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Journal ArticleDOI

Mechanism of hydroxylamine-induced inhibition of β-galactosidase synthesis in Escherichia coli

30 Nov 1967-Biochemical and Biophysical Research Communications (Academic Press)-Vol. 29, Iss: 4, pp 463-468
TL;DR: It is demonstrated that in some strains, the HA effect involves catabolite repression in addition to interference with translation of the specific m-RNA, and the use of HA as a specific inhibitor of chain initiation is thus limited in such strains.

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Abstract: Kepes and Beguin (1966) demonstrated an inhibition of induced β-galatosidase peptide synthesis by hydroxylamine (HA) without effect on messenger RNA (m-RNA) formation. The mechanism by which HA inhibits chain initiation is unclear, as is the question whether the effect is general or only specific for the β-galactosidase peptide. Experiments with E. coli strains K12 (λ) and 113-3 (a met, B12 auxotroph, Davis and Mingioli, 1950 ) indicated more severe inhibition of β-galactosidase synthesis by HA if glycerol, or similar metabolizable carbon sources are present in the medium. This report demonstrates that in some strains, the HA effect involves catabolite repression in addition to interference with translation of the specific m-RNA. The use of HA as a specific inhibitor of chain initiation is thus limited in such strains.

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Topics: Catabolite repression (56%), Hydroxylamine (53%), Escherichia coli (52%)
Citations
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Book ChapterDOI
Kenneth Paigen1, Beverly Williams1Institutions (1)
TL;DR: This chapter focuses on three devices: catabolite repression, transient repression, and catabolites inhibition, which regulate the utilization of many carbohydrates, which influences many aspects of microbial growth and metabolism.

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Abstract: Publisher Summary This chapter focuses on three devices: catabolite repression, transient repression, and catabolite inhibition, which regulate the utilization of many carbohydrates. Catabolite repression is a reduction in the rate of synthesis of certain enzymes, particularly those of degradative metabolism, in the presence of glucose or other readily metabolized carbon sources. Catabolite inhibition is a control exerted by glucose on enzyme activity rather than on enzyme formation, analogous to feedback inhibition in biosynthetic pathways. Catabolite repression influences many aspects of microbial growth and metabolism. In addition to the well known repressions of carbohydrate utilization and amino-acid degradation in bacteria and yeast, catabolite repression affects the formation of enzymes that function in the tricarboxylic acid cycle, glyoxylate cycle, fatty acid degradation, carbon dioxide fixation, and the respiratory chain. In higher organisms, catabolite repression has been observed in sugar cane, rats, and man. The question of whether catabolite repression acts to inhibit the transcription of DNA into m-RNA or to inhibit translation of messenger into protein has received conflicting answers. Catabolite repression is a control system that usually affects catabolic enzymes. If catabolite repression and transient repression are not mediated by the specific apo-repressor of each operon, there must be another protein that recognizes the low molecular-weight effector. The significance of a control mechanism, which influences the activity as opposed to the concentration of a carbohydrate-metabolizing enzyme is readily appreciated because bacteria have a limited ability to change enzyme concentrations.

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163 citations


Journal ArticleDOI
01 Oct 2014-Chemosphere
TL;DR: Based on the activities of BPAF, BPF, BPA and MBP, the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti-androgenic assays that are reported in the literature.

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Abstract: The present study investigated and compared the estrogenic and androgenic activities of the three different classes of environmental pollutants and their metabolites using the XenoScreen XL YES/YAS assay, which has advantages compared with the original YES/YAS protocol. Contrary to the parent brominated flame retardants TBB and TBPH, which demonstrated no or very weak (anti)estrogenic or (anti)androgenic activities, their metabolites, TBBA and TBMEPH, exhibited anti-estrogenic (IC50 for TBBA=31.75 μM and IC50 for TBMEPH=0.265 μM) and anti-androgenic (IC50 for TBBA=73.95 μM and IC50 for TBMEPH=2.92 μM) activities. These results reveal that metabolism can enhance the anti-estrogenic and anti-androgenic effects of these two novel brominated flame retardants. Based on the activities of BPAF, BPF, BPA and MBP, we can conclude that the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti)androgenic assays that are reported in the literature. For BPA, it was confirmed previously that the metabolite formed after an ipso-reaction (hydroxycumyl alcohol) exhibited higher estrogenic activity compared with the parent BPA, but this was not confirmed for BPAF and BPF ipso-metabolites, which were not active in the XenoScreen YES/YAS assay. Among the substituted BPA analogues, bis-GMA exhibited weak anti-estrogenic activity, BADGE demonstrated weak anti-estrogenic and anti-androgenic activities (IC50=13.73 μM), and the hydrolysed product BADGE·2H2O demonstrated no (anti)estrogenic or (anti)androgenic activities.

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45 citations


Journal ArticleDOI
TL;DR: Iptakalim has exerted protective effects against hypertensive damage to target organs in rats and improves endothelial dysfunction associated with cardiovascular diseases by selective activation of the SUR2B/Kir6.1 subtype of KATP channels expressed in the endothelium.

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Abstract: Hypertension is the most common cardiovascular disease. The discovery of the antihypertensive action of adenosine triphosphate-sensitive potassium (K(ATP)) channel openers was a significant advance in the treatment of hypertension. Iptakalim is a novel K(ATP) channel opener with a unique chemical structure that differs from other K(ATP) openers. Among the 3 different subtypes of K(ATP) channels heterologously expressed in human embryonic kidney cells and Xenopus oocytes, iptakalim exhibits significant selectivity for SUR2B/Kir6.1 channels, mild effects on SUR2A/Kir6.2 channels, and fails to open SUR1/Kir6.2 channels. Iptakalim is a more potent activator of the SUR2B/Kir6.1 subtype of K(ATP) channels than diazoxide and pinacidil, the 2 most commonly studied K(ATP) channel openers. Iptakalim selectively produces arteriolar vasodilation with essentially no effect on the capacitance vessels. It can preferentially relax arterioles and small arteries, without affecting large arteries. Furthermore, iptakalim strongly lowers the blood pressure of hypertensive rodents and humans but has little effect on normotensive rodents and humans. Selective antihypertensive action is not observed with pinacidil or diazoxide and may be due to the high selectivity of iptakalim for the SUR2B/Kir6.1 subtype of K(ATP) channels, as well as its selective relaxation of resistance vessels. In pulmonary arterial smooth muscle cells, iptakalim inhibits the increase of cytoplasmic free Ca2+ concentration, as well as cell proliferation induced by endothelin-1. Furthermore, iptakalim has exerted protective effects against hypertensive damage to target organs in rats and improves endothelial dysfunction associated with cardiovascular diseases by selective activation of the SUR2B/Kir6.1 subtype of K(ATP) channels expressed in the endothelium. Clinical trials of iptakalim in the treatment of mild-moderate hypertension have been completed in China. In additional to strong antihypertensive efficacy, iptakalim seems to have a favorable safety and tolerability profile. Iptakalim is a promising new generation antihypertensive drug.

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39 citations



References
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Journal ArticleDOI
TL;DR: Certain biochemical properties of auxotrophicl mutants of Escherichia coli with specific growth requirements for most of the known water-soluble vitamins, as well as of others responding to methionine but not to B,2 are described.

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Abstract: The penicillin method (Davis, 1948; Lederberg and Zinder, 1948) has permitted convenient isolation of auxotrophicl mutants of Escherichia coli with specific growth requirements for most of the known water-soluble vitamins, as well as amino acids, purines, and pyrimidines. Accordingly, when crystalline vitamin B12 became available, a search was made for mutants requiring this nutrilite. Several strains of the desired type were promptly recovered. In all cases methionine, but not homocysteine, could be substituted for the vitamin. This paper describes certain biochemical properties of these mutants, as well as of others responding to methionine but not to B,2.

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2,819 citations


Journal ArticleDOI
Daisuke Nakada1, Boris Magasanik1Institutions (1)
TL;DR: During the induction phase a messenger RNA specific for β-galactosidase is produced which directs the subsequent synthesis of the enzyme in the inducer-free medium, which largely prevents the subsequent production of normal enzyme and causes the formation of an altered protein serologically related to β-Galactosids.

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Abstract: The mechanism of the induction of β-galactosidase in Escherichia coli at 30°C was investigated by separating the phase of enzyme induction from the phase of enzyme production. This separation was accomplished by removing the inducer by filtration after 3 to 4 min of contact with the cells, that is before enzyme had been formed; the cells then produced the enzyme in inducer-free medium at an exponentially declining rate for approximately 10 min. It was found that interference with the synthesis of normal RNA by 5-fluorouracil during the phase of enzyme induction largely prevents the subsequent production of normal enzyme and causes the formation of an altered protein serologically related to β-galactosidase; on the other hand, such an interference with RNA synthesis during the phase of enzyme production does not affect the formation of the enzyme by previously induced cells. Conversely, interference with protein synthesis by starvation for an amino acid or by chloramphenicol during the phase of induction does not prevent the subsequent production of normal enzyme; but such an interference with protein synthesis during the phase of enzyme production inhibits the formation of the enzyme by previously induced cells to the same extent as that of other protein. These results indicate that during the induction phase a messenger RNA specific for β-galactosidase is produced which directs the subsequent synthesis of the enzyme in the inducer-free medium. No additional β-galactosidase messenger RNA is formed after removal of the inducer and that present in the cell decays exponentially with a half-life of approximately 2·5 min. The rate of this decay is independent of the rate of protein synthesis or the rate of energy metabolism. An excess of intracellular catabolites, produced either by the rapid catabolism of glucose or by the restriction of protein synthesis in the presence of a utilizable energy source, inhibits the induction of the enzyme but does not affect the production of the enzyme by previously induced cells. Apparently, the catabolite repressor inhibits and the inducer stimulates the synthesis of the unstable messenger RNA specific for β-galactosidase.

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260 citations



Journal ArticleDOI
Adam Kepes1, Simone Beguin1Institutions (1)
TL;DR: The residual enzyme-synthesizing ability under these conditions arises from the ability of preformed messenger to bind new ribosomes and to initiate and terminate new complete peptide chains, which decreases exponentially with time whereas process (b) has a linear time course.

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Abstract: In a growing bacterial population in the steady state of induced enzyme synthesis, the synthesis of messenger RNA can be stopped by various means. The residual enzyme-synthesizing ability under these conditions arises (a) from the ability of preformed messenger to bind new ribosomes and to initiate and terminate new complete peptide chains and (b) from the continuation and termination of growing peptide chains present on the polysomes. In the case of the induced synthesis of β-galactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) in Escherichia coli the contributions from (a) and (b) are approximately equal. Potentiality (a) decreases exponentially with time whereas process (b) has a linear time course; the growth of the peptide chain of one β-galactosidase subunit of molecular weight 130 000 takes 80 to 90 sec at 37°. The products from (a) and (b) can be distinguished by appropriate labeling of the enzyme with an amino acid analogue and by the preferential inhibition of (a) by hydroxylamine (5 · 10 −5 M). The implications of the rate of peptide chain growth measured by this method are discussed.

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59 citations


Journal ArticleDOI
Kenneth Paigen1Institutions (1)
TL;DR: It is concluded that under normal growth conditions the total rate of macromolecular synthesis can be a primary factor in regulating the formation of enzymes feeding the intermediary pool, and the physiological role of catabolite repression may be expanded to include not only the activities of compounds which affect the rate of carbon entry into the pool, but also the activities which modify the rates of utilization of the intermediary Pool for synthetic purposes.

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Abstract: The induction of galactokinase (EC 2.7.1.6) is initially inhibited in Escherichia coli growing in complex media. The inhibition was shown to arise from the presence in broth and casein hydrolysate of repressors of galactokinase induction which are consumed during growth; in particular leucine, valine, histidine, and serine. These amino acids have in common an inhibitory effect on the growth of E. coli. It is suggested that they block anabolic processes and thus cause an accumulation of the intermediary pool. The enlarged intermediary pool then represses the formation of galactokinase through catabolite repression. This interpretation was supported by the following observations: (a) a similar amino acid repression is exerted on β-galactosidase (EC 3.2.1.23) induction; (b) other growth inhibitors which block the synthesis of macromolecules also preferentially inhibit galactokinase and β-galactosidase induction; and (c) iodoacetate, which inhibits growth by blocking metabolism of the carbon source, enhances induction. It is concluded that under normal growth conditions the total rate of macromolecular synthesis can be a primary factor in regulating the formation of enzymes feeding the intermediary pool. The physiological role of catabolite repression may therefore be expanded to include not only the activities of compounds which affect the rate of carbon entry into the pool, but also the activities of compounds which modify the rate of utilization of the intermediary pool for synthetic purposes.

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39 citations