scholarly journals Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylation

2019 ◽  
Author(s):  
Po-Hsiang Wang ◽  
Yi-Lung Chen ◽  
Sean Ting-Shyang Wei ◽  
Kan Wu ◽  
Tzong-Huei Lee ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Specific Inhibitor ◽  
Steroid Metabolism ◽  
Cell Extracts ◽  
Propyl Iodide ◽  
Steroid Estrogens

AbstractSteroid estrogens modulate physiology and development of vertebrates. Biosynthesis of C18 estrogens from C19 androgens by the O2-dependent aromatase is thought to be irreversible. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster emtABCD differentially transcribed under estrogen-fed conditions. emtABCD encodes a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; emtA-disrupted strain DHT3 can catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, indicate the occurrence of a cobalamin-mediated estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell-extracts requires methylcobalamin and is inhibited by propyl-iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-mediated estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.

scholarly journals Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylation

2019 ◽  
Vol 117 (3) ◽  
pp. 1395-1403 ◽  
Author(s):  
Po-Hsiang Wang ◽  
Yi-Lung Chen ◽  
Sean Ting-Shyang Wei ◽  
Kan Wu ◽  
Tzong-Huei Lee ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Specific Inhibitor ◽  
Steroid Metabolism ◽  
Irreversible Reaction ◽  
Cell Extracts ◽  
Propyl Iodide ◽  
Steroid Estrogens

Steroid estrogens modulate physiology and development of vertebrates. Conversion of C19 androgens into C18 estrogens is thought to be an irreversible reaction. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster, emtABCD, differentially transcribed under estrogen-fed conditions and predicted to encode a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; an emtA-disrupted DHT3 derivative could catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, suggested the occurrence of a cobalamin-dependent estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell extracts requires methylcobalamin and is inhibited by propyl iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-dependent estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.

scholarly journals Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1309-1313 ◽  
Author(s):  
EG Levin
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Specific Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Active Inhibitor ◽  
Latent Form ◽  
Cell Extracts

Abstract Human endothelial cells release two forms of a plasminogen activator- specific inhibitor: an active form that readily binds to and inhibits plasminogen activators and an inactive or latent form that has no anti- activator activity but which can be activated by denaturation. Latent and active forms of plasminogen activator-specific inhibitor were measured in cultures of human umbilical vein endothelial cells. Latent inhibitor was activated by treatment with 1% sodium dodecyl sulfate (SDS), and both forms were assayed by the 125I-fibrin plate method. After 16 hours, the conditioned medium contained 104.6 U/mL latent inhibitor activity and 2.6 U/mL active inhibitor. The level of each form in the culture medium increased with time although the activity associated with the latent form rose more rapidly: the ratio of latent to active inhibitor activity was 12 at four hours (10.3 U/mL v 0.86 U/mL) and reached 56 at 24 hours (155.3 U/mL v 2.80 U/mL). Intracellular inhibitor activity was associated with the active form only; no additional inhibitor activity was observed following SDS treatment of cell extracts. A decline in active inhibitor activity occurred during incubation at 37 degrees C with a 50% reduction in activity occurring in two hours. Treatment of conditioned medium with 10 U/mL thrombin also resulted in a loss of active inhibitor activity. The latent inhibitor, however, was not affected by either of these conditions. The inhibitor activity lost during incubation at 37 degrees C or thrombin treatment could be regenerated by SDS treatment, suggesting that the loss of the active inhibitor activity represented a conversion of this form to its latent counterpart. Thus, the concentration, stability, and regulation of these two forms of plasminogen activator inhibitor in human endothelial cell cultures differ significantly.

scholarly journals Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

2013 ◽  
Vol 79 (19) ◽  
pp. 6176-6179 ◽  
Author(s):  
Shuning Wang ◽  
Haiyan Huang ◽  
Jörg Kahnt ◽  
Rudolf K. Thauer
Keyword(s):  
Uric Acid ◽  
Gene Cluster ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Enzyme Complex ◽  
Content Type ◽  
Cell Extracts

ABSTRACTCell extracts of uric acid-grownClostridium aciduricicatalyzed the coupled reduction of NAD+and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene clusterhylCBA-fdhF2.

Cloning of a Gene Cluster Responsible for the Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α

2004 ◽  
Vol 68 (1) ◽  
pp. 146-152 ◽  
Author(s):  
Tomonobu TOYOMASU ◽  
Kentaro NAKAMINAMI ◽  
Hiroaki TOSHIMA ◽  
Takashi MIE ◽  
Kenji WATANABE ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Polymerase ◽  
Specific Inhibitor ◽  
Dna Polymerase Α

scholarly journals Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3311-3315 ◽  
Author(s):  
T Kaneko ◽  
T Watanabe ◽  
M Oishi
Keyword(s):  
Protein Synthesis ◽  
Early Stage ◽  
Mitochondrial Protein ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Mitochondrial Protein Synthesis ◽  
Cell Extracts ◽  
Factor Ii ◽  
Mouse Erythroleukemia

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

scholarly journals Degradation of Morpholine by an EnvironmentalMycobacterium Strain Involves a Cytochrome P-450

1998 ◽  
Vol 64 (1) ◽  
pp. 159-165 ◽  
Author(s):  
P. Poupin ◽  
N. Truffaut ◽  
B. Combourieu ◽  
P. Besse ◽  
M. Sancelme ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Direct Analysis ◽  
Difference Spectrum ◽  
Sole Source ◽  
Spectrophotometric Analysis ◽  
Treatment Unit ◽  
H Nmr ◽  
Cell Extracts ◽  
Cytochrome P 450

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3311-3315
Author(s):  
T Kaneko ◽  
T Watanabe ◽  
M Oishi
Keyword(s):  
Protein Synthesis ◽  
Early Stage ◽  
Mitochondrial Protein ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Mitochondrial Protein Synthesis ◽  
Cell Extracts ◽  
Factor Ii ◽  
Mouse Erythroleukemia

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

scholarly journals Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

2010 ◽  
Vol 192 (19) ◽  
pp. 5237-5244 ◽  
Author(s):  
Diliana Dancheva Simeonova ◽  
Marlena Marie Wilson ◽  
William W. Metcalf ◽  
Bernhard Schink
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Electron Donor ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Strictly Anaerobic ◽  
Oxidation Activity ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Phosphorus Source

ABSTRACT Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

scholarly journals Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism

2006 ◽  
Vol 188 (23) ◽  
pp. 8062-8069 ◽  
Author(s):  
Jeffrey M. Boyd ◽  
Ashley Ellsworth ◽  
Scott A. Ensign
Keyword(s):  
Specific Inhibitor ◽  
Time Dependent ◽  
Coenzyme M ◽  
Carboxylase Activity ◽  
Alkene Oxidation ◽  
Cell Extracts ◽  
Degradation Activity ◽  
Dependent Growth ◽  
Dependent Pathway

ABSTRACT Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO2 fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO2-fixing enzyme 2-KPCC.

Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27

1997 ◽  
Vol 110 (3) ◽  
pp. 357-368 ◽  
Author(s):  
J. Guay ◽  
H. Lambert ◽  
G. Gingras-Breton ◽  
J.N. Lavoie ◽  
J. Huot ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Kinase ◽  
Heat Shock Protein ◽  
Chinese Hamster ◽  
Strong Support ◽  
Specific Inhibitor ◽  
Heat Shock Protein 27 ◽  
Cell Extracts ◽  
Hsp27 Phosphorylation ◽  
Homeostatic Function

We have studied the contribution of the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase), JNK/SAPK (c-JUN NH2-terminal kinase/stress-activated protein kinase) and p38, to activation of the HSP27 (heat shock protein 27) kinase MAPKAP kinase-2/3 and to HSP27 phosphorylation in Chinese hamster CCL39 cells stimulated by either growth factors, cytokines or stressing agents. In vitro assays using fractionated cell extracts or immunoprecipitates indicated that only fractions containing ERK or p38, and not those containing JNK/SAPK, had the capacity to activate MAPKAP kinase-2/3. In vivo, however, it appeared that only p38 is an upstream activator of HSP27 phosphorylation after both stress or growth factor stimulation: expression of an interfering mutant of ras, which blocked the activation of ERK by both types of inducers, had no effect on HSP27 phosphorylation and p38 activation; and the cell-permeant specific inhibitor of 038, SB203580, blocked MAPKAP-kinase2/3 activation and HSP27 phosphorylation. HSP27 has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. This raises the possibility that p38 might be directly involved in mediating actin responses to external stimuli. Accordingly, we observed that a prior activation of p38 increased the stability of the actin microfilaments in cells exposed to cytochalasin D. The effect was dependent on the expression of HSP27 and was totally annihilated by blocking the p38 activity with SB203580. The results provide strong support to the idea that activation of p38 during adverse environmental conditions serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. Its activation during normal agonist stimulation may constitute an additional actin signaling pathway, the importance of which depends on the level of expression of HSP27.

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