Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have developed a method for the quantitative, exhaustive sequence specificity determination of DNA-binding proteins. The QuESSD method overcomes the limitations inherent in other published in vitro selection methods, not only defining the consensus sequence, but also quantifying the effect on DNA-protein affinity of replacing each base in the recognition domain with every other base. The features distinguishing this method from other in vitro selection approaches are: (1) instead of synthesizing one target oligonucleotide population containing a long randomized domain, we synthesize several oligonucleotide populations, each randomized at two positions. (2) Instead of carrying out several cycles of selection and amplification, we carry out a single cycle. (3) We have developed data collection and analysis procedures that eliminate artifacts and allow generation of quantitative results. The QuESSD method yields accurate measures of: (a) the selectivity of the protein for each base at each position within the recognition domain (normalized relative selectivity), (b) the contributions of individual sites within the recognition domain to the binding affinity (selectivity variance), (c) the relative binding affinity of any given sequence (global selectivity). We confirmed results by (1) tabulating directly the frequency of appearance of individual species in the pool of protein-bound oligonucleotides by cloning and sequencing individual oligonucleotides, and (2) competition EMSA analysis of oligonucleotides designed on the basis of QuESSD data. We have used this method to map the sequence specificity of the nuclear protein XF1 and to distinguish the sequence specificities of XF1 and the AH receptor complex, both of which bind to XRE1, a xenobiotic responsive element (XRE) located upstream of the CYP1A1 gene. Using data obtained by the QuESSD method, we designed oligonucleotides specific for XF1 or for the AH receptor, and prepared CAT reporter gene constructs carrying these oligonucleotides, or wild-type XRE1, upstream of a minimal promoter. Transfection studies using these constructs indicated that XF1 can function as a weak activator of basal transcription, and can, under some circumstances, compete with the AH receptor for binding to XRE1.
J Mol Biol 1997 Feb 28
PMID:Mapping sequence specific DNA-protein interactions: a versatile, quantitative method and its application to transcription factor XF1. 906 5

Introduced microorganisms are potentially powerful agents for manipulation of processes and/or components in soil. Fields of application include enhancement of crop growth, protection of crops against plant-pathogenic organisms, stimulation of biodegradation of xenobiotic compounds (bioaugmentation), and improvement of soil structure. Inoculation of soils has already been applied for decades, but it has often yielded inconsistent or disappointing results. This is caused mainly by a commonly observed rapid decline in inoculant population activity following introduction into soil, i.e., a decline of the numbers of inoculant cells and/or a decline of the (average) activity per cell. In this review, we discuss the available information on the effects of key factors that determine the fate and activity of microorganisms introduced into soil, with emphasis on bacteria. The factors addressed include the physiological status of the inoculant cells, the biotic and abiotic interactions in soil, soil properties, and substrate availability. Finally, we address the possibilities available to effectively manipulate the fate and activity of introduced microorganisms in relation to the main areas of their application.
Microbiol Mol Biol Rev 1997 Jun
PMID:Fate and activity of microorganisms introduced into soil. 918 7

Wood preserving waste (WPW) sites contain numerous toxic compounds, including phenols, polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins, and dibenzofurans. Previous in vitro and in vivo 32P-postlabeling studies showed the induction of multiple carcinogen-DNA adducts by WPW extracts. We now have tested the hypothesis in a mouse skin bioassay that a WPW extract not only causes the formation of exogenous, xenobiotic-derived DNA adducts, but also alters the levels of endogenous DNA modifications. Skin DNA of female ICR mice treated topically with an organic WPW extract was found by 32P-postlabeling to contain significantly increased levels of bulky oxidative DNA lesions (type II I-compounds), in addition to exogenous PAH-derived adducts. The mechanism of this increase is postulated to proceed through electrophilic quinoid compounds, which presumably were formed from phenols by chemical reactions of waste material or biologically by oxidative metabolism. On the other hand, the levels of another class of endogenous DNA adducts (type I I-compounds) were reduced significantly in exposed skin DNA. This effect was explained by the presence of cytochrome P450 inducers in the extract. All three types of DNA alterations observed may play a significant role in carcinogenesis. Our results imply that in addition to exogenous carcinogen-DNA adducts, alterations of endogenous DNA modifications may need to be considered in evaluating carcinogenic risk from toxic chemical wastes and the effects of remediation measures.
Environ Mol Mutagen 1997
PMID:Comparative 32P-postlabeling analysis of exogenous and endogenous DNA adducts in mouse skin exposed to a wood-preserving waste extract, a complex mixture of polycyclic and polychlorinated chemicals. 921 88

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds and activates the aryl hydrocarbon receptor (Ah-R), an endogenous transcription factor that is expressed in the thymus. TCDD exposure leads, among other effects, to thymus atrophy and immunosuppression. We previously analyzed the interference of TCDD with differentiation processes in fetal thymus organ cultures and found that in the presence of TCDD, the proliferation rate of immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes is inhibited, whereas the maturation along the CD4/CD8 path is accelerated. Moreover, the differentiation of thymocytes is skewed by TCDD at < or = 40% (compared with approximately 15% without TCDD) of the CD8 single-positive subset of future cytotoxic T cells, and apparently more cells audition for and pass positive selection. The fetal murine thymus expresses functional Ah-R mRNA, as shown by reverse transcription-polymerase chain reaction and TCDD-inducible CYP1A1 and CYP1B1 expression. Because the differentiation of thymocytes is to a considerable extent controlled by cytokines and many cytokine genes are potential targets of the Ah-R due to Ah-R-binding elements (xenobiotic response elements) in their promoters, we analyzed the cytokine expression in fetal thymus organ culture exposed to TCDD. Fetal thymi were cultured from gestation day 15 for < or = 8 days, thus covering ex vivo the period after population of the thymus anlage until birth. We show with semiquantitative reverse transcription-polymerase chain reaction that more interleukin (IL)-1beta, IL-2, IL-6, tumor growth factor (TGF)-beta3, and tumor necrosis factor-alpha are produced in TCDD-exposed thymi, whereas other cytokines (e.g., TGF-beta1, PAI-2, or IL-4) are only slightly up- and down-modulated during the culture period or not modulated at all (e.g., IL-1beta, IL-7, interferon-gamma, and TGF-beta2).
Mol Pharmacol 1997 Jul
PMID:Cytokine gene expression during ontogeny in murine thymus on activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin. 922 9

The human Dubin-Johnson syndrome is an autosomal recessive liver disease characterized by a chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of many endogenous and xenobiotic compounds. A similar disease phenotype has been described for a naturally occurring mutant Wistar rat strain, the TR- rat, which is defective in the, functionally defined, canalicular multispecific organic anion transporter (cMOAT). The complementary DNA encoding this protein has been cloned from rat and recently from human liver. cMOAT is a new member of the ATP-binding cassette transporter family, and homologous to the multidrug resistance-associated protein 1. A mutation in the cMOAT gene is responsible for the phenotype observed in TR- rats. This information should soon lead tc a complete genetic characterization of the human Dubin-Johnson syndrome.
J Mol Med (Berl) 1997 Jun
PMID:The canalicular multispecific organic anion transporter and conjugated hyperbilirubinemia in rat and man. 923 82

Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS.
Mol Cell Biol 1997 Aug
PMID:The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents. 923 35

Preliminary studies of a partial cDNA clone of the Eig17-1 gene from Drosophila melanogaster have shown that it encodes a probable cytochrome P450 of unknown function. To further characterize the Eig17-1 gene product, a full-length cDNA clone was isolated from a late-larval cDNA library and sequenced. Eig17-1 encodes a protein of 538 amino acids. The predicted protein is a cytochrome P450 that has been assigned to a new family, CYP18. The CYP18 protein is most closely related to steroid and xenobiotic metabolizing P450s of family CYP2 (30-33% identity), and to vertebrate steroidogenic P450s of families CYP17 and CYP21 (25-28% identity). Developmental Northern blot analysis revealed five distinct periods of Cyp18 expression during postembryonic development. Each period lasted 12-15 h, and was tightly correlated with reported ecdysteroid pulses in the first, second and third larval instars, at the time of pupariation and in pupae. This pattern of expression is consistent with the known induction of Cyp18 transcription by 20-hydroxyecdysone at the time of pupariation and suggests that ecdysteroids are major regulators of Cyp18 expression throughout postembryonic development. Northern blot analysis of RNA isolated from different prepupal tissues indicates that Cyp18 is differentially expressed in various ecdysteroid-responsive tissues. High Cyp18 expression was observed in body wall and gut while negligible expression was observed in salivary glands and fat body.
Mol Cell Endocrinol 1997 Jul 04
PMID:Sequence and developmental expression of Cyp18, a member of a new cytochrome P450 family from Drosophila. 925 62

Human pulmonary tissue are known to contain enzymes mediating procarcinogen activation. Peripheral blood lymphocytes and bronchoalveolar macrophages (BAMs) have been used as surrogates for the lung in studies involving cytochrome P450 (CYP) parameters, including CYP1A1 inducibility in relation to susceptibility to lung cancer. In this study, a comprehensive view of the expression patterns of xenobiotic-metabolizing CYP forms in human BAMs and peripheral blood lymphocytes was obtained by using gene-specific reverse transcriptase-polymerase chain reaction analysis. These patterns were compared with that in the whole lung. mRNAs of CYP2B6/7, CYP2C, CYP2E1, CYP2F1, CYP3A5, and CYP4B1 were detected in all seven BAM samples studied; however, only the mRNA of CYP2E1 was found consistently in all eight lymphocyte samples. The amounts of amplification products of CYP2B6/7, CYP2C, CYP3A5, and CYP4B1 were low and inconsistent, indicating low levels of expression in lymphocytes. Consistent with previous knowledge, mRNAs of CYP1A1, CYP2B6/7, CYP2E1, CYP2F1, CYP3A5, and CYP4B1 were detected in whole-lung tissue. These results give an overall picture of the expression of CYP genes in the xenobiotic-metabolizing families CYP1, CYP2, and CYP3 in BAMs, peripheral blood lymphocytes, and whole-lung tissue and will aid in directing future studies on the respective protein products. The differences in the CYP gene expression patterns between lung and lymphocytes cast additional doubt on the use of lymphocytes as a surrogate for the lung.
Mol Carcinog 1997 Oct
PMID:Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in human bronchoalveolar macrophages and peripheral blood lymphocytes. 936 12

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.
J Mol Biol 1997 Nov 07
PMID:Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase. 936 77

Recent reports indicate that the cytochrome P450 isozyme, CYP2D16, is expressed at high levels in the inner regions of the guinea pig adrenal cortex and may contribute to xenobiotic and/or steroid metabolism in the gland. In the present studies, immunohistochemical and in situ hybridization techniques were employed to definitively establish the localization of CYP2D16 within the adrenal cortex. In male guinea pigs of various ages, CYP2D16 protein and mRNA were highly localized to the zona reticularis (ZR); none was detectable in the zona fasciculata (ZF), zona glomerulosa (ZG) or the medulla. In contrast, the steroidogenic P450 isozyme, CYP17, was distributed throughout the ZF and ZR. From the earliest stages of development of the ZR, CYP2D16 staining was intense. As guinea pigs aged, the ZR progressively enlarged and comprised a proportionately greater amount of the cortex. At all ages, CYP2D16 was uniformly distributed throughout only the ZR. Coinciding with the age-related growth of the ZR and increase in adrenal CYP2D16 content was an increase in adrenal xenobiotic-metabolizing activity. The results establish that CYP2D16 has an intraadrenal localization that is unique among P450 isozymes, suggesting novel regulatory mechanisms and indicating that CYP2D16 may serve as a specific marker for ZR cells. The increase in CYP2D16 expression with age probably accounts for increasing levels of xenobiotic metabolism and may also contribute to an increase in intraadrenal cortisol degradation in older animals.
Mol Cell Endocrinol 1997 Nov 15
PMID:Localization of CYP2D16 in the guinea pig adrenal cortex by immunohistochemistry and in situ hybridization. 942 57


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