AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines ...AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.展开更多
Upon infection into human red cell,Plasmodium falciparum differentiates into asexual and sexual(gametocyte) stages.The mitochondrion is a tubular-cristate organelle,functionally and structurally different between the ...Upon infection into human red cell,Plasmodium falciparum differentiates into asexual and sexual(gametocyte) stages.The mitochondrion is a tubular-cristate organelle,functionally and structurally different between the two stages.Genes and proteins involving metabolic and functional roles,protein targeting and import to this organelle, are comprehensively reviewed.The genes and proteins of the electron transport system are identified, partially characterized in human and rodent malaria parasites consisting of a single subunit of NADH dehydrogenase, two subunits of succinate dehydrogenase,cytochrome C reductase and cytochrome Coxidase.One of the primary functional roles of the mitochondrion in the parasite is the coordination of pyrimidine biosynthesis, the electron transport system and oxygen utilization through dihydroorotate dehydrogenase.All enzymes of tricarboxylic acid cycle,pyruvate dehydrogenase complex and some enzymes of ATP synthase,are identified and partially characterized using the completed P.falciparum genome.Some metabolic and functional roles of the organelle include oxidative phosphorylation,ubiquinone and heme biosynthesis,antioxidant defense and redox balance.Recent physiological studies involve membrane potential maintenance,cellular signaling and cation homeostasis.The organelle is a target for antimalarial drug,i.e.atovaquone.Based on the lines of evidence, we hypothesize that the parasite exhibits metabolic adaptation of the underdeveloped mitochondrial organelle to life in the mosquito vector and the human host.展开更多
"Juemingzi", a source of traditional Chinese herbal medicine, has been demonstrated to play a role in decreasing serum cholesterol concentration. In this study, a novel protein, which has shown an in-hibitor..."Juemingzi", a source of traditional Chinese herbal medicine, has been demonstrated to play a role in decreasing serum cholesterol concentration. In this study, a novel protein, which has shown an in-hibitory effect on cholesterol biosynthesis, was isolated from Senna obtusifolia L. seed by gel filtration and ion exchange chromatography. The novel protein’s molecular mass was 19.7 kD and its pI was 4.80. Both SDS-PAGE and isoelectric-focusing (IEF) revealed a single Coomassie brilliant blue stained band, indicating that the novel protein was a single peptide. The N-terminal amino acid sequence of the pro-tein was IPYISASFPLNIEFLPSE, which had no similarity with any other protein sequences in the NCBI protein database. Circular dichroism (CD) signals indicated that S. obtusifolia seed protein contained 12.5% α-helix, 55.6% β-sheet, and 31.9% random coil.展开更多
Proteins are one of the major classes of biomolecules that execute biological functions for maintenance of life.Various kinds of nanostructures self-assembled from proteins have been created in nature over millions of...Proteins are one of the major classes of biomolecules that execute biological functions for maintenance of life.Various kinds of nanostructures self-assembled from proteins have been created in nature over millions of years of evolution,including protein nanowires,layers and nanocages.These protein nanostructures can be reconstructed and equipped with desired new functions.Learning from and manipulating the self-assembly of protein nanostructures not only help to deepen our understanding of the nature of life but also offer new routes to fabricate novel nanomaterials for diverse applications.This review summarizes the recent research progress in this field,focusing on the characteristics,functionalization strategies,and applications of protein nanostructures.展开更多
Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a nati...Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan(L-4-nitro-Trp)synthesis using an Escherichia coli-based cell-free protein synthesis(CFPS)system.Naturally,a nitric oxide(NO)synthase(TxtD)and a cytochrome P450 enzyme(TxtE)are responsible for synthesizing L-4-nitro-Trp,which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A.Recombinant coexpression of TxtD and TxtE in a heterologous host like E.coli for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD.Using CFPS,TxtD and TxtE were successfully expressed in vitro,enabling the formation of L-4-nitro-Trp.After optimization,the cell-free system was able to synthesize approximately 360μM L-4-nitro-Trp within 16 h.Overall,this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds,which are important building blocks widely used in pharmaceuticals,agrochemicals,and industrial chemicals.展开更多
The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was o...The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.展开更多
Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific...Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific protein-protein interactions formed between the peptidyl carrier protein(PCP)and its partner enzymes,NRPS enzymology and control remains fertile ground for discovery.This review focuses on the recent efforts within structural biology by compiling high-resolution structural data that shed light into the various protein-protein interfaces formed between the PCP and its partner enzymes,including the phosphopantetheinyl transferase(PPTase),adenylation(A)domain,condensation(C)domain,thioesterase(TE)domain and other tailoring enzymes within the synthetase.Integrating our understanding of how the PCP recognizes partner proteins with the potential to use directed evolution and combinatorial biosynthetic methods will enhance future efforts in discovery and production of new bioactive compounds.展开更多
Terpenoids constitute the largest class of natural products with complex structures,essential functions,and versatile applications.Creation of new building blocks beyond the conventional five-carbon(C_(5))units,dimeth...Terpenoids constitute the largest class of natural products with complex structures,essential functions,and versatile applications.Creation of new building blocks beyond the conventional five-carbon(C_(5))units,dimethylallyl diphosphate(DMAPP)and isopentenyl diphosphate,expands significantly the chemical space of terpenoids.Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate(GPP)C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C_(6) unit,2-methyl-DMAPP.Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP.Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond.Furthermore,using Escherichia coli as the chassis,we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans-and cis-prenyl diphosphates(C_(5n+1))and terpenoids.This work provides insights into substrate recognition of prenyl diphosphate methyltransferases,and strategies to diversify terpenoids by expanding the building block portfolio.展开更多
基金Project supported by the National Natural Science Foundation of China,No.39770861.and JANSSEN Science Research Foundation.
文摘AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.
基金the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases(CHEMAL)the National Science and Technology Development Agency of Thailand (Career Development Award)the Thailand Research Fund(Basic Research)
文摘Upon infection into human red cell,Plasmodium falciparum differentiates into asexual and sexual(gametocyte) stages.The mitochondrion is a tubular-cristate organelle,functionally and structurally different between the two stages.Genes and proteins involving metabolic and functional roles,protein targeting and import to this organelle, are comprehensively reviewed.The genes and proteins of the electron transport system are identified, partially characterized in human and rodent malaria parasites consisting of a single subunit of NADH dehydrogenase, two subunits of succinate dehydrogenase,cytochrome C reductase and cytochrome Coxidase.One of the primary functional roles of the mitochondrion in the parasite is the coordination of pyrimidine biosynthesis, the electron transport system and oxygen utilization through dihydroorotate dehydrogenase.All enzymes of tricarboxylic acid cycle,pyruvate dehydrogenase complex and some enzymes of ATP synthase,are identified and partially characterized using the completed P.falciparum genome.Some metabolic and functional roles of the organelle include oxidative phosphorylation,ubiquinone and heme biosynthesis,antioxidant defense and redox balance.Recent physiological studies involve membrane potential maintenance,cellular signaling and cation homeostasis.The organelle is a target for antimalarial drug,i.e.atovaquone.Based on the lines of evidence, we hypothesize that the parasite exhibits metabolic adaptation of the underdeveloped mitochondrial organelle to life in the mosquito vector and the human host.
基金Guangdong Provincial Department of Science and Technology (Grant No. 2003C34409)Guangzhou Civil Department of Science and Technology (Grant No. 2002Z3-85041)
文摘"Juemingzi", a source of traditional Chinese herbal medicine, has been demonstrated to play a role in decreasing serum cholesterol concentration. In this study, a novel protein, which has shown an in-hibitory effect on cholesterol biosynthesis, was isolated from Senna obtusifolia L. seed by gel filtration and ion exchange chromatography. The novel protein’s molecular mass was 19.7 kD and its pI was 4.80. Both SDS-PAGE and isoelectric-focusing (IEF) revealed a single Coomassie brilliant blue stained band, indicating that the novel protein was a single peptide. The N-terminal amino acid sequence of the pro-tein was IPYISASFPLNIEFLPSE, which had no similarity with any other protein sequences in the NCBI protein database. Circular dichroism (CD) signals indicated that S. obtusifolia seed protein contained 12.5% α-helix, 55.6% β-sheet, and 31.9% random coil.
基金supported by the National Natural Science Foundation of China(21890743,31771103,and 91527302)the National Key Research and Development Program of China(2017YFA0205503)+3 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(CAS)(XDB29050100)CAS Emergency Project of ASF Research(KJZD-SWL06 and KJZD-SWL07)Youth Innovation Promotion Association of CAS(2014308)Wuhan Huanghe Talents Program of Science and Technology。
文摘Proteins are one of the major classes of biomolecules that execute biological functions for maintenance of life.Various kinds of nanostructures self-assembled from proteins have been created in nature over millions of years of evolution,including protein nanowires,layers and nanocages.These protein nanostructures can be reconstructed and equipped with desired new functions.Learning from and manipulating the self-assembly of protein nanostructures not only help to deepen our understanding of the nature of life but also offer new routes to fabricate novel nanomaterials for diverse applications.This review summarizes the recent research progress in this field,focusing on the characteristics,functionalization strategies,and applications of protein nanostructures.
基金This work was supported by the National Natural Science Foundation of China(Nos.31971348 and 32171427)the Natural Science Foundation of Shanghai(No.19ZR1477200)J.L.also acknowledges the starting grant from ShanghaiTech University.
文摘Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan(L-4-nitro-Trp)synthesis using an Escherichia coli-based cell-free protein synthesis(CFPS)system.Naturally,a nitric oxide(NO)synthase(TxtD)and a cytochrome P450 enzyme(TxtE)are responsible for synthesizing L-4-nitro-Trp,which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A.Recombinant coexpression of TxtD and TxtE in a heterologous host like E.coli for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD.Using CFPS,TxtD and TxtE were successfully expressed in vitro,enabling the formation of L-4-nitro-Trp.After optimization,the cell-free system was able to synthesize approximately 360μM L-4-nitro-Trp within 16 h.Overall,this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds,which are important building blocks widely used in pharmaceuticals,agrochemicals,and industrial chemicals.
文摘The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.
基金J.C.C.was supported by the National Institute of General Medical Science(NIGMS)of the National Institutes of Health(NIH)under award number 1F31GM13761601A1J.O.S.was supported by the ACS Bridge Program and The Genentech FoundationThis work was supported by the NIGMS of the NIH under award number R01GM095970.
文摘Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific protein-protein interactions formed between the peptidyl carrier protein(PCP)and its partner enzymes,NRPS enzymology and control remains fertile ground for discovery.This review focuses on the recent efforts within structural biology by compiling high-resolution structural data that shed light into the various protein-protein interfaces formed between the PCP and its partner enzymes,including the phosphopantetheinyl transferase(PPTase),adenylation(A)domain,condensation(C)domain,thioesterase(TE)domain and other tailoring enzymes within the synthetase.Integrating our understanding of how the PCP recognizes partner proteins with the potential to use directed evolution and combinatorial biosynthetic methods will enhance future efforts in discovery and production of new bioactive compounds.
基金supported in part by the National Key R&D Program of China (2021YFA0909600 and 2019YFA0909400).
文摘Terpenoids constitute the largest class of natural products with complex structures,essential functions,and versatile applications.Creation of new building blocks beyond the conventional five-carbon(C_(5))units,dimethylallyl diphosphate(DMAPP)and isopentenyl diphosphate,expands significantly the chemical space of terpenoids.Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate(GPP)C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C_(6) unit,2-methyl-DMAPP.Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP.Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond.Furthermore,using Escherichia coli as the chassis,we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans-and cis-prenyl diphosphates(C_(5n+1))and terpenoids.This work provides insights into substrate recognition of prenyl diphosphate methyltransferases,and strategies to diversify terpenoids by expanding the building block portfolio.
