L.Batsch)as materials.RNA-sequencing was employed to investigate the expression profiles of PpTPSs during fruit ripening and in response to hormone and temperature treatments.Enzyme activities of PpTPSs were assessed using different substrates.Results:Here,we show that peach contains 38 TPS genes,with 24 members in the TPS-a cluster.Transcriptome analysis revealed that the expression of PpTPSs in peach fruits was regulated by environmental factors such as UV-B light and low temperature,as well as by phytohormones such as ethylene and methyl jasmonate.After analyzing the expression of 38 PpTPSs in peach fruit developmental stages and different tissues,we screened and cloned six new highly expressed TPS genes.Subcellular localization showed that PpTPS13 and PpTPS23 were localized in the plastid,whereas PpTPS12,PpTPS22,PpTPS25,and PpTPS28 were localized in the cytoplasm.Heterologous expression of PpTPSs in Escherichia coli followed by the enzymatic assays revealed that only fourTPSs(PpTPS12,PpTPS22,PpTPS25,and PpTPS28)were active in vitro.Using GPP and FPP as substrates,these PpTPSs were able to synthesize an array of volatile terpenes,including 15 monoterpenes such as geraniol,camphene,pinene,borneol and phellandrene,and 14 sesquiterpenes such as farnesene,nerolidol and α-bergamotene.Conclusions:Our results identify target genes for engineering to increase the production of volatile terpenes and thereby improve fruit quality.展开更多
Background:Ophiopogon japonicus(L.f)Ker-Gawl.growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix.Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside,a promi...Background:Ophiopogon japonicus(L.f)Ker-Gawl.growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix.Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside,a prominent pharmacologically active compound,serves as a marker distinguishing O.japonicus in Zhejiang from those in other geographical areas.It is synthesized from borneol through glycosylation,with terpene synthase(TPS)being the critical enzyme catalyzing the conversion of terpene precursors into borneol.Objective:The aim of the study was to define key genes involved in biosynthesis of borneol in O.japonicus.Methods:The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O.japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli.Results:This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E.coli.Seven terpene synthase genes(OjTPS1 to OjTPS7)were identified in different terpene synthase subfamilies,including 2 from TPS-a,4 from TPS-b,and 1 from TPS-g.Biochemical analysis using an engineered system E.coli demonstrated that all the 7 terpene synthases produced monoterpenes,and OjTPS3,OjTPS5,and OjTPS6 also yielded sesquiterpenes.Conclusions:These 7 terpene synthases produced 13 monoterpenes and 8 sesquiterpenes.Notably,OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.展开更多
Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung ...Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung cancer potential.We aimed to explore the anti–lung cancer effect of BBR and related mechanisms by targeting the glycogen synthase kinase 3β(GSK3β)/β-catenin pathway.Methods:Lung adenocarcinoma(LUAD)cells A549 and NCI-H1975 were treated with BBR.Results:Our results showed that BBR inhibited cell proliferation by decreasing c-Myc levels and induced cel cycle arrest in the G0/G1 phase by lowering cyclin D1 levels.BBR induced apoptosis by upregulating cleaved caspase 3 levels.BBR inhibited cell migration and invasion by decreasing N-cadherin levels.Furthermore,BBR upregulated the expression of GSK3βprotein and phospho-β-catenin proteins in the cytoplasm,while decreasing the expression ofβ-catenin protein.Next,LUAD cel s were exposed to CHIR-99021(a GSK3βinhibitor).This treatment led to an increase in c-Myc,cyclin D1,andβ-catenin levels at specific concentrations.BBR partially reversed the effects of CHIR-99021.Finally,LUAD cells were treated with CHIR-99021(4μmoL/L)combined with BBR(30 and 60μmoL/L)for 24 h.The expression of programmed death ligand 1(PD-L1)was assessed by Western blot analysis.Jurkat T cells and A549 cel s were cocultured for 24 h to examine the lactate dehydrogenase release rate.Results suggested that BBR suppressed the expression of PD-L1 and heightened the immune lethality of T cells.Conclusions:BBR suppressed the proliferative activity of LUAD cell lines A549 and NCI-H1975 in vitro,induced cell cycle arrest and cancer cel apoptosis in the G0/G1 stage,and repressed the migration and invasion of cancer cells.BBR reduced the PD-L1 protein expression and enhanced T-cell–mediated cytotoxicity.These effects appear to be related to BBR's regulation of the GSK3β/β-catenin pathway.展开更多
With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic...With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.展开更多
Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which mi...Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.展开更多
Interferon regulatory factor 7 plays a crucial role in the innate immune response.However,whether interferon regulatory factor 7-mediated signaling contributes to Parkinson's disease remains unknown.Here we report...Interferon regulatory factor 7 plays a crucial role in the innate immune response.However,whether interferon regulatory factor 7-mediated signaling contributes to Parkinson's disease remains unknown.Here we report that interferon regulatory factor 7 is markedly up-regulated in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease and co-localizes with microglial cells.Both the selective cyclic guanosine monophosphate adenosine monophosphate synthase inhibitor RU.521 and the stimulator of interferon genes inhibitor H151 effectively suppressed interferon regulatory factor 7 activation in BV2 microglia exposed to 1-methyl-4-phenylpyridinium and inhibited transformation of mouse BV2 microglia into the neurotoxic M1 phenotype.In addition,si RNA-mediated knockdown of interferon regulatory factor 7 expression in BV2 microglia reduced the expression of inducible nitric oxide synthase,tumor necrosis factorα,CD16,CD32,and CD86 and increased the expression of the anti-inflammatory markers ARG1 and YM1.Taken together,our findings indicate that the cyclic guanosine monophosphate adenosine monophosphate synthase-stimulator of interferon genes-interferon regulatory factor 7 pathway plays a crucial role in the pathogenesis of Parkinson's disease.展开更多
Nitric oxide(NO)is a gaseous molecule produced by 3 different NO synthase(NOS)isoforms:Neural/brain NOS(nNOS/bNOS,type 1),endothelial NOS(eNOS,type 3)and inducible NOS(type 2).Type 1 and 3 NOS are constitutively expre...Nitric oxide(NO)is a gaseous molecule produced by 3 different NO synthase(NOS)isoforms:Neural/brain NOS(nNOS/bNOS,type 1),endothelial NOS(eNOS,type 3)and inducible NOS(type 2).Type 1 and 3 NOS are constitutively expressed.NO can serve different purposes:As a vasoactive molecule,as a neurotransmitter or as an immunomodulator.It plays a key role in cerebral ischemia/reperfusion injury(CIRI).Hypoxic episodes simulate the production of oxygen free radicals,leading to mitochondrial and phospholipid damage.Upon reperfusion,increased levels of oxygen trigger oxide synthases;whose products are associated with neuronal damage by promoting lipid peroxidation,nitrosylation and excitotoxicity.Molecular pathways in CIRI can be altered by NOS.Neuroprotective effects are observed with eNOS activity.While nNOS interplay is prone to endothelial inflammation,oxidative stress and apoptosis.Therefore,nNOS appears to be detrimental.The interaction between NO and other free radicals develops peroxynitrite;which is a cytotoxic agent.It plays a main role in the likelihood of hemorrhagic events by tissue plasminogen activator(t-PA).Peroxynitrite scavengers are currently being studied as potential targets to prevent hemorrhagic transformation in CIRI.展开更多
Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration ...Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.展开更多
基金This work was supported by the National Natural Science Foundation of China(grant numbers 81560301 and 81160012)the Natural Science Foundation of Qinghai Province(grant number 2022-ZJ-905)‘2022 Qinghai Province Kunlun Talents High-end Innovation and Entrepreneurship Talents’Outstanding Talent Project.
文摘Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.
基金supported by the National Natural Science Foundation of China(Grant Nos.31991185,31902019,32102384)National Key Research and Development Program of China(Grant No.2021YFF1000103)+2 种基金Key Research and Development Program of Guangdong Province(Grant No.2021B0707010005)Taishan Scholars Program of Shandong Province,China(2016-2020)supported by the Youth innovation Program of Chinese Academy of Agricultural Sciences(Grant No.Y2023QC05)。
文摘The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecular biology experiments to reveal that tomato phytoene synthase 1(SlPSY1)is responsible for the accumulation of an important flavor chemical,6-methyl-5-hepten-2-one(MHO).To dissect the function of SlPSY1 in regulating fruit quality,we generated and analyzed a dataset encompassing over 2000 compounds detected by GC-MS and LC-MS/MS along with transcriptomic data.The combined results illustrated that SlPSY1 deficiency imparts novel flavor to yellow tomatoes with 236 volatiles significantly changed and improves fruit firmness,possibly due to accumulation of seven cutins.Further analysis indicated SlPSY1 is essential for carotenoid-derived metabolite biosynthesis by catalyzing prephytoene-PP(PPPP)to 15-cis-phytoene.Notably,we showed that SlPSY1 can influence the metabolic flux between carotenoid and flavonoid pathways,and this metabolic flux was confirmed by silencing SlCHS1.Our study provided insights into the multiple effects of SlPSY1 on tomato fruit metabolome and highlights the potential to produce high-quality fruit by rational design of SlPSY1 expression.
基金Supported by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo grant 04/15650-1
文摘AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS： In situ hybridization of EBV-encoded small RNA-1 （EBER-1） and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS： 12% of the cases of GC （25/208） showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION： Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.
文摘In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.
基金supported financially by the National Natural Science Foundation of China(Nos.21937006 and 22107103)the Yunnan Key Research and Development Program(No.2019ZF011–2)the“Western Light”Program of the CAS(to Y.Liu)。
文摘Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa TPS1 generated a 5/8/6/5 tetracyclic sesteraltererol(1)and its absolute stereochemistry was determined by X-ray crystallographic analysis of its derivative 10,11-epoxysesteraltererol(2),which enabled revision of the absolute configuration of C7 of sesterfisherol produced by Nf SS and PTTS014 characterized previously and its derivative 10,11-epoxysesterfisherol.Aa TPS2 produced a 5/15 bicyclic preterpestacin I(3).Site-directed mutagenesis suggested that F192 in Aa TPS1 was likely involved in controlling of the hydroxylation of C12,and eight amino acids were important for the enzyme activity of Aa TPS1 and Aa TPS2.The engineered Escherichia coli and Saccharomyces cerevisiae strains were constructed for the productions of compounds 1 and 3,and the highest titer of compound 1 reached 62.3 mg/L in shake-flask culture.Both compounds 1 and 2 showed anti-adipogenic activity.
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
文摘To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.
文摘Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm species. In this study, enzymes and starch were extracted from P. edulis seedlings germinated in the dark at room temperature. Granule_bound proteins of 58 kD and 91 kD were recognized by a pea SS Ⅱ antiserum. The 58 kD granule_bound protein was purified and identified as granule_bound starch synthase Ⅰ by alignment of the N_terminal sequence with that of granule_bound starch synthase Ⅰ from several angiosperms. Elution of soluble starch synthase activity from a DEAE_Sepharose column showed two starch synthase activity peaks, indicating at least two isoforms of soluble starch synthases. Primer affinities of soluble starch synthases were investigated. Glycogen from rabbit was the best primer for soluble starch synthase. The enzymological properties of Pinus starch synthases appear to be similar to those reported for angiosperms.
基金This work was supported by the National Natural Science Foundation of China(31772176,31672038 and 31621064)and the National Key Research and Development Program of China(2017YFDO201900 and 2017YFD0200400).
文摘Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.
文摘Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles in determining the size and shape of cells throughcontrolling the rate and direction of cell expansion·Wallsare also involved in ahsorption of nutrients,transductionof sigmals and initial resistance to pathogen attack oncells.Wall polysaccharides from different plants are alsofood sources,used as building and paper-making materi-als and have other industrial applications.
基金supported by grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.
基金supported by the National Key R&D Program of China(2020YFA0908000)the National Natural Science Foundation of China(31901015)Science and Technology Partnership Program,Ministry of Science and Technology of China(KY202001017).
文摘Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted in a few plant species.MTSs are responsible for the complex cyclization of monoterpene precursors,resulting in the production of diverse monoterpene products.These enzymatic reactions are considered exceptionally complex in nature.Therefore,it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products.In our study,we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products.By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic,monocyclic and bicyclic products in MTSs,we observed that even a single amino acid mutation can change the specificity and diversity of MTS products,which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing.Notably,residues N340,I448,and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations.Alterations in these residues,either directly or indirectly,can impact the synthesis of single monoterpenes or their mixtures.By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs,it will be possible to reasonably design and engineer different catalytic activities into existing MTSs,laying a foundation for the artificial design and industrial application of MTSs.
基金supported by the Zhejiang Provincial Natural Science Foundation(No.LD22C150001)the Ningbo Key Research and Development Program(No.2022Z179)the Fundamental Research Funds for the Zhejiang Provincial Universities(No.2021XZZX026),China.
文摘Objectives:Terpenes are important volatile organic compounds that impact fruit aroma and flavor quality.Terpene synthases(TPSs)are the key enzymes responsible for the biosynthesis of basic backbone structure of terpenes.The identification and characterization of TPSs are critical for comprehending the biosynthesis of terpenes in fruit.Materials and Methods:The present study utilized cultivated peach(Prunus persica L.Batsch)as materials.RNA-sequencing was employed to investigate the expression profiles of PpTPSs during fruit ripening and in response to hormone and temperature treatments.Enzyme activities of PpTPSs were assessed using different substrates.Results:Here,we show that peach contains 38 TPS genes,with 24 members in the TPS-a cluster.Transcriptome analysis revealed that the expression of PpTPSs in peach fruits was regulated by environmental factors such as UV-B light and low temperature,as well as by phytohormones such as ethylene and methyl jasmonate.After analyzing the expression of 38 PpTPSs in peach fruit developmental stages and different tissues,we screened and cloned six new highly expressed TPS genes.Subcellular localization showed that PpTPS13 and PpTPS23 were localized in the plastid,whereas PpTPS12,PpTPS22,PpTPS25,and PpTPS28 were localized in the cytoplasm.Heterologous expression of PpTPSs in Escherichia coli followed by the enzymatic assays revealed that only fourTPSs(PpTPS12,PpTPS22,PpTPS25,and PpTPS28)were active in vitro.Using GPP and FPP as substrates,these PpTPSs were able to synthesize an array of volatile terpenes,including 15 monoterpenes such as geraniol,camphene,pinene,borneol and phellandrene,and 14 sesquiterpenes such as farnesene,nerolidol and α-bergamotene.Conclusions:Our results identify target genes for engineering to increase the production of volatile terpenes and thereby improve fruit quality.
基金supported by National Natural Science Foundation of China(U23A20513)the Fundamental Research Funds for the Central Public Welfare Research Institutes(ZZ14-YQ-049)+1 种基金National Natural Science Foundation of China(82274054)Key project at central government level:the ability to establish sustainable use of valuable Chinese Medicine Resources(2060302).
文摘Background:Ophiopogon japonicus(L.f)Ker-Gawl.growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix.Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside,a prominent pharmacologically active compound,serves as a marker distinguishing O.japonicus in Zhejiang from those in other geographical areas.It is synthesized from borneol through glycosylation,with terpene synthase(TPS)being the critical enzyme catalyzing the conversion of terpene precursors into borneol.Objective:The aim of the study was to define key genes involved in biosynthesis of borneol in O.japonicus.Methods:The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O.japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli.Results:This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E.coli.Seven terpene synthase genes(OjTPS1 to OjTPS7)were identified in different terpene synthase subfamilies,including 2 from TPS-a,4 from TPS-b,and 1 from TPS-g.Biochemical analysis using an engineered system E.coli demonstrated that all the 7 terpene synthases produced monoterpenes,and OjTPS3,OjTPS5,and OjTPS6 also yielded sesquiterpenes.Conclusions:These 7 terpene synthases produced 13 monoterpenes and 8 sesquiterpenes.Notably,OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.
基金Supported by a grant from the National Natural Science Foundation of China(no.82174457)。
文摘Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung cancer potential.We aimed to explore the anti–lung cancer effect of BBR and related mechanisms by targeting the glycogen synthase kinase 3β(GSK3β)/β-catenin pathway.Methods:Lung adenocarcinoma(LUAD)cells A549 and NCI-H1975 were treated with BBR.Results:Our results showed that BBR inhibited cell proliferation by decreasing c-Myc levels and induced cel cycle arrest in the G0/G1 phase by lowering cyclin D1 levels.BBR induced apoptosis by upregulating cleaved caspase 3 levels.BBR inhibited cell migration and invasion by decreasing N-cadherin levels.Furthermore,BBR upregulated the expression of GSK3βprotein and phospho-β-catenin proteins in the cytoplasm,while decreasing the expression ofβ-catenin protein.Next,LUAD cel s were exposed to CHIR-99021(a GSK3βinhibitor).This treatment led to an increase in c-Myc,cyclin D1,andβ-catenin levels at specific concentrations.BBR partially reversed the effects of CHIR-99021.Finally,LUAD cells were treated with CHIR-99021(4μmoL/L)combined with BBR(30 and 60μmoL/L)for 24 h.The expression of programmed death ligand 1(PD-L1)was assessed by Western blot analysis.Jurkat T cells and A549 cel s were cocultured for 24 h to examine the lactate dehydrogenase release rate.Results suggested that BBR suppressed the expression of PD-L1 and heightened the immune lethality of T cells.Conclusions:BBR suppressed the proliferative activity of LUAD cell lines A549 and NCI-H1975 in vitro,induced cell cycle arrest and cancer cel apoptosis in the G0/G1 stage,and repressed the migration and invasion of cancer cells.BBR reduced the PD-L1 protein expression and enhanced T-cell–mediated cytotoxicity.These effects appear to be related to BBR's regulation of the GSK3β/β-catenin pathway.
基金supported by the National Key R&D Program of China,No.2019YFE0121200(to LQZ)the National Natural Science Foundation of China,Nos.82325017(to LQZ),82030032(to LQZ),82261138555(to DL)+2 种基金the Natural Science Foundation of Hubei Province,No.2022CFA004(to LQZ)the Natural Science Foundation of Jiangxi Province,No.20224BAB206040(to XZ)Research Project of Cognitive Science and Transdisciplinary Studies Center of Jiangxi Province,No.RZYB202201(to XZ).
文摘With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.
基金supported by grants from the National Key Research and Development Program of China,No.2017YFA0105400(to LR)the Key Research and Development Program of Guangdong Province,No.2019B020236002(to LR)the National Natural Science Foundation of China,Nos.81972111(to LZ),81772349(to BL).
文摘Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.
基金supported by the National Natural Science Foundation of China,Nos.82171429,81771384a grant from Wuxi Municipal Health Commission,No.1286010241190480(all to YS)。
文摘Interferon regulatory factor 7 plays a crucial role in the innate immune response.However,whether interferon regulatory factor 7-mediated signaling contributes to Parkinson's disease remains unknown.Here we report that interferon regulatory factor 7 is markedly up-regulated in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease and co-localizes with microglial cells.Both the selective cyclic guanosine monophosphate adenosine monophosphate synthase inhibitor RU.521 and the stimulator of interferon genes inhibitor H151 effectively suppressed interferon regulatory factor 7 activation in BV2 microglia exposed to 1-methyl-4-phenylpyridinium and inhibited transformation of mouse BV2 microglia into the neurotoxic M1 phenotype.In addition,si RNA-mediated knockdown of interferon regulatory factor 7 expression in BV2 microglia reduced the expression of inducible nitric oxide synthase,tumor necrosis factorα,CD16,CD32,and CD86 and increased the expression of the anti-inflammatory markers ARG1 and YM1.Taken together,our findings indicate that the cyclic guanosine monophosphate adenosine monophosphate synthase-stimulator of interferon genes-interferon regulatory factor 7 pathway plays a crucial role in the pathogenesis of Parkinson's disease.
文摘Nitric oxide(NO)is a gaseous molecule produced by 3 different NO synthase(NOS)isoforms:Neural/brain NOS(nNOS/bNOS,type 1),endothelial NOS(eNOS,type 3)and inducible NOS(type 2).Type 1 and 3 NOS are constitutively expressed.NO can serve different purposes:As a vasoactive molecule,as a neurotransmitter or as an immunomodulator.It plays a key role in cerebral ischemia/reperfusion injury(CIRI).Hypoxic episodes simulate the production of oxygen free radicals,leading to mitochondrial and phospholipid damage.Upon reperfusion,increased levels of oxygen trigger oxide synthases;whose products are associated with neuronal damage by promoting lipid peroxidation,nitrosylation and excitotoxicity.Molecular pathways in CIRI can be altered by NOS.Neuroprotective effects are observed with eNOS activity.While nNOS interplay is prone to endothelial inflammation,oxidative stress and apoptosis.Therefore,nNOS appears to be detrimental.The interaction between NO and other free radicals develops peroxynitrite;which is a cytotoxic agent.It plays a main role in the likelihood of hemorrhagic events by tissue plasminogen activator(t-PA).Peroxynitrite scavengers are currently being studied as potential targets to prevent hemorrhagic transformation in CIRI.
文摘Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.
