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Etienne Hollande Marjorie Fanjul Nawal Houti Jean-Charles Faye Philippe Courriere 《In vitro cellular & developmental biology. Animal》1998,34(7):593-599
Summary In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an
attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic
duct cells, the expression of estrogen receptor (ERα) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they
acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1
cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features
that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERα and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERα (236 ± 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%).
At the onset of the stationary phase (Day 5), ERα levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 ± 0.9
fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol
to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen.
Immunocytochemical analysis with anti-ERα-1D5 antibodies showed nuclear and cytoplasmic localization of ERα in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular
multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement
of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERα is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in
nontarget tissues. 相似文献
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Cornelissen S Julsing MK Schmid A Bühler B 《Journal of industrial microbiology & biotechnology》2012,39(2):275-287
Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis.
In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression
levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U gCDW−1, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol gCDW−1). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in
order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical
production, but are sufficient for preparative-scale drug metabolite synthesis. 相似文献
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Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress 总被引:2,自引:0,他引:2
Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability
of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the
tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes.
Primers targeting each gene (18S rRNA, EF-1α, Ntubc2, α- and β-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set
of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses.
L25 and EF-1α demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1α was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach.
These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco. 相似文献
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Liliya Horbal Yuriy Rebets Maria Rabyk Andriy Luzhetskyy Bogdan Ostash Elisabeth Welle Tatsunosuke Nakamura Victor Fedorenko Andreas Bechthold 《Applied microbiology and biotechnology》2010,85(4):1069-1079
Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the
lipReg1–4 and lipX1 showed that they encode components of two-component signal transduction systems (LipReg1 and LipReg2), multiple antibiotics
resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease
(LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies,
and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in α-lipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity
of LipReg1, which in its turn govern the expression of the α-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4. 相似文献
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To enhance our understanding of brassinosteroid (BR) biosynthesis in rice, we attempted to identify putative rice homologs
of Arabidopsis CYP90A1/ CPD and related mutants. Two candidate genes, designated CYP90A3/OsCPD1 and CYP90A4/OsCPD2, are located on chromosomes 11 (2.0 cM) and 12 (1.9 cM), respectively. Based on sequence similarity with the Arabidopsis CYP90A1/CPD gene, we predict that the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 gene products function as C-23α hydroxylases in the BR biosynthesis pathway. Both are broadly expressed in wild-type rice,
and their expression is regulated by a feedback mechanism. A retrotransposon insertion mutant of CYP90A3/OsCPD1, oscpd1-1, did not produce any BR-deficient phenotype or feedback upregulation of genes for BR biosynthesis enzymes. These results
indicate that if, as predicted, the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 genes do function in the BR biosynthesis pathway, they may each have enough capacity to catalyze BR biosynthesis on their
own. As a consequence, the oscpd1-1 mutant may not be deficient in endogenous BRs. Interestingly, BR biosynthesis enzymes except C-6 oxidase are encoded by plural
genes in rice but by single genes in Arabidopsis (again, except C-6 oxidase). On the basis of these findings, we discuss the differences in BR biosynthesis between rice and
Arabidopsis. 相似文献
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Brezna B Kweon O Stingley RL Freeman JP Khan AA Polek B Jones RC Cerniglia CE 《Applied microbiology and biotechnology》2006,71(4):522-532
Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were ≥80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherchia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[α]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycylic aromatic hydrocarbons.An erratum to this article can be found at 相似文献
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Voltage-dependent L-type Ca+ channels of the C2C12 line myoblasts of mice have been studied at the stage of proliferation and 24 h after the beginning
of differentiation. The expression of genesCacna1s, Cacna1c, Cacna1d, and Cacna1f, which encode channel forming subunits α1S, α1C, α1D, and α1F, respectively, has been assessed. The expression of genes Cacna2d and Cacn1g, which encode the α2, δ, and γ regulatory subunits, has been studied as well. For the first time, the expression of Cacna1d, which is typical for nerve cells, has been revealed in proliferating myoblasts, whereas in differentiating mononuclear myoblasts
the expression of this gene was significantly decreased. On the contrary, the low level of expression of Cacna1S, which encodes the specific α1S channel forming subunit of skeletal muscles, has been observed in proliferating myoblasts,
whereas in differentiating mononuclear myoblasts it has been shown to increase multifold. No considerable changes in expression
of Cacna2d and Cacn1g have been revealed in proliferating and differentiating myoblasts. No traces of expression of Cacna1c and Cacna1f have been revealed in myoblasts. 相似文献
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Importin αs are import receptors for nuclear localization signal-containing proteins. Most animal importin αs assort into
α1, α2, and α3 groups. Studies in Drosophila melanogaster, Caenorhabditis elegans, and mouse suggest that the animal importin α gene family evolved from ancestral plant-like genes to serve paralog-specific
roles in gametogenesis. To explore this hypothesis we extended the phylogenetic analysis of the importin α gene family to
nonbilateral animals and investigated whether animal-like genes occur in premetazoan taxa. Maximum likelihood analysis suggests
that animal-like importin α genes occur in the Choanoflaggelate Monosiga brevicollis and the amoebozoan Dictyostelium; however, both of these results are caused by long-branch attraction effects. The absence of animal-like α genes in premetazoan
taxa is consistent with the hypothesis that they duplicated and then specialized to function in animal gametogenesis. The
gene structures of the importin αs provide insight into how the animal importin α gene family may have evolved from the most
likely ancestral gene. Interestingly, animal α1s are more similar to plant and fungal α1-like sequences than they are to animal
α2s or α3s. We show that animal α1 genes share most of their introns with plant α1-like genes, and α2s and α3s share many
more intron positions with each other than with the α1s. Together, phylogenetics and gene structure analysis suggests a parsimonious
path for the evolution of the mammalian importin α gene family from an ancestral α1-like progenitor. Finally, these results
establish a rational basis for a unified nomenclature of the importin α gene family. 相似文献
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Functional genomics of PPAR-γ in human immunomodulatory cells 总被引:1,自引:0,他引:1
Keeping in view the fact that peroxisome-proliferators activated receptors-PPARs (α,γ) play a crucial role in atherogenic
inflammation, the present study was addressed to explore as to how selective and specific PPAR-γ gene silencing within human
mononuclear cells affects genes involved in lipid metabolism and innate immune process. Such a study revealed that with respect
to control cells, the PPAR-γ knock-out cells exhibited significant reduction in the expression of genes coding for PPAR- α,
CD-36, LDL-R as well as significant increase in the expression of genes coding for IL-4, IL-8, IFN-γ, CX3CR1, hTERT. However,
the expression of genes coding for LXR-α and Receptor-C
k
could not be detected in PPAR-γ knock-out cells. Based on these results, we propose that PPAR-γ gene has the inherent capacity
to influence the lipid mediated inflammation process in atherosclerotic lesions. 相似文献
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Background
Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense. 相似文献19.
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N. Kitamoto J. Matsui Y. Kawai A. Kato S. Yoshino K. Ohmiya N. Tsukagoshi 《Applied microbiology and biotechnology》1998,50(1):85-92
For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase
genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other
fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal
protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A
and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded
a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately
100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature
optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C.
Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998 相似文献