cyp7b1 catalyses the 7α-hydroxylation of dehydroepiandrosterone and25-hydroxycholesterol in rat prostate

2001 ◽  
Vol 355 (2) ◽  
pp. 509-515 ◽  
Author(s):  
Cécile MARTIN ◽  
Rhona BEAN ◽  
Ken ROSE ◽  
Fouad HABIB ◽  
Jonathan SECKL
Keyword(s):  
Prostate Gland ◽  
Northern Blotting ◽  
Sex Steroid ◽  
Cytochrome P450 Enzyme ◽  
Steroid Synthesis ◽  
Peripheral Tissues ◽  
Reverse Transcription Pcr ◽  
P450 Enzyme ◽  
Rat Prostate

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3β-steroids at the 7α-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7α-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7α-hydroxylation conversion in rat prostate. NADPH-dependent 7α-hydroxylation was confirmed for 3β-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent Km of 14±1µM for 7α-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6µM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The Vmax value of the prostate was 46±2pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7α-hydroxylation in the prostate gland.

THE BINDING OF [1,2-3H]TESTOSTERONE WITHIN NUCLEI OF THE RAT PROSTATE

1969 ◽  
Vol 44 (3) ◽  
pp. 323-333 ◽  
Author(s):  
W. I. P. MAINWARING
Keyword(s):  
Molecular Weight ◽  
Equilibrium Dialysis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Rat Tissues ◽  
Receptor Complex ◽  
Rat Prostate ◽  
Binding Component

SUMMARY The specificity of the binding of [1,2-3H]testosterone to nuclei of various rat tissues in vivo has been studied. A significant amount of radioactivity was retained in the nuclei of androgen-dependent tissues only, particularly the ventral prostate gland. The bound radioactivity was only partially recovered as [1,2-3H]testosterone; the remainder was identified as [3H]5α-dihydrotestosterone. Efforts were made to characterize the binding component, or 'receptor', in prostatic nuclei. On digestion of nuclei labelled in vivo with [1,2-3H]testosterone, with enzymes of narrow substrate specificity, only trypsin released tritium, suggesting that the receptor is a protein. On the basis of subfractionation studies of labelled nuclei, the receptor is an acidic protein. The androgen—receptor complex could be effectively extracted from the prostatic nuclei in 1 m-NaCl and from the results of fractionations on a calibrated agarose column, the complex has a molecular weight 100,000–120,000. The specificity of the binding of steroids to such 1 m-NaCl extracts in vitro was investigated by the equilibrium dialysis procedure. Under these conditions, the specificity of the binding of [1,2-3H]testosterone demonstrated in vivo could not be simulated. The receptor is probably part of the chromatin complex but its precise intranuclear localization cannot be determined by biochemical procedures alone.

Inhibition of aldosterone biosynthesis by staurosporine

2005 ◽  
Vol 386 (7) ◽  
pp. 663-669 ◽  
Author(s):  
Matthias Bureik ◽  
Alexander Mion ◽  
Christopher J. Kenyon ◽  
Rita Bernhardt
Keyword(s):  
Kinase Inhibitor ◽  
Therapeutic Potential ◽  
Hypotensive Effect ◽  
Cytochrome P450 Enzyme ◽  
Aldosterone Synthase ◽  
Kinase C ◽  
Steroid Hydroxylase ◽  
P450 Enzyme ◽  
Adrenodoxin Reductase

Abstract Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50=11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 μg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p=0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.

scholarly journals Chlorogenic Acid as a Model Compound for Optimization of an In Vitro Gut Microbiome-Metabolism Model

Proceedings ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 31 ◽  
Author(s):  
Olivier Mortelé ◽  
Elias Iturrospe ◽  
Annelies Breynaert ◽  
Christine Lammens ◽  
Xavier Basil Britto ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Gut Microbiome ◽  
Model Compound ◽  
In Vitro Models ◽  
Cytochrome P450 Enzyme ◽  
Flight Mass Spectrometry ◽  
P450 Enzyme ◽  
Analytical Phase

It has been believed that the metabolism of xenobiotics occurred mainly by the cytochrome P450 enzyme system in the liver. However, recent data clearly suggest a significant role for the gut microbiota in the metabolism of xenobiotic compounds. This microbiotic biotransformation could lead to differences on activation, inactivation and possible toxicity of these compounds. In vitro models are generally used to study the colonic biotransformation as they allow easy dynamic and multiple sampling over time. However, to ensure this accurately mimics communities in vivo, the pre-analytical phase requires optimization. Chlorogenic acid, a polyphenolic compound abundantly present in the human diet, was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform. Samples of the in vitro gastrointestinal dialysis-model with colon stage were analyzed by liquid chromatography coupled to high resolution time-of-flight mass spectrometry. Complementary screening approaches were also employed to identify the biotransformation products.

scholarly journals Involvement of the C-Terminal Extension of the α Polypeptide and of the PucC Protein in LH2 Complex Biosynthesis in Rubrivivax gelatinosus

2004 ◽  
Vol 186 (10) ◽  
pp. 3143-3152 ◽  
Author(s):  
Anne-Soisig Steunou ◽  
Soufian Ouchane ◽  
Françoise Reiss-Husson ◽  
Chantal Astier
Keyword(s):  
Purple Bacteria ◽  
Growth Conditions ◽  
Initiation Site ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Rubrivivax Gelatinosus

ABSTRACT The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the β and α polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the α polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2−, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Inhibin-related proteins in rat prostate

1996 ◽  
Vol 149 (1) ◽  
pp. 93-99 ◽  
Author(s):  
G P Risbridger ◽  
T Thomas ◽  
C J Gurusinghe ◽  
J R McFarlane
Keyword(s):  
Transforming Growth Factor ◽  
Prostate Gland ◽  
Transforming Growth Factor Β ◽  
Subunit Mrna ◽  
Prostate Weight ◽  
Reverse Transcription Pcr ◽  
Androgen Withdrawal ◽  
Rat Prostate ◽  
Related Proteins ◽  
First Time

Abstract Inhibin and activin are members of the transforming growth factor β (TGFβ) family which can regulate cell proliferation in a number of tissues. The presence of inhibins and the related proteins, activins, in the prostate has been implicated by the detection of activin type II receptors. The aim of this study was to determine whether or not immunoactive (ir) inhibin and ir-activin are present in the rat prostate and to study the acute regulation by androgens. The results showed that mRNAs for the α and β inhibin subunits were detected in rat prostate by reverse transcription-PCR together with ir-inhibin and ir-activin in prostate cytosols. The levels of ir-activin in the prostate (223 ± 44 ng/gland) were greater than the levels of ir-inhibin (6·89 ng/gland), and activin immunoreactivity was localised to the epithelial cells. The presence of these proteins and the subunit mRNAs suggests that these proteins are produced in the prostate and may have a role in prostate function. The study of the effect of androgen withdrawal on the levels of ir-activin and ir-inhibin in these tissues showed no change in the content of ir-inhibin or ir-activin (ng/g tissue) after 3 days of castration or following the administration of the cytotoxic drug ethane dimethane sulphonate (EDS), although there was a significant (P<0·01) decline in prostate weight. Fourteen days after EDS treatment, as the prostate weight fell significantly lower, the amount of ir-inhibin and ir-activin per prostate gland was significantly (P<0·01) reduced although the concentration was unaffected. These data demonstrate, for the first time, that inhibin α and β subunit mRNA and ir-inhibin and ir-activin are present in the prostate; the role of these proteins in prostate function remains to be established. Journal of Endocrinology (1996) 149, 93–99

scholarly journals Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maximilian Frey ◽  
Iris Klaiber ◽  
Jürgen Conrad ◽  
Aylin Bersch ◽  
Irini Pateraki ◽  
...  
Keyword(s):  
Helianthus Annuus ◽  
Chemical Shifts ◽  
Sesquiterpene Lactones ◽  
2D Nmr ◽  
Cytochrome P450 Enzyme ◽  
The Core ◽  
Pathway Reconstruction ◽  
P450 Enzyme ◽  
Sunflower Genome

Abstract Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

scholarly journals Towards Accurate Genotype–Phenotype Correlations in the CYP2D6 Gene

2020 ◽  
Vol 10 (4) ◽  
pp. 158 ◽  
Author(s):  
Angel Pey
Keyword(s):  
Large Scale ◽  
Phenotypic Variability ◽  
Genetic Diseases ◽  
Individual Response ◽  
Cytochrome P450 Enzyme ◽  
Cyp2d6 Gene ◽  
Human Genes ◽  
P450 Enzyme ◽  
Simple Activity

Establishing accurate and large-scale genotype–phenotype correlations and predictions of individual response to pharmacological treatments are two of the holy grails of Personalized Medicine. These tasks are challenging and require an integrated knowledge of the complex processes that regulate gene expression and, ultimately, protein functionality in vivo, the effects of mutations/polymorphisms and the different sources of interindividual phenotypic variability. A remarkable example of our advances in these challenging tasks is the highly polymorphic CYP2D6 gene, which encodes a cytochrome P450 enzyme involved in the metabolization of many of the most marketed drugs (including SARS-Cov-2 therapies such as hydroxychloroquine). Since the introduction of simple activity scores (AS) over 10 years ago, its ability to establish genotype–phenotype correlations on the drug metabolizing capacity of this enzyme in human population has provided lessons that will help to improve this type of score for this, and likely many other human genes and proteins. Multidisciplinary research emerges as the best approach to incorporate additional concepts to refine and improve such functional/activity scores for the CYP2D6 gene, as well as for many other human genes associated with simple and complex genetic diseases.

scholarly journals Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence

2001 ◽  
Vol 280 (1) ◽  
pp. E120-E129 ◽  
Author(s):  
Fabien Van Coppenolle ◽  
Christian Slomianny ◽  
Françoise Carpentier ◽  
Xuefen Le Bourhis ◽  
Ahmed Ahidouch ◽  
...  
Keyword(s):  
Hormonal Regulation ◽  
Prostate Gland ◽  
In Vivo Model ◽  
Histological Changes ◽  
Prostate Hyperplasia ◽  
Prostate Development ◽  
Rat Prostate ◽  
Prostate Growth ◽  
Benign Prostate

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg · kg−1 · day−1). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.

scholarly journals A Homologue of aliB Is Found in the Capsule Region of Nonencapsulated Streptococcus pneumoniae

2004 ◽  
Vol 186 (12) ◽  
pp. 3721-3729 ◽  
Author(s):  
Lucy J. Hathaway ◽  
Patricia Stutzmann Meier ◽  
Patrick Bättig ◽  
Suzanne Aebi ◽  
Kathrin Mühlemann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Northern Blotting ◽  
Clonal Population ◽  
Reverse Transcription Pcr ◽  
Group I ◽  
Invasive Diseases ◽  
Additional Sequence ◽  
Group Ii ◽  
Clonal Population Structure

ABSTRACT The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.

scholarly journals Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum

2006 ◽  
Vol 188 (2) ◽  
pp. 409-423 ◽  
Author(s):  
Michal Letek ◽  
Noelia Valbuena ◽  
Angelina Ramos ◽  
Efrén Ordóñez ◽  
José A. Gil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Northern Blotting ◽  
Receptor Protein ◽  
Chromosomal Dna ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr

ABSTRACT The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

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