Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(...Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.展开更多
The full-length genomic DNA of MCAT (Malonyl-CoA:acyl carrier protein transacylase) in Brassica napus was cloned. BnMCAT shares very high identity with AtMCAT in gene sequence and gene structure. A multiple alignment ...The full-length genomic DNA of MCAT (Malonyl-CoA:acyl carrier protein transacylase) in Brassica napus was cloned. BnMCAT shares very high identity with AtMCAT in gene sequence and gene structure. A multiple alignment of the protein sequence showed that BnMCAT shares high identity with other MCATs from E. coli and plants. BnMCAT was expressed in all tissues, such as roots, stems, leaves, flowers, and seeds, and no significant differences in the expression level were found in different embryo stages after pollination. According to an in vitro relative activity analysis, purified recombinant BnMCAT expressed in E. coli had transacylase activity. Although the relative activities of BnMCAT in crude extracts isolated from different staged embryos were similar and showed little variation, a higher relative activity was found in a crude extract isolated from embryos in comparison to leaves. Different relative activities of BnMCAT in crude extracts isolated from cultivars with different oil content were also found, suggesting that the activity of BnMCAT might be a decisive factor for a high oil content. Together, these results showed that BnMCAT is an important enzyme in the FAS system and indicate that BnMCAT might be a new target enzyme for future crop improvement through genetic engineering.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
The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. T...The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.展开更多
AIM: To evaluate the relationship between the expression of lipopolyaaccharides (LPS) binding protein (LBP) and CD14mRNA and the severity of liver injury in alcohol-fed rats.METHODS: Twenty Wietar rats were divided i...AIM: To evaluate the relationship between the expression of lipopolyaaccharides (LPS) binding protein (LBP) and CD14mRNA and the severity of liver injury in alcohol-fed rats.METHODS: Twenty Wietar rats were divided into two groups: ethanol-fed group (group E) and control group (group C). Group E was fed with ethanol( 5-12g. Kg- 1. D-1)and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 weeks and 8 weeks.Levels of endotoxin and alanine transeminase (ALT) inblood were measured, and liver pathology was observed under light and electronic microscopy. Expressions of LBP and CD14 mRNA in liver tissues were determined by RT-PCR analysis.RESULTS: Plasma endotoxin levels were increased more significantly in group E( 129 ± 21) ng. L- 1 and ( 187 ± 35) ng.L- 1 at 4 and 8 wk than in control rats(48 ± 9) ng. L- 1 and (53±11) ng.L-1, respectively (P< 0.05). Mean values of plasma ALT levels were (1867 ± 250) nkat. L-1 and (2450 ±367) nkat. L- 1 in Group E. The values were increased more dramatically in ethanol-fed rats than in Group C after 4 and 8weeks. In liver section from ethanol-fed rats, there were marked pathological changes (steatosis, cell infiltration and necrosis). In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels compared with the control group ( P< 0.05).CONCLUSION: Ethanol administration led to a significant increase in endotoxin levels in serum and LBP and CD14mRNA expressions in liver tissues. The increase of LBP and CD14 mRNA expression might wake the liver more sensitive to endotoxin and liver injury.展开更多
MOLECULAR PHYSIOLLGY OF HEPATOCELLULAR TRANSPORT PROTEINS Basolaferal transport systems Na+-dependent bile salt uptake Uptake of bile salts into the liver was first isolated perfused rat liver[1],isolated hepatocyte...MOLECULAR PHYSIOLLGY OF HEPATOCELLULAR TRANSPORT PROTEINS Basolaferal transport systems Na+-dependent bile salt uptake Uptake of bile salts into the liver was first isolated perfused rat liver[1],isolated hepatocyte cultures and basolateral plasma membrane vesicles [2,4].展开更多
Stabilization of proteins in delivery devices and design of appropriate protein carriers are major research issues due to the extreme sensitivity of proteins.Previously,negatively charged nanoparticles,consisting of p...Stabilization of proteins in delivery devices and design of appropriate protein carriers are major research issues due to the extreme sensitivity of proteins.Previously,negatively charged nanoparticles,consisting of poly(lactic-co-glycolic acid)(PLGA)and poly(styrene-co-4–styrene-sulfonate)(PSS),showed considerably high loading capacity for positively charged model protein lysozyme depending on the surface charge density of nanoparticles.展开更多
Objective To observe influence of fibrin seala nt (FS)on osteoinductive ability of inject-type BMP.Method The inject-type BMP power was dissolved in the main glue p art or thrombin part of FS,then mixed with the main ...Objective To observe influence of fibrin seala nt (FS)on osteoinductive ability of inject-type BMP.Method The inject-type BMP power was dissolved in the main glue p art or thrombin part of FS,then mixed with the main glue part or thrombin pa rt of FS into gel,observe coagulating time,then implant comp osite into the thigh muscle pouch of m ice to evaluate their capacity to induce new bone formation,and compared to the single BMP implant gr oup.Result There was no difference in the coagul ating time between two mixing method,the osteoin-ductive ability of implants BMP dissolved in the main glue part or thrombin part of FS group was higher than that of simply BMP implant group.Conclusion FS was perfect carrier to inject-type BMP.展开更多
文摘Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.
文摘The full-length genomic DNA of MCAT (Malonyl-CoA:acyl carrier protein transacylase) in Brassica napus was cloned. BnMCAT shares very high identity with AtMCAT in gene sequence and gene structure. A multiple alignment of the protein sequence showed that BnMCAT shares high identity with other MCATs from E. coli and plants. BnMCAT was expressed in all tissues, such as roots, stems, leaves, flowers, and seeds, and no significant differences in the expression level were found in different embryo stages after pollination. According to an in vitro relative activity analysis, purified recombinant BnMCAT expressed in E. coli had transacylase activity. Although the relative activities of BnMCAT in crude extracts isolated from different staged embryos were similar and showed little variation, a higher relative activity was found in a crude extract isolated from embryos in comparison to leaves. Different relative activities of BnMCAT in crude extracts isolated from cultivars with different oil content were also found, suggesting that the activity of BnMCAT might be a decisive factor for a high oil content. Together, these results showed that BnMCAT is an important enzyme in the FAS system and indicate that BnMCAT might be a new target enzyme for future crop improvement through genetic engineering.
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 by a grant from the National High Tech R&D Program(863 Program)of China(2001AA2121).
文摘The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.
基金Supported by the National Natural Science Foundation of China(No.39970719).
文摘AIM: To evaluate the relationship between the expression of lipopolyaaccharides (LPS) binding protein (LBP) and CD14mRNA and the severity of liver injury in alcohol-fed rats.METHODS: Twenty Wietar rats were divided into two groups: ethanol-fed group (group E) and control group (group C). Group E was fed with ethanol( 5-12g. Kg- 1. D-1)and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 weeks and 8 weeks.Levels of endotoxin and alanine transeminase (ALT) inblood were measured, and liver pathology was observed under light and electronic microscopy. Expressions of LBP and CD14 mRNA in liver tissues were determined by RT-PCR analysis.RESULTS: Plasma endotoxin levels were increased more significantly in group E( 129 ± 21) ng. L- 1 and ( 187 ± 35) ng.L- 1 at 4 and 8 wk than in control rats(48 ± 9) ng. L- 1 and (53±11) ng.L-1, respectively (P< 0.05). Mean values of plasma ALT levels were (1867 ± 250) nkat. L-1 and (2450 ±367) nkat. L- 1 in Group E. The values were increased more dramatically in ethanol-fed rats than in Group C after 4 and 8weeks. In liver section from ethanol-fed rats, there were marked pathological changes (steatosis, cell infiltration and necrosis). In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels compared with the control group ( P< 0.05).CONCLUSION: Ethanol administration led to a significant increase in endotoxin levels in serum and LBP and CD14mRNA expressions in liver tissues. The increase of LBP and CD14 mRNA expression might wake the liver more sensitive to endotoxin and liver injury.
基金supported by"H+Die Spitaler der Schweiz" the Swiss Agency for Development and Cooperation(DEZA)by the University Hospital Zurich/Switzerland
文摘MOLECULAR PHYSIOLLGY OF HEPATOCELLULAR TRANSPORT PROTEINS Basolaferal transport systems Na+-dependent bile salt uptake Uptake of bile salts into the liver was first isolated perfused rat liver[1],isolated hepatocyte cultures and basolateral plasma membrane vesicles [2,4].
文摘Stabilization of proteins in delivery devices and design of appropriate protein carriers are major research issues due to the extreme sensitivity of proteins.Previously,negatively charged nanoparticles,consisting of poly(lactic-co-glycolic acid)(PLGA)and poly(styrene-co-4–styrene-sulfonate)(PSS),showed considerably high loading capacity for positively charged model protein lysozyme depending on the surface charge density of nanoparticles.
文摘Objective To observe influence of fibrin seala nt (FS)on osteoinductive ability of inject-type BMP.Method The inject-type BMP power was dissolved in the main glue p art or thrombin part of FS,then mixed with the main glue part or thrombin pa rt of FS into gel,observe coagulating time,then implant comp osite into the thigh muscle pouch of m ice to evaluate their capacity to induce new bone formation,and compared to the single BMP implant gr oup.Result There was no difference in the coagul ating time between two mixing method,the osteoin-ductive ability of implants BMP dissolved in the main glue part or thrombin part of FS group was higher than that of simply BMP implant group.Conclusion FS was perfect carrier to inject-type BMP.
