Biochemistry, Genetics and Molecular Biology Molecular Biology

Steroid Chemistry and Biochemistry

Description

This cluster of papers focuses on the microbial transformation and degradation of steroids, particularly cholesterol, by various microorganisms such as Mycobacterium tuberculosis and actinobacteria. It explores the enzymatic processes involved in steroid metabolism, including the role of cholesterol oxidase and 3-ketosteroid dehydrogenases. The cluster also delves into the biotechnological applications and potential biotransformation of steroids for drug development.

Keywords

Microbial Transformation; Steroid Degradation; Cholesterol Oxidase; Biotransformation; Mycobacterium tuberculosis; 3-Ketosteroid Dehydrogenase; Actinobacteria; Enzymatic Hydroxylation; Rhodococcus erythropolis; Metagenomes

Research Article| January 01 1952 Methods of paper chromatography of steroids applicable to the study of steroids in mammalian blood and tissues I. E. Bush I. E. Bush 1National Institute … Research Article| January 01 1952 Methods of paper chromatography of steroids applicable to the study of steroids in mammalian blood and tissues I. E. Bush I. E. Bush 1National Institute for Medical Research, Mill Hill, London, N.W. 7 Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1952) 50 (3): 370–378. https://doi.org/10.1042/bj0500370 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation I. E. Bush; Methods of paper chromatography of steroids applicable to the study of steroids in mammalian blood and tissues. Biochem J 1 January 1952; 50 (3): 370–378. doi: https://doi.org/10.1042/bj0500370 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search © 1952 CAMBRIDGE UNIVERSITY PRESS1952 Article PDF first page preview Close Modal You do not currently have access to this content.
This chapter contains sections titled: Principles and Applications Experimental Procedures Appraisal of Enzymic Methods This chapter contains sections titled: Principles and Applications Experimental Procedures Appraisal of Enzymic Methods
Mater&--Radioactive steroids were obtained from Kew England Nuclear.Nonradioactive steroids, purchased from Steraloid (Pawling, New York), were recrystallized from organic solvents, and their infrared spectra were taken to assure the 1 … Mater&--Radioactive steroids were obtained from Kew England Nuclear.Nonradioactive steroids, purchased from Steraloid (Pawling, New York), were recrystallized from organic solvents, and their infrared spectra were taken to assure the 1 The terminology used is: 5ol-dihydrotestosterone, 17&hydroxy-L-androstan-3-one; androstenedione, A4-androstene-3,17-dione; cyproterone, 1,2a-methylene-6-chloro-A4se-pregnadien-l7a -01-3, 20-dione.
The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion … The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion of the 15-hydro-peroxide of prostaglandin G1 to a 15-hydroxyl group, producing prostaglandin H1. The latter enzymatic reaction required heme and was stimulated by a variety of compounds, including tryptophan, epinephrine, and guaiacol, but not by glutathione. A peroxidatic dehydrogenation was demonstrated with epinephrine or guaiacol in the presence of various hydroperoxides, including hydrogen peroxide and prostaglandin G1. Higher activity and affinity were observed with the 15-hydroperoxide of eicosapolyenoic acid, especially those with the prostaglandin structure. Both the dehydrogenation of epinephrine or guaiacol and the 15-hydroperoxide reduction of prostaglandin G1 were demonstrated in nearly stoichiometric quantities. With tryptophan, however, such a stoichiometric transformation was not observed. The peroxidase activity as followed with guaiacol and hydrogen peroxide and the tryptophan-stimulated conversion of prostaglandin G1 to H1 were not dissociable as examined by isoelectric focusing, heat treatment, pH profile, and heme specificity. The results suggest that the peroxidase with a broad substrate specificity is an integral part of prostaglandin endoperoxide synthetase which is responsible for the conversion of prostaglandin G1 to H1.
Magnetic nanoparticles (Fe3O4) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of … Magnetic nanoparticles (Fe3O4) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe3O4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.
Abstract An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration … Abstract An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration curve is linear to 600 mg/dl. Cholesterol esters are hydrolyzed to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol produced is oxidized by cholesterol oxidase to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann—Burchard procedures (AA-2 and SMA 12/60) and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.
The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth … The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size ...Read More
Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining … Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. After 3 weeks of culture, the oligodendrocytes were also highly immunoreactive to monospecific polyclonal antibodies against cytochrome P450SCC, an enzyme involved in the conversion of cholesterol to pregnenolone (P). Biosynthesis of [3H]cholesterol, [3H]P, and [3H]Pregn-5- ene-3β,20α-diol was demonstrated in these primary cultures by incubating cells with [3H]mevalonolactone in the presence of mevinolin and trilostane. The activity of the 2',3'-cyclic nucleotide 3'-phosphodiesterase enzyme, a documented indicator of oligodendrocyte differentiation, increased rapidly after day 10 of culture, together with the onset of steroid biosynthetic activity. Both reached a maximum at 3 weeks of culture and remained stable up to 6.5 weeks. In the absence of trilostane, [3H]P was converted by glial cell cultures to [3H]progesterone, [3H]5αpregnane- 3,20-dione, and [3H]3α-hydroxy-5α-pregnan-20-one. The demonstration of P, pregn-5-ene-3β,20α-diol, and progesterone synthesis by normal rat glial cells, once oligodendrocytes have undergone their differentiation process, brings additional support to the concept of “neurosteroids.”
Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad … Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNovel Steroidal Inhibitors of Human Cytochrome P45017.alpha.-Hydroxylase-C17,20-lyase): Potential Agents for the Treatment of Prostatic CancerGerard A. Potter, S. Elaine Barrie, Michael Jarman, and Martin G. RowlandsCite this: … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNovel Steroidal Inhibitors of Human Cytochrome P45017.alpha.-Hydroxylase-C17,20-lyase): Potential Agents for the Treatment of Prostatic CancerGerard A. Potter, S. Elaine Barrie, Michael Jarman, and Martin G. RowlandsCite this: J. Med. Chem. 1995, 38, 13, 2463–2471Publication Date (Print):June 1, 1995Publication History Published online1 May 2002Published inissue 1 June 1995https://pubs.acs.org/doi/10.1021/jm00013a022https://doi.org/10.1021/jm00013a022research-articleACS PublicationsRequest reuse permissionsArticle Views2892Altmetric-Citations311LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Terbinafine (Lamisil) has primarily fungicidal action against many fungi as a result of its specific mechanism of squalene epoxidase inhibition. Treated fungi accumulate squalene while becoming deficient in ergosterol, an … Terbinafine (Lamisil) has primarily fungicidal action against many fungi as a result of its specific mechanism of squalene epoxidase inhibition. Treated fungi accumulate squalene while becoming deficient in ergosterol, an essential component of fungal cell membranes. The cidal action is closely associated with the development of high intracellular squalene concentrations, which are believed to interfere with fungal membrane function and cell wall synthesis. In the case of Candida albicans, growth inhibition with terbinafine appears to result from the ergosterol deficiency. The filamentous form of this fungus is more susceptible than the yeast form. Measurement of ergosterol biosynthesis by incorporation of radiolabelled precursors indicates a correlation between inhibition of growth and ergosterol biosynthesis in a range of pathogenic fungi. Terbinafine is a potent non-competitive inhibitor of squalene epoxidase from Candida (Ki = 30 nM). In contrast, inhibition of rat liver squalene epoxidase only occurs at higher drug concentrations (Ki = 77 microM), and is competitive with squalene. Thus, terbinafine has no effect on cholesterol biosynthesis in vivo. Squalene epoxidase is not an enzyme of the cytochrome P-450 type, thereby avoiding potential inhibition of this class of enzymes.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNotes - The Direct Conversion of Steroidal Δ5-3β-Alcohols to Δ5- and Δ4-3-KetonesCarl. Djerassi, R. R. Engle, and A. BowersCite this: J. Org. Chem. 1956, 21, 12, 1547–1549Publication … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNotes - The Direct Conversion of Steroidal Δ5-3β-Alcohols to Δ5- and Δ4-3-KetonesCarl. Djerassi, R. R. Engle, and A. BowersCite this: J. Org. Chem. 1956, 21, 12, 1547–1549Publication Date (Print):December 1, 1956Publication History Published online21 February 2003Published inissue 1 December 1956https://pubs.acs.org/doi/10.1021/jo01118a627https://doi.org/10.1021/jo01118a627research-articleACS PublicationsRequest reuse permissionsArticle Views1538Altmetric-Citations356LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis , degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted … Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis , degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC , supAB , and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC − mutant accumulated 3,4-DHSA, indicating that HsaC RHA1 , formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette–Guérin. M. bovis bacillus Calmette–Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC H37Rv and HsaD H37Rv transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities ≈40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis 's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.
Different short‐chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C‐terminal third of their … Different short‐chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C‐terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15‐hydroxyprostaglandin, 17β‐hydroxysteroid and 11β‐hydroxy‐steroid dehydrogenases. In addition, species variants, isozyme‐like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme‐binding domain, but central‐chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14–58%). Only six of the 250‐odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N‐terminal coenzyme‐binding domain similar to that of other dehydrogenases, and a C‐terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues, Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium‐chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme‐binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter‐domain, active‐site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short‐chain family.
The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth … The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size ...Read More
THE INTRACELLULAR receptor proteins for steroid hormones are a group of transcriptional regulatory proteins with several common features, acting by direct interaction with DNA. The protein must first be activated … THE INTRACELLULAR receptor proteins for steroid hormones are a group of transcriptional regulatory proteins with several common features, acting by direct interaction with DNA. The protein must first be activated by the binding of the appropriate class of steroid. The steroid hormone receptor proteins, in particular the glucocorticoid receptor, and their role in gene regulation represent the best characterized eukaryotic gene regulating systems today, with major recent advances in knowledge of structure and function of both the protein and DNA components involved. During the 20 yr of research in this field, there are several major landmarks that have contributed to our understanding of gene regulation by steroid hormones, particularly by glucocorticoids, namely: 1) the synthesis of tritium-labeled steroids with high specific activity (1); 2) the demonstration of a high affinity, low capacity binding species, the putative receptor protein, for glucocorticoids (for review, see Refs. 2–5); 3) the interaction of the receptor with the nucleus or DNA (2–5); 4) the demonstration that glucocorticoids can regulate specific genes, their messenger RNAs (mRNAs) and their transcription (2–5); 5) the purification of the glucocorticoid-receptor complex (GR) (6–8); 6) the demonstration of specific GRbinding sequences within the genome (9); 7) the demonstration by gene transfer of glucocorticoid-responsive sequences (10); 8) the cloning of complementary DNA (cDNA) for the glucocorticoid (11–13), estrogen receptor (ER) (14–16), and progesterone (17–19) receptors (PR).
Abstract We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with … Abstract We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.
An enzymatic, fluorometric method for the determination of €ree and total cholesterol in cells in culture is presented.The method is simple, requiring one reagent that includes all of the enzymes … An enzymatic, fluorometric method for the determination of €ree and total cholesterol in cells in culture is presented.The method is simple, requiring one reagent that includes all of the enzymes and a second reagent that increases the pH, which enhances the fluorescence of the product.The method is based on the enzymatic hydrolysis of cholesteryl esters to free cholesterol, the oxidation of cholesterol with the liberation of hydrogen peroxide, and the reaction of this peroxide with a fluorogen to form a fluorescent product in the presence of a peroxidase.It is rapid, in that free cholesterol can be read in 5 minutes and total cholesterol after 20 minutes.The precision of the method is greater than that obtained from gas-liquid chromatography.
Eine einfache enzymatische Methode zur Bestimmung des Gesamt-Cholesterins im Serum wird beschrieben.Die im Serum vorhandenen Cholcsterinester werden mittels Cholcstcrinesterase quantitativ in freies Cholestcrin und Fettsäuren gespalten.Das freie Cholesterin wird in … Eine einfache enzymatische Methode zur Bestimmung des Gesamt-Cholesterins im Serum wird beschrieben.Die im Serum vorhandenen Cholcsterinester werden mittels Cholcstcrinesterase quantitativ in freies Cholestcrin und Fettsäuren gespalten.Das freie Cholesterin wird in Gegenwart von Sauerstoff und Cholesterinoxydase in A 4 -Cholestenon umgewandelt.Das bei dieser Reaktion entstehende Wasserstoffperoxyd oxydiert bei Anwesenheit von Katalase Methanol zu Formaldchyd.Dieses reagiert mit Ammoniumionen und Acetylaceton unter Bildung von 3,5-Diacctyl-l,4-dihydrolutidin, dessen Farbintcnsität bei 405 nm gemessen wird.Richtigkeit und Präzision der Methode sind sehr gut.Die Proportionalität ist bis 25,
The mechanism regulating the production of steroids in response to trophic hormone stimulation has been the subject of investigation for over three decades. When considering the effects of trophic hormones … The mechanism regulating the production of steroids in response to trophic hormone stimulation has been the subject of investigation for over three decades. When considering the effects of trophic hormones on the steroidogenic process it is necessary to first distinguish between acute effects and chronic effects. Acute effects are those which result in the very rapid (within minutes) synthesis and secretion of steroids in response to hormone stimulation and involve the rapid translocation of intracellular cholesterol to the site of its cleavage, as will be discussed later. Chronic effects are those which occur on the order of hours to tens of hours and involve increased gene transcription and translation of the proteins involved in the biosynthesis of steroids. This chapter will focus on studies designed to elucidate the mechanisms involved in the acute regulation of steroid production in response to hormone stimulation. Overviews of the effects of chronic stimulation on steroidogenic enzymes have appeared in several excellent review articles (Simpson and Waterman 1983; Miller 1988; Hanukoglu 1992).
Procedures for the determination of free and total cholesterol in lipid extracts or sonicates of lo4 cultured human skin fibroblasts are described.The method for free cholesterol employs cholesterol oxidase to … Procedures for the determination of free and total cholesterol in lipid extracts or sonicates of lo4 cultured human skin fibroblasts are described.The method for free cholesterol employs cholesterol oxidase to generate H202 and peroxidase to catalyze the reaction of H,OZ with phydroxyphenylacetic acid to yield a stable fluorescent product.Cholesterol ester hydrolase is included for determination of total cholesterol.When samples of' sonified cell suspensions are used directly, the extraction of lipids is avoided, permitting one person to carry out analyses of 30 o r more subcultures in one day.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTApproaches to the Total Synthesis of Adrenal Steroids.1 V. 4b-Methyl-7- ethylenedioxy-1,2,3,4,4aα,4b,5,6,7,8,10,10a β-dodecahydrophenanthrene-4 β-ol-1-one and Related Tricyclic DerivativesG. I. Poos, G. E. Arth, R. E. Beyler, and L. … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTApproaches to the Total Synthesis of Adrenal Steroids.1 V. 4b-Methyl-7- ethylenedioxy-1,2,3,4,4aα,4b,5,6,7,8,10,10a β-dodecahydrophenanthrene-4 β-ol-1-one and Related Tricyclic DerivativesG. I. Poos, G. E. Arth, R. E. Beyler, and L. H. SarettCite this: J. Am. Chem. Soc. 1953, 75, 2, 422–429Publication Date (Print):January 1, 1953Publication History Published online1 May 2002Published inissue 1 January 1953https://pubs.acs.org/doi/10.1021/ja01098a049https://doi.org/10.1021/ja01098a049research-articleACS PublicationsRequest reuse permissionsArticle Views1364Altmetric-Citations442LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Total Synthesis of Steroids1R. B. Woodward, Franz Sondheimer, David Taub, Karl Heusler, and W. M. McLamoreCite this: J. Am. Chem. Soc. 1952, 74, 17, 4223–4251Publication Date … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Total Synthesis of Steroids1R. B. Woodward, Franz Sondheimer, David Taub, Karl Heusler, and W. M. McLamoreCite this: J. Am. Chem. Soc. 1952, 74, 17, 4223–4251Publication Date (Print):September 1, 1952Publication History Published online1 May 2002Published inissue 1 September 1952https://doi.org/10.1021/ja01137a001RIGHTS & PERMISSIONSArticle Views8164Altmetric-Citations329LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts
In August, 1899, I presented a memoir to the Royal Society on the inheritance of coat-colour in the horse and of eye-colour in man, which was read November, 1899, and … In August, 1899, I presented a memoir to the Royal Society on the inheritance of coat-colour in the horse and of eye-colour in man, which was read November, 1899, and ultimately ordered to be published in the 'Phil. Trans.’ Before that memoir was printed, Mr. Yule’s valuable memoir on Association was read, and, further, Mr. Leslie Bramley-Moore showed me that the theory of my memoir as given in § 6 of the present memoir led to somewhat divergent results according to the methods of proportioning adopted. We therefore undertook a new investigation of the theory of the whole subject, which is embodied in the present memoir. The data involved in the paper on coat-colour in horses and eye-colour in man have all been recalculated, and that paper is nearly ready for presentation. But it seemed best to separate the purely theoretical considerations from their application to special cases of inheritance, and accordingly the old memoir now reappears in two sections. The theory discussed in this paper was, further, the basis of a paper on the Law of Reversion with special reference to the Inheritance of Coat-colour in Basset Hounds recently communicated to the Society, and about to appear in the ‘ Proceedings. While I am responsible for the general outlines of the present paper, the rough draft of it was taken up and carried on in leisure moments by Mr. Leslie Bramley-Moore, Mr. L. N. G. Filon, M. A., and Miss Alice Lee, D. Sc. Mr. Bramley-Moore discovered the u -functions ; Mr. Filon proved most of their general properties and the convergency of the series; I alone am responsible for sections 4, 5, and 6. Mr. Leslie Bramley-Moore sent me, without proof, on the eve of his departure for the Cape, the general expansion for z on p. 26. I am responsible for the present proof and its applications. To Dr. Alice Lee we owe most of the illustrations and the table on p. 17. Thus the work is essentially a joint memoir in which we have equal part, and the use of the first personal pronoun is due to the fact that the material had to be put together and thrown into form by one of our number.—K. P.
Designed for undergraduates, graduate students, and industry practitioners, Bioseparations Science and Engineering fills a critical need in the field of bioseparations. Current, comprehensive, and concise, it covers bioseparations unit operations … Designed for undergraduates, graduate students, and industry practitioners, Bioseparations Science and Engineering fills a critical need in the field of bioseparations. Current, comprehensive, and concise, it covers bioseparations unit operations in unprecedented depth. In each of the chapters, the authors use a consistent method of explaining unit operations, starting with a qualitative description noting the significance and general application of the unit operation. They then illustrate the scientific application of the operation, develop the required mathematical theory, and finally, describe the applications of the theory in engineering practice, with an emphasis on design and scaleup. Unique to this text is a chapter dedicated to bioseparations process design and economics, in which a process simular, SuperPro Designer® is used to analyze and evaluate the production of three important biological products. New to this second edition are updated discussions of moment analysis, computer simulation, membrane chromatography, and evaporation, among others, as well as revised problem sets. Unique features include basic information about bioproducts and engineering analysis and a chapter with bioseparations laboratory exercises. Bioseparations Science and Engineering is ideal for students and professionals working in or studying bioseparations, and is the premier text in the field.
An excessively high serum cholesterol (CHOL) level in humans can easily lead to cardiovascular diseases (CVDs), including hypertension and coronary heart disease. In this study, a CHOL-lowering bacterium, Cellulosimicrobium cellulans … An excessively high serum cholesterol (CHOL) level in humans can easily lead to cardiovascular diseases (CVDs), including hypertension and coronary heart disease. In this study, a CHOL-lowering bacterium, Cellulosimicrobium cellulans YS01, was isolated from healthy human intestinal microbiota and identified via average nucleotide identity (ANI) analysis. The cells of YS01 degraded 74.00% of CHOL within 5 d, which decreased from the initial 1.00 g/L to 0.26 g/L. And its extracellular crude enzymes achieved equivalent efficiency within 24 h, which decreased from the initial 0.50 g/L to 0.13 g/L. The results indicated that YS01 indeed has a strong ability in the biodegradation of CHOL. Furthermore, the whole genome analysis of YS01 revealed that cholesterol oxidase and choloylglycine hydrolase encoded by gene choD and gene cbh, respectively, may play key roles in the conversion of CHOL. Cholest-4-ene-3-one was produced from CHOL through the catalysis by cholesterol oxidase, and choloylglycine hydrolase was also involved in another biodegradation pathway of CHOL. The results provide scientific insights into the mechanisms of biodegrading CHOL using C. cellulans YS01 and lay a solid foundation for the development of new CHOL-lowering strategies based on microbial therapy.
<title>Abstract</title> Background Carotol, a major sesquiterpene alcohol found in carrot essential oil, exhibits promising biological activities including cytotoxic effects against various cancer cell lines. Despite its bioactivity, the metabolic fate … <title>Abstract</title> Background Carotol, a major sesquiterpene alcohol found in carrot essential oil, exhibits promising biological activities including cytotoxic effects against various cancer cell lines. Despite its bioactivity, the metabolic fate and biotransformation pathways of carotol remain largely unexplored, particularly through microbial systems that can offer novel insights into its structural modifications and potential pharmacological applications. Results In this study, seventeen microbial strains were screened for their ability to biotransform carotol, with <italic>Absidia coerulea</italic> ATCC 6647 identified as the most effective strain. Preparative-scale fermentation using this strain led to the isolation and purification of three metabolites (CM1, CM2, and CM3). Spectroscopic analysis, including 1D and 2D NMR, HRMS, and single crystal X-ray diffraction, elucidated the structures of these metabolites as 9α-hydroxydaucol (CM1), 9α,13-dihydroxydaucol (CM2), and a diol derivative of daucol (CM3). Cytotoxicity evaluation against human liver (HepG-2), colon (HCT-116), breast (MCF-7), and lung (A-549) carcinoma cell lines, alongside normal lung fibroblasts (MRC-5), revealed that carotol exhibited the highest anticancer activity followed by CM2, CM3, and CM1. Molecular docking studies against human NADPH oxidase demonstrated that carotol and CM2 have stronger binding affinities and more stable interactions compared to the other metabolites, suggesting NADPH oxidase inhibition as a possible mechanism for their anticancer effects. Conclusion This study provides the first comprehensive microbial biotransformation pathway for carotol, leading to the identification of novel hydroxylated metabolites with varying cytotoxic activities. The findings highlight the potential of <italic>Absidia coerulea</italic> as a biocatalyst for producing bioactive carotol derivatives and underscore the relevance of NADPH oxidase inhibition in their anticancer mechanism. These results lay a foundation for future pharmacokinetic and drug development research involving carotol and its metabolites.
This article describes the development of novel, hydrolytically stable cardiotonic steroid analogs featuring a 3β‐amine moiety instead of the commonly found 3β‐carbohydrates such as oleandrose. To establish the desired 3β‐configuration … This article describes the development of novel, hydrolytically stable cardiotonic steroid analogs featuring a 3β‐amine moiety instead of the commonly found 3β‐carbohydrates such as oleandrose. To establish the desired 3β‐configuration stereoselectively, a new method based on chiral phosphoric acid‐controlled diastereoselective reductive amination with Hantzsch esters was developed. This method utilizes readily available unsubstituted (S)‐BINOL‐based hydrogen phosphate as the catalyst, enabling the synthesis of 13 different 5β‐androsterone and digitoxigenin analogs with up to 36:1β:α diastereoselectivity. Additionally, this strategy was applied to generate two novel oleandrigenin analogs 15a and 15gin 3 steps from readily available gitoxigenin. The synthetic analogs were subjected to the NCI‐60 human tumor cell lines screen, and several different digitoxigenin derivatives with tumor cell growth inhibitory power in submicromolar range were identified. The subsequent in vitro evaluation of digitoxigenin and oleandrin derivatives 13a, 13g, 15a, and 15gdemonstrated that these four analogs reduced steady‐state ATP1A1 levels in T98G cells in the 12‐96 nM range. Interestingly, only the oleandrin analog 15g also lowered also steady‐state levels of the cellular prion protein (PrPC), the main therapeutic target for the treatment of prion diseases.
Introduction and Objective: Incretin mimetic drugs are widely used to treat type 2 diabetes and obesity and have recently gained popularity for weight loss in healthy individuals. These drugs act … Introduction and Objective: Incretin mimetic drugs are widely used to treat type 2 diabetes and obesity and have recently gained popularity for weight loss in healthy individuals. These drugs act as agonists of the glucagon-like peptide 1 receptor (GLP-1R), enhancing the effects of the natural hormone glucagon-like peptide 1 (GLP-1). The therapeutic benefits of these medications, including improved glucose control and weight loss, require continued usage and wane with time. The molecular mechanisms underlying this loss of effect to incretin drugs remain unknown. Methods: After activation by an agonist and signaling to G protein, GLP-1R recruits arrestins and undergoes endocytosis. This study investigate the role of G Protein Coupled Receptor Associated Sorting Protein 1 (GASP1), which regulates the post-endocytic trafficking of GLP-1R, in incretin drug tolerance. Results: We found that disrupting the GASP1 protein specifically in pancreatic islet beta cells prevented incretin drug tolerance at the cellular, tissue, and whole-animal levels. Conclusion: These findings suggest that post-endocytic sorting of GLP-1R contributes to the reduced effectiveness of incretin drugs with prolonged use and propose a potential strategy to prevent tolerance by favoring G protein engagement over arrestin signaling. Disclosure A. Gaur: None.
Noor N. Rafo , Hadil B. Al-Sabaawy | ˜Al-œmağallaẗ al-ʻirāqiyyaẗ li-l-ʻulūm al-bayṭariyyaẗ/Iraqi journal of veterinary sciences
By exploring the use of plasmids to confer Rhodococcus jostii RHA1 the possibility of utilizing xylose to produce lipids we have observed that the plasmid used was not always maintained … By exploring the use of plasmids to confer Rhodococcus jostii RHA1 the possibility of utilizing xylose to produce lipids we have observed that the plasmid used was not always maintained in the transformants as expected. Instead, we observed an illegitimate integration of the antibiotic resistance gene from the plasmid into the recombinant cells. Genome sequencing of the transformants has provided evidence that this illegitimate integration is not size-, site-or sequence-specific. But even more surprising, genome sequencing revealed the presence of an unexpected circular multicopy replicative element (75-80 kb) that appears to be excised from the chromosome as a consequence of the stress generated by the antibiotic used in the selection process. The excised fragment does not contain any of the typical features of genomic islands. These results provide evidence that the genome of this oleaginous strain is more plastic than initially anticipated and our findings open the option of developing new ways to genetically modify this strain by using illegitimate recombinant approaches. But even more remarkably, the discovery of this atypical replicative element raises new questions about the existence of novel mechanisms of evolution in bacteria.
T.M. Penning , Jerzy Adamski | The Journal of Steroid Biochemistry and Molecular Biology
ABSTRACT Progesterone (PROG) is one of the most ubiquitous sexual hormones found as a pollutant in soil and water systems. Despite the fact that PROG can be degraded by various … ABSTRACT Progesterone (PROG) is one of the most ubiquitous sexual hormones found as a pollutant in soil and water systems. Despite the fact that PROG can be degraded by various bacterial species, the pathways leading to its complete oxic mineralization remain unknown. In this study, we investigated bacterial progesterone catabolism using the steroid-degrading bacterium Caenibius tardaugens as a model, in which we demonstrated its capacity to degrade progestogens. The transcriptomic analyses in the presence of PROG showed the overexpression of EGO55_13845 and EGO55_13860 genes, coding a Baeyer-Villiger monooxygenase (BVMO) and a luciferase-like monooxygenase (LLM), respectively. Both genes are located next to two regulatory proteins forming a small gene cluster (named pdc ) that can be found in other steroid-degrading bacteria. Mutagenic analyses and gene complementation allowed ascertaining that EGO55_13845 and EGO55_13860 genes are involved in PROG degradation. To assess their enzymatic activities, both proteins were overexpressed in Escherichia coli, showing that they catalyze a Baeyer-Villiger monooxygenation of PROG, resulting in the production of testosterone acetate. They are also active on 1,2-dehydroprogesterone, an intermediate in PROG degradation, converting it into boldenone acetate. BVMO and LLM enzymes are functionally redundant, as each can replace the other in metabolizing PROG. The presence of two functionally redundant BVMO and LLM enzymes in C. tardaugens can be explained by their distinct substrate preferences. Our results settle for the first time the genetic and biochemical basis for explaining how PROG is recognized and channeled into the 9,10-seco degradation pathway in a PROG-degrading bacterium. IMPORTANCE This study investigates for the first time the key steps in bacterial progesterone (PROG) degradation, revealing new insights into the process. The main stages of PROG degradation were examined in the bacterium Caenibius tardaugens . The conducted transcriptomic analysis allows identifying the progesterone degradation cluster pdc, which is also present in other related bacteria. We demonstrated that Baeyer-Villiger monooxygenase and luciferase-like monooxygenase encoded within the pdc cluster catalyze the PROG Baeyer-Villiger monooxygenation, producing testosterone acetate. The activity redundancy can be explained by the difference in substrate specificity of each enzyme.
Burkholderia oklahomensis LMG 23618 T is a Burkholderia pseudomallei -like bacterium originally isolated in 1973 from a wound infection caused by a farming accident in Oklahoma. Metabolic profiling of an … Burkholderia oklahomensis LMG 23618 T is a Burkholderia pseudomallei -like bacterium originally isolated in 1973 from a wound infection caused by a farming accident in Oklahoma. Metabolic profiling of an organic extract from cultures of B. oklahomensis LMG 23618 T using UHPLC-ESI-Q-ToF-MS led to identification of three known metabolites, betulinan A, yersiniabactin and ulbactin B, in addition to a novel polycholorinated compound. Mass-directed purification enabled isolation of the novel specialized metabolite, which was shown by X-ray crystallography and NMR spectroscopic analysis to be 4,4ʹ-dihydroxy-3,3ʹ,5,5ʹ-tetrachlorobenzophenone. Feeding experiments with stable isotope-labelled precursors established that the carbon skeleton of this unusual metabolite derives from two molecules of tyrosine. This led us to propose a plausible biosynthetic pathway via decarboxylative condensation of 3, 5-dichloro-4-hydroxybenzoic acid with its coenzyme A thioester derivative. The absolute configuration of ulbactin B was also established as 4ʹR, 3ʹʹS, 7ʹʹS, 8ʹʹR using X-ray crystallography and NMR spectroscopy.
22‐Hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) and 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (PDCE) are useful precursors for the synthesis of steroidal active pharmaceutical ingredients. In this study, we identify the sterol metabolism‐related genes, which encode … 22‐Hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) and 3‐oxo‐4,17‐pregnadiene‐20‐carboxylic acid methyl ester (PDCE) are useful precursors for the synthesis of steroidal active pharmaceutical ingredients. In this study, we identify the sterol metabolism‐related genes, which encode the aldolases (Ltp2 and Thl) and carboxylic acid reductases (CAR) in Mycolicibacterium neoaurum NRRL B‐3805 (B3805), by analysis of the metabolites from phytosterols biotransformation. Based on these results, a genetically modified strain is constructed by disrupting the kstD, ltp2, and hsd4A genes and overexpressing the aldolase gene ( thl ) in the strain B3805. This recombinant strain (B3805V) is able to transform 5 g L −1 phytosterols to 2.0 g L −1 4‐HBC without detectable AD by‐product. Additionally, by disrupting the ltp2 and car genes, a strain (strain B3805VI) is obtained to transform phytosterols to PDCE with 1.44 g L −1 titer. The PDCE concentration is further increased by about 42% to 2.1 g L −1 without 4‐HBC by‐product by deleting thl gene (strain B3805VII). On the preparative scale, the strain B3805VII transforms 10 g L −1 of phytosterols into PDCE with 5.1 g L −1 . This study presents one‐step bioproduction of pharmaceutically important 4‐HBC and PDCE with high yield and purity from bio‐renewable phytosterols, which are readily available as a by‐product from the plant oil industry.
ABSTRACT Bile salts are steroids with distinctive hydroxylation patterns that are produced and excreted by vertebrates. In contrast to common human bile salts, ursodeoxycholate (UDCA) has a 7-hydroxy group in … ABSTRACT Bile salts are steroids with distinctive hydroxylation patterns that are produced and excreted by vertebrates. In contrast to common human bile salts, ursodeoxycholate (UDCA) has a 7-hydroxy group in β-configuration and is used in large amounts for the treatment of diverse gastrointestinal diseases. We isolated the 7β-hydroxysteroid dehydratase Hsh3 that is involved in UDCA degradation by Sphingobium sp. strain Chol11. Hsh3 eliminates the 7β-hydroxy group as water, leading to a double bond in the B-ring. This is similar to 7α-hydroxysteroid dehydratases in this and other strains, but Hsh3 is evolutionarily different from these. Purified Hsh3 accepted steroids with and without side chains as substrates and had minor activity with 7α-hydroxy groups. The deletion mutant strain Chol11 Δ hsh3 had impacted growth with UDCA and accumulated a novel compound. The compound was identified as 3′,5-dihydroxy-H-methyl-hexahydro-5-indene-1-one-propanoate, consisting of steroid rings C and D with a C 3 -side chain carrying the former 7β-hydroxy group, indicating that Hsh3 activity is important especially for the later stages of bile salt degradation. Hsh3 homologs were found in other sphingomonads that were also able to degrade UDCA as well as in environmental metagenomes. Thus, Hsh3 adds to the bacterial enzyme repertoire for degrading a variety of differently hydroxylated bile salts. IMPORTANCE The bacterial degradation of different bile salts is not only important for the removal of these steroidal compounds from the environment but also harbors interesting enzymes for steroid biotechnology. The 7β-hydroxy bile salt ursodeoxycholate (UDCA) naturally occurs in high concentration in the feces of black bears and has important pharmaceutical relevance for the treatment of different liver-related diseases, for which it is administered in high and increasing amounts. Additionally, it is present in the bile salt pool of humans in trace amounts. While UDCA degradation is environmentally important, the enzyme Hsh3 modifies the hydroxy group that confers the medically relevant properties and thus might be interesting for microbiome analyses and biotechnological applications.
In 2020, there were more than 2.3 million new breast cancer cases globally, with 665,684 recorded deaths. Despite advances in diagnoses and treatment of early-stage breast cancer, metastatic breast cancer … In 2020, there were more than 2.3 million new breast cancer cases globally, with 665,684 recorded deaths. Despite advances in diagnoses and treatment of early-stage breast cancer, metastatic breast cancer remains almost incurable and the cause of 90% of breast cancer-related deaths. As ion channels are implicated in processes that promote or inhibit cancer progression, my lab investigated a role for the Epithelial Sodium Channel (ENaC) in breast cancer progression. Preliminary results from bioinformatic analysis of published datasets, followed by in vitro cell line studies, revealed that the alpha subunit of ENaC (αENaC) was important in breast cancer proliferation and metastasis. This current study is therefore aimed at further characterizing the role of αENaC and determining the biological mechanisms underlying its effects in breast cancer progression. We hypothesized that: (1) αENaC protein is highly expressed in breast cancer cells characterized by slower rates of proliferation and migration, (2) higher expression of αENaC protein alters global gene expression, especially those involved in processes underlying cell growth and migration, and (3) expression of αENaC protein changes at different phases of the cell division cycle to alter the regulation and transitioning through the cell cycle phases. To further characterize αENaC’s role in breast cancer, bioinformatic analyses were performed on publicly available gene expression datasets from breast cancer and normal breast tissues. These showed that repression of αENaC expression significantly correlates with increased probabilities for developing metastasis and relapse in breast cancer patients. To investigate the mechanisms underlying αENaC’s effects, RNA sequencing was performed on three monoclonal populations of the MDA-MB-231 breast cancer cell line transfected to stably overexpress αENaC and a control. Analysis of the transcriptomes of these cell lines uncovered 386 genes that were differentially expressed across all three clones due to overexpression of αENaC. Gene Set Enrichment Analysis was then performed, which showed significant underrepresentation of gene sets and corresponding leading-edge genes involved in pathways/processes affecting cell adhesion, migration, immune modulation among others. The MDA-MB-231 breast cancer cell lines were subsequently synchronized to the early S phase of the cell cycle by double thymidine block, then followed through progression along the cell cycle by live fluorescence imaging and western blotting. These show that expression of αENaC oscillates as the cells progress through the different phases of the cell cycle. This study has identified molecular mechanisms and targets which promote breast cancer proliferation and metastasis when αENaC expression is repressed. These could be targeted in the future to improve survival. This study is funded by public grants. University of Otago Doctoral Scholarships, Department of Physiology, University of Otago, BMS Strategic Kickstarter, OMS BMS Bequest, University of Otago Research Grants. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Cholesterol oxidation leads to the development of several oxysterols such as 7-ketocholesterol (7KC), which are linked to various age-related conditions. An approach to reduce their toxicity is proposed using enzymes … Cholesterol oxidation leads to the development of several oxysterols such as 7-ketocholesterol (7KC), which are linked to various age-related conditions. An approach to reduce their toxicity is proposed using enzymes from microbial sources to degrade them. Our earlier studies identified Pseudomonas aeruginosa PseA and Rhodococcus erythropolis MTCC 3951 as potential strains capable of using 7KC as their sole carbon source. These strains produced cholesterol oxidase as the primary enzyme in the degradation pathway. To enhance applicability, cholesterol oxidase (ChOx) enzymes from P. aeruginosa PseA (ChOxP), R. erythropolis MTCC 3951 (ChOxR), and a commercial variant from Streptomyces sp. (ChOxS) were immobilized on silane functionalized silica nanoparticles (SNP) using covalent-coupling methods. The immobilization efficiency was 68%, 86%, and 83% for ChOxP, ChOxR, and ChOxS respectively. The catalytic efficiency of the immobilized enzyme was nearly twice that of the free enzyme, with increased stability across a wide range of temperatures (10-70°C) and pH levels (4.0-9.0), although the optimum pH (7.5) and temperature (30°C) remained unchanged. The nano-immobilized cholesterol oxidases were reusable up to 10 cycles. Further, enzyme immobilization on nanoparticles was confirmed by FTIR, SEM, and TEM. Biotransformation of cholesterol and 7KC using the nanobioconjugates produced pharmaceutically important molecules 4-cholesten-3-one and 4-cholesten-3,7-dione respectively.
The aerobic catabolism of steroids in bacteria is highly conserved, and the mechanism of steroid degradation in mycobacteria has been extensively studied. However, the branching modification pathways of steroids in … The aerobic catabolism of steroids in bacteria is highly conserved, and the mechanism of steroid degradation in mycobacteria has been extensively studied. However, the branching modification pathways of steroids in mycobacteria remain a mystery, including the likely roles of cytochromes P450. In this study, we unraveled the CYP105S17 converting androst-4-ene-3,17-dione (AD) to 17β-hydroxy-4-androstene-3,16-dione (16-oxo-TS), which was subsequently reduced to 16α,17β-dihydroxy-androst-4-ene-3-one (16α-OH-TS) under reductive conditions in Mycolicibacterium neoaurum. By applying this modification pathway, the genetically modified strains overexpressing CYP105S17 were able to produce 16α-OH-TS at titers 13.0 g/L with a conversion rate of 91.9% (supplemented with 20 g/L phytosterols as the substrate) through a two-stage biotransformation process. This is the first instance of utilizing the native P450 of Mycobacterium to produce 16-hydroxylated steroid intermediates at the 10 g scale. This work provides invaluable perspectives and guidance to researchers seeking to understand the complexities of steroid metabolism in bacteria, and also highlights the great potential of Mycobacterium as a production platform for hydroxylated steroid intermediates or pharmaceuticals.
Biosynthesis of steroids by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the unclear mechanisms of electron transfer from … Biosynthesis of steroids by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the unclear mechanisms of electron transfer from regeneration to the final delivery to the NADPH-dependent active centers limit systematically engineering electron transfer to improve steroids production. Here, we elucidate the electron transfer mechanisms of NADPH-dependent enzymes for systematically engineer electron transfer of Saccharomyces cerevisiae, including step-by-step engineering the electron transfer residues of 7-Dehydrocholesterol reductase (DHCR7) and P450 sterol side chain cleaving enzyme (P450scc), electron transfer components for directing carbon flux, and NADPH regeneration pathways, for high-level production of the cholesterol (1.78 g/L) and pregnenolone (0.83 g/L). The electron transfer engineering (ETE) process makes the electron transfer chains shorter and more stable which significantly accelerates deprotonation and proton coupled electron transfer process. This study underscores the significance of ETE strategies in steroids biosynthesis and expands synthetic biology approaches.
CsCYP21A, a steroid 21-hydroxylase from Bufo bufo gargarizans, exhibits unprecedented sequential oxidations. Optimizing Pichia pastoris biotransformation conditions enhanced C21-hydroxylation selectivity, converting 14 substrates to 21-hydroxylated products, with 10 conversions of … CsCYP21A, a steroid 21-hydroxylase from Bufo bufo gargarizans, exhibits unprecedented sequential oxidations. Optimizing Pichia pastoris biotransformation conditions enhanced C21-hydroxylation selectivity, converting 14 substrates to 21-hydroxylated products, with 10 conversions of >80% and 4 yields of >80%. Hydrocortisone production reached 1.5 g L-1 day-1 with 100 g/L wet biomass. CsCYP21A's versatility enables integration into the synthesis of over 10 steroidal drugs, offering a sustainable biocatalytic platform for green pharmaceutical manufacturing.