Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.
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PMID:Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter. 820 May 8

The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell. To further investigate the mode of regulation of the gene (sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied. The mutant strains expressed beta-galactosidase under anaerobic conditions, whereas the wild-type did not. Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2'-dipyridyl, or to the redox cycling compound, paraquat. The wild-type, however, did respond to these effectors. In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5). Replacement of the wild-type 5'-regulatory region with either of the mutants' cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase. Sequence and restriction analysis revealed the presence of an IS2 insertion element in the promoter region of one of the mutants (NC5). This insertion caused the displacement of the 5'-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident-10 region from sodA and -35 from IS2. The second mutation (from NC4) was similarly analyzed, and an IS5 element was identified. The insertion site of IS5 (in NC4) was 6 bp (5'-TTAATT-3') upstream from the IS2 site (in NC5). Anaerobic expression of sodA in NC4 was lower than in NC5. This difference was almost eliminated in an arc- background, suggesting that the sequence 5'-TTAATT-3' might be essential for negative regulation by ArcA.
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PMID:Characterization of cis-acting regulatory mutations causing anaerobic expression of the sodA gene in Escherichia coli. 838 43

The purification by affinity chromatography of beta-galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L-serine deaminase 1. We conclude that sdaA is the structural gene for the latter enzyme. The purified L-serine deaminase 1 obtained after collagenase treatment of an sdaA-collagen-lacZ fusion differs from the native enzyme by the addition of several amino acids at the C-terminal. Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.
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PMID:Use of gene fusions of the structural gene sdaA to purify L-serine deaminase 1 from Escherichia coli K-12. 843 13

The Fur protein, which regulates iron uptake in Escherichia coli, also represses the biosynthesis of the manganese-containing superoxide dismutase (MnSOD). A strain of E. coli bearing a lacZ fusion to the aerobactin operon was used to compare the metal specificities of the regulation of MnSOD and of aerobactin by Fur. Iron, but not manganese, acted as a corepressor of the Fur-dependent inhibition of MnSOD biosynthesis. The iron-mediated inhibition of MnSOD biosynthesis was dependent upon an intact fur locus, indicating that the effect of iron is mediated by the fur gene product. The suppression of the accumulation of MnSOD by iron, but not by manganese, was not due to destabilization of the MnSOD polypeptide by iron. Thus this effect of iron was also seen in a sodA::lacZ operon fusion in which the production of beta-galactosidase was regulated by the sodA promoter. In contrast, both iron and manganese served as corepressors of aerobactin biosynthesis. It thus appears that the effectiveness of specific metal cations to act as corepressors with Fur varies with the gene being regulated by the Fur-metal complex.
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PMID:Iron specificity of the Fur-dependent regulation of the biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli. 844 93

The nifBQ transcriptional unit of Azotobacter vinelandii has been previously shown to be required for activity of the three nitrogenase systems, Mo nitrogenase, V nitrogenase, and Fe nitrogenase, present in this organism. We studied regulation of expression and the role of the nifBQ region by means of translational beta-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orf3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orf5 was never observed. Genes nifB and fdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was approximately 20- and approximately 400-fold lower than that of fdxN, respectively. Without Mo, expression of nifB dropped three- to fourfold and that of nifQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nifB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as beta-galactosidase fusion proteins with the expected molecular masses. The NifB- fusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxN- mutation resulted in reduced nitrogenase activities, especially when V was present. Dinitrogenase activity in extracts was similarly affected, suggesting a role of FdxN in iron-molybdenum cofactor synthesis. The NifO(-)-producing mutation did not affect any of the nitrogenases under standard diazotrophic conditions. The NifQ(-)-producing mutation resulted in an increased (approximately 1,000-fold) Mo requirement for Mo nitrogenase activity, a phenotype already observed with Klebsiella pneumoniae. No effect of the NifQ(-)-producing mutation on V or Fe nitrogenase was found; this is consistent with its very low expression under those conditions. Mutations in orf5 had no effect on nitrogenase activity.
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PMID:Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity. 849 13

In this study we investigated the intra-arterial delivery of viral and nonviral particles to experimental brain tumors. A herpes simplex virus (HSV) vector and monocrystalline iron oxide nanoparticles (MION) were injected into the internal carotid artery of Fisher 344 rats harboring intracerebral 9L gliosarcomas, using bradykinin to disrupt the blood-tumor barrier. Brain and internal organs were stained both for virus-mediated gene expression and for iron. Quantitative comparisons of gene expression and MION uptake with and without blood-tumor barrier disruption were performed in the center and at the periphery of the tumor mass, as well as in normal brain. In addition, MION distribution was traced in vivo by MR imaging. Delivery of HSV into 9L gliosarcoma cells was greatly enhanced by intra-carotid bradykinin infusion. Virus-mediated expression of the HSV-thymidine kinase (TK) and beta-galactosidase gene products was highest at the tumor periphery as compared to the tumor center. Selective HSV infection of multiple tumor foci was achieved in both hemispheres without affecting normal brain. MION uptake was high at the tumor periphery even without blood-tumor barrier disruption. Bradykinin increased MION uptake predominantly in the center of the tumor with virtually no effect at the periphery. These findings show that selective blood-tumor barrier disruption by bradykinin can be used to enhance HSV-mediated gene delivery to tumor cells in the periphery of brain tumors. A crucial aspect in the treatment of malignant brain tumors is the eradication of tumor cells infiltrating the brain; bradykinin may facilitate access of vectors to these areas by selective disruption of their neovasculature.
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PMID:Selective uptake of viral and monocrystalline particles delivered intra-arterially to experimental brain neoplasms. 866 79

We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC). The expression of beta-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene. Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells. Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene. We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP. In addition, we showed that the CpdA protein hydrolyzed cAMP to 5'-adenosine monophosphate and that its activity was activated by iron. Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase.
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PMID:Identification of the cpdA gene encoding cyclic 3',5'-adenosine monophosphate phosphodiesterase in Escherichia coli. 881 Mar 11

The characteristics of the binding of human lactoferrin (LF) to the cells of a human monocytic leukemia cell line, THP-1, were investigated. 125I-Labeled LF (125I-LF) bound to THP-1 cells, and the binding increased markedly as the cells matured into macrophages (THP-1 macrophages) by stimulation with phorbol 12-myristate 13-acetate. Scatchard analysis of the binding of 125I-LF to THP-1 macrophages indicated that high and low affinity receptor sites (Kd = 0.57 x 10(-6) and 3.7 x 10(-6) M, respectively) are present on the cells. The number of these high and low affinity receptor sites were 2.4 x 10(6), and 2.5 x 10(6) per cell, respectively. Removal of iron from 125I-LF did not affect its binding to THP-1 macrophages, indicating that the binding is not dependent on Fe(III) ion. The binding of the labeled LF to THP-1 macrophages was markedly decreased following acetylation, suggesting that the amino residues of the polypeptide portion of LF play a major role in the binding. The binding of labeled LF was partially inhibited by the isolated whole oligosaccharides of LF, and by the isolated whole oligosaccharides of band 3 glycoprotein of human erythrocyte membrane which contain poly-N-acetyllactosaminyl saccharide chains, like the LF oligosaccharides. Their inhibitory activity did not depend on the terminal sialyl residues of the saccharide chains. Lacto-N-fucopentaose III and lacto-N-neotetraose, an analogous structure being present in the poly-N-acetyllactosaminyl chains of LF, also artially inhibited the binding of 125I-LF to the THP-1 macrophages. When poly-N-acetyllactosaminyl saccharide chains of 125I-LF were cleaved by endo beta-galactosidase, the binding of 125I-LF was partially reduced. These results suggest that binding of LF to THP-1 macrophages is primarily mediated by its protein component, but a short oligosaccharide structure, possibly Gal beta 1-4GlcNAc beta 1-3Gal, which is contained in the nonreducing terminal region of poly-N-acetyllactosaminyl saccharide chains of LF and band 3, and in lacto-N-fucopentaose III and lacto-N-neotetraose is also recognized by THP-1 macrophages, and this recognition partly contributes to the binding of LF to cells.
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PMID:Binding characteristics of human lactoferrin to the human monocytic leukemia cell line THP-1 differentiated into macrophages. 885 Mar

The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114. However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P. fluorescens M114. This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114. Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium. This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested. Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants. This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself. In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.
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PMID:Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response. 887 Feb 53

Legionella pneumophila, a parasite of alveolar macrophages, requires iron for intra- and extracellular growth. Although its mechanisms for iron assimilation are poorly understood, this bacterium produces Fur, a protein that can repress gene transcription in response to iron concentration. Because iron- and Fur-regulated genes are important for infection in other bacteria, the identification of similar genes in L. pneumophila was undertaken. A wild-type strain of L. pneumophila was randomly mutated with a mini-Tn10' lacZ transposon, and the resulting gene fusions were tested for iron regulation by assessing beta-galactosidase production in the presence and absence of iron chelators. Of the initial six mutants with iron-repressed lacZ fusions, two strains, NU229 and NU232, possessed fusions that were stably iron regulated. To assay for Fur regulation, the levels of beta-galactosidase were measured in strains no longer producing Fur. As in a number of pathogenic bacteria, L. pneumophila fur could not be insertionally inactivated, but spontaneous Fur- derivatives were generated by selecting for manganese resistance. Strain NU229 contained a Fur-repressed fusion based on derepression of lacZ expression in its manganese-resistant derivative. Extracellular growth of NU229 in bacteriological media was similar to that of wild-type strain 130b. To assess the role of an iron- and Fur-regulated (frgA) gene in intracellular infection, the ability of NU229 to grow within U937 cell monolayers was tested. Quantitative infection assays demonstrated that intracellular growth of NU229 was impaired as much as 80-fold. Reconstruction of the mutant by allelic exchange proved that the infectivity defect in NU229 was due to the inactivation of frgA and not to a second-site mutation. Subsequently, complementation of the interrupted gene by an intact plasmid-encoded gene demonstrated that the infectivity defect was due to the loss of frgA and not to a polar effect. Nucleotide sequence analysis revealed that the 63-kDa FrgA protein has homology with the aerobactin synthetases IucA and IucC of Escherichia coli, raising the possibility that L. pneumophila encodes a siderophore which is required for optimal intracellular replication. Southern hybridization analysis determined that frgA is specific to L. pneumophila.
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PMID:An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases. 897 3


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