BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA...BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA remains unclear.AIM To explore the potential role and underlying mechanisms of NOS1 in ESCA.METHODS Survival rates were analyzed using GeneCards and Gene Expression Profiling Interactive Analysis.The effects and mechanisms of NOS1 on ESCA cells were evaluated via the Cell Counting Kit-8 assay,scratch assay,Transwell assay,flow cytometry,quantitative polymerase chain reaction,western blotting,and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining.The protein interaction network was used to screen the interacting proteins of NOS1 and validate these interactions through co-immuno-precipitation and dual luciferase assays.Additionally,a nude mouse xenograft model was established to evaluate the effect of NOS1 in vivo.RESULTS The survival rate of patients with ESCA with high NOS1 expression was higher than that of patients with low NOS1 expression.NOS1 expression in ESCA cell lines was lower than that in normal esophageal epithelial cells.Overexpression of NOS1(oe-NOS1)inhibited proliferation,invasion,and migration abilities in ESCA cell lines,resulting in decreased autophagy levels and increased apoptosis,pyroptosis,and ferroptosis.Protein interaction studies confirmed the interaction between NOS1 and NOS1 adaptor protein(NOS1AP).Following oe-NOS1 and the silencing of NOS1AP,levels of P62 and microtubule-associated protein 1 light chain 3 beta increased both in vitro and in vivo.Furthermore,the expression levels of E-cadherin,along with the activation of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT),were inhibited in ESCA cell lines.CONCLUSION NOS1 and NOS1 proteins interact to suppress autophagy,activate the PI3K/AKT pathway,and exert anti-cancer effects in ESCA.展开更多
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
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
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
[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purifi...[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purified and assayed with FPP as the substrate.The reaction products were detected by GC-MS.An FPP-overproducing E.coli strain was engineered for heterologous expression of orf2064.The fermentation products were analyzed by GC-MS,and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy(NMR).In addition,orf2064 was heterologously expressed in Streptomyces,and the fermentation products were analyzed by GC-MS.[Results]GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E.coli and Streptomyces consistently produced a compound with identical retention time and[M+]of m/z 204.Subsequent isolation,purification,and NMR analysis confirmed this compound as calarene.[Conclusion]The FPP cyclase encoded by orf2064 in S.exfoliatus is identified as an calarene synthase.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
Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in suga...Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in sugarcane(Saccharum spp.).In this study,18 SsICSs,42 ShICSs,and 36 SzICSs were identified from the genomes of sugarcane AP85-441(Saccharum spontaneum),XTT22(Saccharum spp.hybrid cultivar),and ZZ1(Saccharum spp.hybrid cultivar),respectively.These were phylogenetically divided into three groups,forming distinct clades that were evolutionarily divergent from those in dicotyledonous species.The evolutionary profile of the ICS gene family suggested expansion through whole-genome duplication/segmental events and strong purifying selection.Promoter cis-element and transcriptome analyses indicated that the ICS gene family responded to disease stress.We cloned the ScICS(isochorismate synthase)gene from sugarcane cultivar XTT22 leaves,and found it was localized in chloroplasts.In vivo and in vitro interaction studies revealed an interaction between ScICS and an ScMYB transcription factor.We showed that ScWRKY28 positively regulated ScICS expression by binding to its promoter.ScICS overexpression in transgenic tobacco confirmed its effectiveness in enhancing disease resistance.There was a significant increase in SA content following pathogen infection along with activation of downstream signaling pathways and defense mechanisms.This study establishes the groundwork for functional studies of sugarcane ICS genes and enhances our understanding of the mechanisms of disease resistance in sugarcane.展开更多
Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,...Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.展开更多
African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(...African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(IPP)with allylic diphosphates.Recently we solved the crystal structure pB318L lacking N-terminal transmembrane region and performed a preliminary structural analysis.In this study,structure-based mutagenesis study and geranylgeranyl pyrophosphate(GGPP)production assay further revealed the key residues for the GGPPS activity.Structural comparison showed pB318L displays a strong similarity to typical GGPPSs instead of protein prenyltransferases.The phylogenetic analysis indicated pB318L may share a common ancestor with the GGPPSs from Brassicaceae plants rather than from its natural host.The subcellular localization analysis showed pB318L is localized in both nucleus and cytoplasm(including the endoplasmic reticulum membrane and mitochondria outer membrane).A unique N-terminal nuclear localization signal(NLS)following the transmembrane region was discovered in pB318L and the NLS was confirmed to be required for the nuclear import.We further revealed the NLS plays an essential role in the interaction with nuclear transporter karyopherin subunit alpha 1(KPNA1).Their interaction may suppress signal transducers and activators of transcription 1(STAT1)translocation and subsequently competitively inhibit nuclear import of IFNstimulated gene factor 3(ISGF3)complex.Our biochemical,structural and cellular analyses provide novel insights to pB318L that acts as an essential GGPPS that promotes viral replication and as a nuclear import protein that may be involved in immune evasion of ASFV.展开更多
OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first ...OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.展开更多
Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine...Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.展开更多
AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible i...AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.展开更多
AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence ...AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.展开更多
AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibo...AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies.RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillariesrunning in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed.CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype-specific differences in the subcellular distributions of NOS isoforms.展开更多
During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the ...During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.展开更多
Neuronal nitric oxide synthase （nNOS） is mainly expressed in neurons,to some extent in astrocytes and neuronal stem cells.The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS,including nNOS-,nNOS-,nNOS...Neuronal nitric oxide synthase （nNOS） is mainly expressed in neurons,to some extent in astrocytes and neuronal stem cells.The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS,including nNOS-,nNOS-,nNOS-,nNOS-and nNOS-2.Monomer of nNOS is inactive,and dimer is the active form.Dimerization requires tetrahydrobiopterin （BH 4）,heme and L-arginine binding.Regulation of nNOS expression relies largely on cAMP response element-binding protein （CREB） activity,and nNOS activity is regulated by heat shock protein 90 （HSP90）/HSP70,calmodulin （CaM）,phosphorylation and dephosphorylation at Ser847 and Ser1412,and the protein inhibitor of nNOS （PIN）.There are primarily 9 nNOS-interacting proteins,including post-synaptic density protein 95 （PSD95）,clathrin assembly lymphoid leukemia （CALM）,calcium/calmodulindependent protein kinase II alpha （CAMKIIA）,Disks large homolog 4 （DLG4）,DLG2,6-phosphofructokinase,muscle type （PFK-M）,carboxy-terminal PDZ ligand of nNOS （CAPON） protein,syntrophin and dynein light chain （LC）.Among them,PSD95,CAPON and PFK-M are important nNOS adapter proteins in neurons.The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death.nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states,and negatively regulates neurogenesis under physiological and pathological conditions.展开更多
Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthas...Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.展开更多
A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 3...A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.展开更多
The molecular composition and evolution of the chalcone synthase (CHS) gene family from five species in Camellia (Theaceae) are explored in this study. Sixteen CHS exon 2 from four Camellia species were amplified from...The molecular composition and evolution of the chalcone synthase (CHS) gene family from five species in Camellia (Theaceae) are explored in this study. Sixteen CHS exon 2 from four Camellia species were amplified from total DNA by PCR method. Three sequences of the fifth species in Camellia and two sequences of Glycine max as the designated outgroups were obtained from GenBank. Our results indicated that CHS gene family in Camellia was differentiated to three subfamilies (A, B, C) during the evolutionary history with six groups (A1, A2, A3, BI, B2, C). Among them, only group A2 was possessed by all five species in this study. However, the other five groups were detected only in some species of the plants studied. All members of CHS gene family in this study had high sequence similarity, more than 90% among the members in the same subfamily and more than 78% among different subfamilies at nucleotide level., According to the estimated components of amino acids, the function of CHS genes in Camellia had been diverged. The nucleotide substitutions of the different groups were not identical. Based on phylogenetic analyse inferred from sequences of CHS genes and their deduced amino acid sequences, we concluded that the CHS genes with new function in this genus were evolved either by mutations on several important sites or by accumulation of the mutations after the gene duplication. A further analysis showed that the diversification of CHS genes in Camellia still occurred recently, and the evolutionary models were different to some extant among different species. So we assumed that the different evolutionary models resulted from the impacts of variable environmental elements after the events of speciation.展开更多
基金Supported by the National Natural Science Foundation of China,No.81000201.
文摘BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA remains unclear.AIM To explore the potential role and underlying mechanisms of NOS1 in ESCA.METHODS Survival rates were analyzed using GeneCards and Gene Expression Profiling Interactive Analysis.The effects and mechanisms of NOS1 on ESCA cells were evaluated via the Cell Counting Kit-8 assay,scratch assay,Transwell assay,flow cytometry,quantitative polymerase chain reaction,western blotting,and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining.The protein interaction network was used to screen the interacting proteins of NOS1 and validate these interactions through co-immuno-precipitation and dual luciferase assays.Additionally,a nude mouse xenograft model was established to evaluate the effect of NOS1 in vivo.RESULTS The survival rate of patients with ESCA with high NOS1 expression was higher than that of patients with low NOS1 expression.NOS1 expression in ESCA cell lines was lower than that in normal esophageal epithelial cells.Overexpression of NOS1(oe-NOS1)inhibited proliferation,invasion,and migration abilities in ESCA cell lines,resulting in decreased autophagy levels and increased apoptosis,pyroptosis,and ferroptosis.Protein interaction studies confirmed the interaction between NOS1 and NOS1 adaptor protein(NOS1AP).Following oe-NOS1 and the silencing of NOS1AP,levels of P62 and microtubule-associated protein 1 light chain 3 beta increased both in vitro and in vivo.Furthermore,the expression levels of E-cadherin,along with the activation of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT),were inhibited in ESCA cell lines.CONCLUSION NOS1 and NOS1 proteins interact to suppress autophagy,activate the PI3K/AKT pathway,and exert anti-cancer effects in ESCA.
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 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 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.
文摘[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purified and assayed with FPP as the substrate.The reaction products were detected by GC-MS.An FPP-overproducing E.coli strain was engineered for heterologous expression of orf2064.The fermentation products were analyzed by GC-MS,and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy(NMR).In addition,orf2064 was heterologously expressed in Streptomyces,and the fermentation products were analyzed by GC-MS.[Results]GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E.coli and Streptomyces consistently produced a compound with identical retention time and[M+]of m/z 204.Subsequent isolation,purification,and NMR analysis confirmed this compound as calarene.[Conclusion]The FPP cyclase encoded by orf2064 in S.exfoliatus is identified as an calarene synthase.
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.
基金supported by the National Natural Science Foundation of China(32472166,32172063,and 31771861)the Innovative Team Construction Project of the Modern Agricultural Industry Technology System in Guangdong Province by Agricultural Product Units(Sugarcane and Sisal Industry Technology System,2024CXTD03-06)South China Agricultural University Students Innovation and Entrepreneurship Training Program(2024105641195)。
文摘Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in sugarcane(Saccharum spp.).In this study,18 SsICSs,42 ShICSs,and 36 SzICSs were identified from the genomes of sugarcane AP85-441(Saccharum spontaneum),XTT22(Saccharum spp.hybrid cultivar),and ZZ1(Saccharum spp.hybrid cultivar),respectively.These were phylogenetically divided into three groups,forming distinct clades that were evolutionarily divergent from those in dicotyledonous species.The evolutionary profile of the ICS gene family suggested expansion through whole-genome duplication/segmental events and strong purifying selection.Promoter cis-element and transcriptome analyses indicated that the ICS gene family responded to disease stress.We cloned the ScICS(isochorismate synthase)gene from sugarcane cultivar XTT22 leaves,and found it was localized in chloroplasts.In vivo and in vitro interaction studies revealed an interaction between ScICS and an ScMYB transcription factor.We showed that ScWRKY28 positively regulated ScICS expression by binding to its promoter.ScICS overexpression in transgenic tobacco confirmed its effectiveness in enhancing disease resistance.There was a significant increase in SA content following pathogen infection along with activation of downstream signaling pathways and defense mechanisms.This study establishes the groundwork for functional studies of sugarcane ICS genes and enhances our understanding of the mechanisms of disease resistance in sugarcane.
基金supported by the National Natural Science Foundation of China(32161133010)the National Key Research and Development Program of China(2022YFD1700200)+2 种基金the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSCB-202302)the Shenzhen Science and Technology Program,China(KQTD20180411143628272)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District,China(PT202101-02).
文摘Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.
基金supported by the grants from the National Natural Science Foundation of China to Heng Zhang(31970152)the Strategic Priority Research Program of CAS to Yu-Hui Dong(XDB37040302)the CAS Emergency Research Project on African Swine Fever to Yu-Hui Dong(Grant KJZDSW-L06-01).
文摘African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(IPP)with allylic diphosphates.Recently we solved the crystal structure pB318L lacking N-terminal transmembrane region and performed a preliminary structural analysis.In this study,structure-based mutagenesis study and geranylgeranyl pyrophosphate(GGPP)production assay further revealed the key residues for the GGPPS activity.Structural comparison showed pB318L displays a strong similarity to typical GGPPSs instead of protein prenyltransferases.The phylogenetic analysis indicated pB318L may share a common ancestor with the GGPPSs from Brassicaceae plants rather than from its natural host.The subcellular localization analysis showed pB318L is localized in both nucleus and cytoplasm(including the endoplasmic reticulum membrane and mitochondria outer membrane).A unique N-terminal nuclear localization signal(NLS)following the transmembrane region was discovered in pB318L and the NLS was confirmed to be required for the nuclear import.We further revealed the NLS plays an essential role in the interaction with nuclear transporter karyopherin subunit alpha 1(KPNA1).Their interaction may suppress signal transducers and activators of transcription 1(STAT1)translocation and subsequently competitively inhibit nuclear import of IFNstimulated gene factor 3(ISGF3)complex.Our biochemical,structural and cellular analyses provide novel insights to pB318L that acts as an essential GGPPS that promotes viral replication and as a nuclear import protein that may be involved in immune evasion of ASFV.
基金The University Collaborative Innovation Project of Anhui:Creation of a Combined Animal Model of Coronary Heart Disease based on the Theory of Xin'an Medicine(No.GXXT-2020-024)Start-up Funding for Doctoral Research at Wannan Medical College(WYRCQD2018009)Horizontal Project of South Anhui Medical College(H202003)。
文摘OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.
基金supported by the National Key Research and Development Program of China(2020YFA0908300)the Natural Science Foundation of China(22138006,22278240).
文摘Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.
文摘AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.
基金Natural Science Foudation of Hebei ProvinceEducation Department Foundation of Hebei Province.No.2002136.
文摘AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.
基金Supported by Hungarian National Grant Agency, Grant#F46201to Bagyánszki M
文摘AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies.RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillariesrunning in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed.CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype-specific differences in the subcellular distributions of NOS isoforms.
文摘During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.
基金supported by the National Natural Science Foundation of China(No. 30971021,81030023 and 30901550)
文摘Neuronal nitric oxide synthase （nNOS） is mainly expressed in neurons,to some extent in astrocytes and neuronal stem cells.The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS,including nNOS-,nNOS-,nNOS-,nNOS-and nNOS-2.Monomer of nNOS is inactive,and dimer is the active form.Dimerization requires tetrahydrobiopterin （BH 4）,heme and L-arginine binding.Regulation of nNOS expression relies largely on cAMP response element-binding protein （CREB） activity,and nNOS activity is regulated by heat shock protein 90 （HSP90）/HSP70,calmodulin （CaM）,phosphorylation and dephosphorylation at Ser847 and Ser1412,and the protein inhibitor of nNOS （PIN）.There are primarily 9 nNOS-interacting proteins,including post-synaptic density protein 95 （PSD95）,clathrin assembly lymphoid leukemia （CALM）,calcium/calmodulindependent protein kinase II alpha （CAMKIIA）,Disks large homolog 4 （DLG4）,DLG2,6-phosphofructokinase,muscle type （PFK-M）,carboxy-terminal PDZ ligand of nNOS （CAPON） protein,syntrophin and dynein light chain （LC）.Among them,PSD95,CAPON and PFK-M are important nNOS adapter proteins in neurons.The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death.nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states,and negatively regulates neurogenesis under physiological and pathological conditions.
文摘Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.
文摘A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.
文摘The molecular composition and evolution of the chalcone synthase (CHS) gene family from five species in Camellia (Theaceae) are explored in this study. Sixteen CHS exon 2 from four Camellia species were amplified from total DNA by PCR method. Three sequences of the fifth species in Camellia and two sequences of Glycine max as the designated outgroups were obtained from GenBank. Our results indicated that CHS gene family in Camellia was differentiated to three subfamilies (A, B, C) during the evolutionary history with six groups (A1, A2, A3, BI, B2, C). Among them, only group A2 was possessed by all five species in this study. However, the other five groups were detected only in some species of the plants studied. All members of CHS gene family in this study had high sequence similarity, more than 90% among the members in the same subfamily and more than 78% among different subfamilies at nucleotide level., According to the estimated components of amino acids, the function of CHS genes in Camellia had been diverged. The nucleotide substitutions of the different groups were not identical. Based on phylogenetic analyse inferred from sequences of CHS genes and their deduced amino acid sequences, we concluded that the CHS genes with new function in this genus were evolved either by mutations on several important sites or by accumulation of the mutations after the gene duplication. A further analysis showed that the diversification of CHS genes in Camellia still occurred recently, and the evolutionary models were different to some extant among different species. So we assumed that the different evolutionary models resulted from the impacts of variable environmental elements after the events of speciation.
