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1.
Cloned cytosine deaminase gene expression of Bifidobacterium longum and application to enzyme/pro-drug therapy of hypoxic solid tumors 总被引:18,自引:0,他引:18
Nakamura T Sasaki T Fujimori M Yazawa K Kano Y Amano J Taniguchi S 《Bioscience, biotechnology, and biochemistry》2002,66(11):2362-2366
Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(4):874-876
In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy. 相似文献
3.
A. V. Babwah C. S. Waddell 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(5):802-809
The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non- toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil
(5-FU). Cytosine deaminase activity is not found in higher plants and Brassica napus seedlings are unaffected by the presence of 5-FC in the growth medium. In codA-transformed B. napus seedlings, expression of cytosine deaminase results in a reduction of root and hypocotyl lengths, and a severe suppression
of true leaf development. This phenotype is dependent on the presence of the 5-FC substrate and no effects are seen in plants
grown in the absence of the substrate or in sibling plants lacking the transgene. The codA transformants have been assessed over three generations of growth and in each generation the transgene is stably inherited
and confers the same 5-FC-sensitive phenotype. Transfer of 5-FC-sensitive seedlings to soil results in the restoration of
normal growth in up to 100% of the seedlings. These results indicate that codA is a versatile dominant marker gene that can be used effectively in B. napus for substrate-dependent negative selection.
Received: 24 June 1999 / Accepted: 22 July 1999 相似文献
4.
Beatriz Mesa‐Pereira Carlos Medina Eva María Camacho Amando Flores Eduardo Santero 《Microbial biotechnology》2015,8(1):169-176
In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. 相似文献
5.
A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria
Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. 相似文献
6.
Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism 相似文献
7.
Rebecca L. Dineen Christophe Penno Philip Kelleher Maxence J. B. Bourin Mary O'Connell-Motherway Douwe van Sinderen 《Microbial biotechnology》2021,14(4):1494-1511
pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum. RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an iteron-regulated plasmid (IRP) under strict auto-regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low-copy Bifidobacterium-E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809. 相似文献
8.
Hidaka A Hamaji Y Sasaki T Taniguchi S Fujimori M 《Bioscience, biotechnology, and biochemistry》2007,71(12):2921-2926
Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy. 相似文献
9.
Hamaji Y Fujimori M Sasaki T Matsuhashi H Matsui-Seki K Shimatani-Shibata Y Kano Y Amano J Taniguchi S 《Bioscience, biotechnology, and biochemistry》2007,71(4):874-883
In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy. 相似文献
10.
Thomas P. West 《Antonie van Leeuwenhoek》1996,69(1):331-335
Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH4)2SO4-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa. 相似文献
11.
Patrick Vandeputte Laurent Pineau Gérald Larcher Thierry Noel Daniel Brèthes Dominique Chabasse Jean-Philippe Bouchara 《Mycopathologia》2011,171(1):11-21
Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility
to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance.
One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease
in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced
susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease,
associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata. 相似文献
12.
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14.
Cytosine deaminase is a non-mammalian enzyme of widespread interest for prodrug enzyme therapy due to its ability to convert
prodrug 5-fluorocytosine into anticancer drug 5-fluorouracil. Cytosine deaminase enzyme has been purified to homogeneity from
E. coli K-12 MTCC 1302 strain. Km values for cytosine and 5-fluorocytosine were found to be 0.26 mM and 1.82 mM, respectively. We developed a chitosan-entrapped
cytosine deaminase nanocomposite. Atomic force microscopy and transmission electron microscopy images showed an elongated
sphere shape nanocomposite with an average size of 80 nm diameter. Fourier transform infrared spectroscopy and X-ray diffraction
results confirmed gel formation and entrapment of cytosine deaminase within the nanocomposite. Sustained release of cytosine
deaminase from the nanocomposite up to one week depicted its potential implication in prodrug inducted enzyme therapy. 相似文献
15.
Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine
and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of
the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers,
such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate.
This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation
of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested
that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine
and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme. 相似文献
16.
Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect
against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated
with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile
fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of
B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream. 相似文献
17.
Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD. 相似文献
18.
Summary The gene specifying cytosine deaminase (cod) is shown to be located at approximately 86 minutes on the linkage map of E. coli. The corresponding gene in S. typhimurium has been reported to have a different location. 相似文献
19.
Bifidobacteria can potentially be used for gene therapy. Here, we reported that 65% of the total hIFN-α2b produced from Bifidobacteria longum transformed with pBAD-SPIFN plasmids encoding a fusion protein of the arabinosidase signal peptide and human IFN-α2b (hIFN-α2b),
was secreted. For B. longum transformed with pBAD-IFN plasmids (hIFN-α2b without the signal peptide), only 15% of the total IFN-α2b was secreted and
western blotting and N-terminal amino-acid sequence analysis revealed cleavage of the arabinosidase signal peptide from the
secreted hIFN-α2b. Moreover, the active level of the secreted hIFN-α2b in the supernatant of B. longum transformed with pBAD-SPIFN plasmids was over 1,000 IU/ml commercial rhIFN-α2b. Hence, the arabinosidase signal peptide can
enhance the secretion efficiency of IFN-α2b from B. longum.
Q. Deng, W. Zeng and Z. Yu contributed equally to this work. 相似文献
20.
We have constructed a constitutive high-level-expression vector for the genus Bifidobacterium and used it to express cholesterol oxidase from Streptomyces coelicola. The promoter region of the 16S rRNA gene was amplified by inverse PCR and used for the construction of pBES16PR. The optimal
ribosome-binding site (RBS) for Bifidobacterium was incorporated in pBES16PR. In order to test the efficacy of this expression vector, we constructed pBES16PR-CHOL with
the structural gene for cholesterol oxidase under the control of the 16S rRNA promoter, and used it to transform Bifidobacterium longum. The gene was successfully expressed and high level of cholesterol oxidase activity was obtained in B. longum. This is the first report of an expression vector for the genus Bifidobacterium using a 16S rRNA gene promoter and successful expression of cholesterol oxidase.
Myeong Soo Park and Bin Kwon contributed equally. 相似文献