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91.
92.
Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus 总被引:1,自引:0,他引:1
Camilo Canel M. Inês Lopes-Cardoso Serap Whitmer Leslie van der Fits Giancarlo Pasquali Robert van der Heijden J. Harry C. Hoge Robert Verpoorte 《Planta》1998,205(3):414-419
Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan
decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures
established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures
transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the
pathway. Two such lines accumulated over 200 mg · L−1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and
tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines
showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the
medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity,
but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and
appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive
over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway
will yield positive results only in the presence of a favorable epigenetic environment.
Received: 12 June 1997 / Accepted: 24 October 1997 相似文献
93.
Jae Young Kim In Kook Park 《The international journal of biochemistry & cell biology》1998,30(12):1337-1344
The effects of 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of Japanese quail were investigated. SDS-polyacrylamide gel electrophoresis showed that the soluble proteins with molecular masses corresponding to 160.4 and 52.5 kDa were either missing or present at lower concentrations in the brain of the 6-AN treated group compared to those in the control group. The soluble liver proteins with molecular masses 200, 120 and 70.5 kDa were missing in the treated group compared to those in the control while those of a molecular mass 15.1 kDa were found to be present at higher concentrations. Similarly, treatment with 6-AN decreased the concentration of soluble proteins in pectoral muscle with molecular masses 92.3, 54.5, 43.5, 41.2, 34.5, 27.5, 20.1 and 17.5 kDa and increased those with molecular masses 96.5, 37.7, 25.0, 19.3, 16.6, 13.8 and 10.8 kDa. In the heart, soluble proteins with molecular mass 84.6 kDa were increased. There was a marked reduction in the treatment group in the concentration of NAD in pectoral muscle but not in other tissues. A similar observation was also made with total RNA levels. The specific activity of malic enzyme was markedly increased by 6-AN treatment in the kidney and pectoral muscle but reduced in the liver. 6-Phosphogluconate dehydrogenase and lactate dehydrogenase activities were markedly reduced in the liver. Glyceraldehyde-3-phosphate dehydrogenase activity was significantly decreased in liver and pectoral muscle. NAD glycohydrolase activity was markedly decreased in pectoral muscle. Acetylcholinesterase activity was markedly reduced in liver but was enhanced in pectoral muscle. The results suggest that the metabolic actions of 6-AN are specific for certain proteins in the liver and muscle with the effect being most pronounced in muscle. The effects are also quite distinct from those shown by its analogue 3-acetylpyridine. 相似文献
94.
Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells 总被引:1,自引:0,他引:1
Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml–1. A maximum production of 4.6 g recombinant tumstatin (107 cells)–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO4. 相似文献
95.
Chung JW Hong SJ Kim KJ Goti D Stins MF Shin S Dawson VL Dawson TM Kim KS 《The Journal of biological chemistry》2003,278(19):16857-16862
Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells. 相似文献
96.
Sphingosine-1-phosphate (S1P) is considered to be an important regulator of diverse biological processes acting as a natural ligand to EDG receptors. As a preliminary study to develop potent and selective agonist and antagonist for EDG receptors, we report synthesis of S1P stereoisomers and analogues and their binding affinities to EDG-1, -3, and -5. 相似文献
97.
98.
Miyakoshi A Yoon WK Jee Y Matsumoto Y 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(12):6371-6378
Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases. 相似文献
99.
Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth. 相似文献
100.
Luciana Gabriel Nogueira Ronaldo Honorato Barros Santos Barbara Maria Ianni Alfredo Inácio Fiorelli Eliane Conti Mairena Luiz Alberto Benvenuti Amanda Frade Eduardo Donadi Fabrício Dias Bruno Saba Hui-Tzu Lin Wang Abilio Fragata Marcelo Sampaio Mario Hiroyuki Hirata Paula Buck Charles Mady Edimar Alcides Bocchi Noedir Antonio Stolf Jorge Kalil Edecio Cunha-Neto 《PLoS neglected tropical diseases》2012,6(10)