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W2097775940 | Biosynthesis of an Escherichia coli cell, with organic compounds as sources of energy and carbon, requires approximately 20 to 60 billion high-energy phosphate bonds [Stouthamer, A. H. (1973) Antonie van Leeuwenhoek 39, 545–565]. A substantial fraction of this energy budget is devoted to biosynthesis of amino acids, the building blocks of proteins. The fueling reactions of central metabolism provide precursor metabolites for synthesis of the 20 amino acids incorporated into proteins. Thus, synthesis of an amino acid entails a dual cost: energy is lost by diverting chemical intermediates from fueling reactions and additional energy is required to convert precursor metabolites to amino acids. Among amino acids, costs of synthesis vary from 12 to 74 high-energy phosphate bonds per molecule. The energetic advantage to encoding a less costly amino acid in a highly expressed gene can be greater than 0.025% of the total energy budget. Here, we provide evidence that amino acid composition in the proteomes of E. coli and Bacillus subtilis reflects the action of natural selection to enhance metabolic efficiency. We employ synonymous codon usage bias as a measure of translation rates and show increases in the abundance of less energetically costly amino acids in highly expressed proteins. |
W2605069562 | Abstract Surfactin, a lipopeptide produced by Bacillus subtilis , is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β‐hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY , which encode for the global transcriptional regulator and the gene lpdV , located in the bkd operon ( lpdV , bkdAA , bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl‐CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP‐HPLC and the surfactin isoforms were characterized using LC‐MS‐MS and GC‐MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C 14 isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using 1 H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C 14 FA chain. |
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W1994981031 | Abstract Background Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level. Results We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism , in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor). Conclusions Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and specialized metabolite stress response in C. acetobutylicum . Since the majority of the transcription factors and their target genes are highly conserved in other organisms of the Clostridium genus, this network would be largely applicable to other Clostridium organisms. The network informs the molecular basis of Clostridium responses to toxic metabolites in natural ecosystems and the microbiome, and will facilitate the construction of genome-scale models with added regulatory-network dimensions to guide the development of tolerant strains. |
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W1975321361 | Abstract Fatty acids are the primary natural starting materials for the production of surfactant and detergent molecules. Derived from vegetable (oil) and animal (fat) triglyceride sources, the fatty acids are linear aliphatic carboxylic acids. Industrial interest in branched‐chain fatty acids is driven by the needs for products with enhanced performance benefits including higher solubility, ease of handling, better hard water tolerance, and improved oxidative stability. Therefore, catalytic processes have been developed for the conversion of linear fatty acids to branched ones. High yields of branched acids are obtained from unsaturated fatty acids over acidic zeolites, particularly those with large pores. Fatty esters also are readily isomerized to branched ones by means of the same catalyst and process. It is postulated that the isomerization of fatty acids proceeds through three‐ and four‐membered ring carbocation intermediates formed inside the largepore zeolites. This is supported by evidence of methyl and ethyl branching in the primary and final products. The total number of carbon atoms in the fatty acid molecule is unchanged. Surfactants derived from branched fatty acids show favorable physical properties, including a lower viscosity and improved handling, even as intended performance characteristics are maintained. |
W2783317487 | ABSTRACT To overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated in Lactococcus lactis . In this study, the transcription levels of eight transcriptional regulators of L. lactis subsp. lactis F44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. Overexpressing ythA , a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. The ythA -overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis ( argG and argH ), arginine deiminase pathway, and polar amino acid transport ( ysfE and ysfF ) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels of nisIPRKFEG genes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA. IMPORTANCE Nisin, produced by Lactococcus lactis subsp. lactis , is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance of L. lactis F44. Overexpressing ythA could significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrial L. lactis strains. |
W2791235950 | Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant or phoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography (FPLC) analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium (LPM) and high-phosphate medium (HPM), respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptional-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions. |
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W1589377261 | Abstract Membrane vesicles of Bacillus subtilis actively transport amino acids in the presence of electron donors. The effectiveness of each physiological electron donor depends on the presence of the necessary membrane proteins that allow substrate oxidation and couple it to amino acid transport. Some of these proteins are present in all membranes while others (e.g. oxidizing glycerol phosphate) appear only in certain growth media (containing glycerol). Physiological electron donors are NADH, NADPH, l-α-glycerol phosphate, l-lactate, and succinate. NADH stimulates amino acid uptake at the highest initial rate whereas glycerol phosphate is the most effective electron donor since it energizes the transport of the largest number of amino acid molecules per nmole of substrate oxidized per min. Reduced phenazine methosulfate (PMS) is a very efficient nonphysiological electron donor which energizes amino acid transport at a high initial rate. At the membrane concentrations usually employed (1 to 5 mg of membrane protein per ml), NADH, NADPH, and reduced PMS consume oxygen so rapidly that special oxygenation is required to maintain the uptake of amino acids at a high rate. This uptake stops when the energy source has been oxidized; the accumulated amino acid then leaks out at a rate of 8% per min. With reduced (by ascorbate) PMS as electron donor, the Km and Vmax values of the uptake of 18 amino acids were determined. The Km values range from 10-5 to 10-6 m whereas the Vmax values vary widely for the different amino acids. Competition experiments have shown the presence of at least nine different amino acid transport carriers in the vesicles. The energy for active transport appears to be produced in the cytochrome-linked electron transport chain: all electron donors effective in amino acid transport cause the reduction of the membrane-bound cytochromes. The electron transport inhibitors antimycin, 2-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and azide also inhibit amino acid transport, for all energy sources. Rotenone and oligomycin inhibit only the lactate-energized transport. Under conditions under which amino acids are transported no net ATP synthesis has been observed. |
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W2945732919 | This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals. |
W2972428201 | Phosphorus is required for many biological molecules and essential functions, including DNA replication, transcription of RNA, protein translation, posttranslational modifications, and numerous facets of metabolism. In order to maintain the proper level of phosphate for these processes, many bacteria adapt to changes in environmental phosphate levels. The mechanisms for sensing phosphate levels and adapting to changes have been extensively studied for multiple organisms. The phosphate response of Escherichia coli alters the expression of numerous genes, many of which are involved in the acquisition and scavenging of phosphate more efficiently. This review shares findings on the mechanisms by which E. coli cells sense and respond to changes in environmental inorganic phosphate concentrations by reviewing the genes and proteins that regulate this response. The PhoR/PhoB two-component signal transduction system is central to this process and works in association with the high-affinity phosphate transporter encoded by the pstSCAB genes and the PhoU protein. Multiple models to explain how this process is regulated are discussed. |
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W2915615881 | Abstract Surfactin is one of the most widely studied biosurfactants due to its many potential applications in different fields. In the present study, Bacillus velezensis BS‐37, initially identified as a strain of Bacillus subtilis , was used to efficiently produce surfactin with the addition of glycerol, an inexpensive by‐product of biodiesel production. After 36 hr of growth in glycerol medium, the total surfactin concentration reached more than 1,000 mg/L, which was two times higher than that in sucrose medium. Moreover, the addition of l ‐ and d ‐Leu to the culture medium had opposite effects on surfactin production by BS‐37. While surfactin production increased significantly to nearly 2,000 mg/L with the addition of 10 mM l ‐Leu, it was dramatically reduced to about 250 mg/L with the addition of 10 mM d ‐Leu. To systemically elucidate the mechanisms influencing the efficiency of this biosynthesis process, we sequenced the genome of BS‐37 and analyzed changes of the transcriptome in glycerol medium in response to d ‐/ l ‐leucine. The RPKM analysis of the transcriptome of BS‐37 showed that the transcription levels of genes encoding modular surfactin synthase, the glycerol utilization pathway, and branched‐chain amino acid (BCAA) synthesis pathways were all at a relatively high level, which may offered an explanation why this strain can efficiently use glycerol to produce surfactin with a high yield. Neither l ‐Leu nor d ‐Leu had a significant effect on the expression of genes in these pathways, indicating that l ‐Leu plays an important role as a precursor or substrate involved in surfactin production, while d ‐Leu appears to act as a competitive inhibitor. The results of the present study provide new insights into the synthesis of surfactin and ways of its regulation, and enrich the genomic and transcriptomic resources available for the construction of high‐producing strains. |
W3005139013 | The Gram-positive model organism and soil bacterium <i>Bacillus subtilis</i> naturally produces a variety of antimicrobial peptides (AMPs), including the ribosomally synthesized and post-translationally modified AMP YydF, which is encoded in the <i>yydFGHIJ</i> locus. The <i>yydF</i> gene encodes the pre-pro-peptide, which is, in a unique manner, initially modified at two amino acid positions by the radical SAM epimerase YydG. Subsequently, the membrane-anchored putative protease YydH is thought to cleave and release the mature AMP, YydF, to the environment. The AMP YydF, with two discreet epimerizations among 17 residues as sole post-translational modification, defines a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs) called epipeptides, for which the mode-of-action (MOA) is unknown. The predicted ABC transporter encoded by <i>yydIJ</i> was previously postulated as an autoimmunity determinant of <i>B. subtilis</i> against its own AMP. Here, we demonstrate that extrinsically added YydF<sup>*</sup> kills <i>B. subtilis</i> cells by dissipating membrane potential via membrane permeabilization. This severe membrane perturbation is accompanied by a rapid reduction of membrane fluidity, substantiated by lipid domain formation. The epipeptide triggers a narrow and highly specific cellular response. The strong induction of <i>liaIH</i> expression, a marker for cell envelope stress in <i>B. subtilis</i>, further supports the MOA described above. A subsequent mutational study demonstrates that LiaIH-and not YydIJ-represents the most efficient resistance determinant against YydF<sup>*</sup> action. Unexpectedly, none of the observed cellular effects upon YydF<sup>*</sup> treatment alone are able to trigger <i>liaIH</i> expression, indicating that only the unique combination of membrane permeabilization and membrane rigidification caused by the epipetide, leads to the observed cell envelope stress response. |
W3133553060 | Surfactin is a lipoheptapeptide produced by several Bacillus species and identified for the first time in 1969. At first, the biosynthesis of this remarkable biosurfactant was described in this review. The peptide moiety of the surfactin is synthesized using huge multienzymatic proteins called NonRibosomal Peptide Synthetases. This mechanism is responsible for the peptide biodiversity of the members of the surfactin family. In addition, on the fatty acid side, fifteen different isoforms (from C12 to C17) can be incorporated so increasing the number of the surfactin-like biomolecules. The review also highlights the last development in metabolic modeling and engineering and in synthetic biology to direct surfactin biosynthesis but also to generate novel derivatives. This large set of different biomolecules leads to a broad spectrum of physico-chemical properties and biological activities. The last parts of the review summarized the numerous studies related to the production processes optimization as well as the approaches developed to increase the surfactin productivity of Bacillus cells taking into account the different steps of its biosynthesis from gene transcription to surfactin degradation in the culture medium. |
W3199953739 | In the current scenario of changing climatic conditions and the rising global population, there is an urgent need to explore novel, efficient, and economical natural products for the benefit of humankind. Biosurfactants are one of the latest explored microbial synthesized biomolecules that have been used in numerous fields, including agriculture, pharmaceuticals, cosmetics, food processing, and environment-cleaning industries, as a source of raw materials, for the lubrication, wetting, foaming, emulsions formulations, and as stabilizing dispersions. The amphiphilic nature of biosurfactants have shown to be a great advantage, distributing themselves into two immiscible surfaces by reducing the interfacial surface tension and increasing the solubility of hydrophobic compounds. Furthermore, their eco-friendly nature, low or even no toxic nature, durability at higher temperatures, and ability to withstand a wide range of pH fluctuations make microbial surfactants preferable compared to their chemical counterparts. Additionally, biosurfactants can obviate the oxidation flow by eliciting antioxidant properties, antimicrobial and anticancer activities, and drug delivery systems, further broadening their applicability in the food and pharmaceutical industries. Nowadays, biosurfactants have been broadly utilized to improve the soil quality by improving the concentration of trace elements and have either been mixed with pesticides or applied singly on the plant surfaces for plant disease management. In the present review, we summarize the latest research on microbial synthesized biosurfactant compounds, the limiting factors of biosurfactant production, their application in improving soil quality and plant disease management, and their use as antioxidant or antimicrobial compounds in the pharmaceutical industries. |
W3163410698 | Iturin, a metabolite produced by <i>Bacillus subtilis</i>, has a broad-spectrum antibacterial effect, and because they are secreted in the rhizosphere of plants, iturins are often mixed with many organic molecules. In recent years, people have improved their separation and purification methods but still cannot achieve simple and effective procedures, making Iturins an ideal biological control agent for insects and bacteria; commercial value still cannot be realized. With the in-depth studies of Iturins, its anti-cancer, hemolysis and other biological activities have gradually been discovered. This article reviews the branches of the Iturin family, structural features of these metabolite, separation and purification methods used for producing it, culture optimization, and various biological activities of the Iturin family, such as insecticidal, antibacterial, hemolytic and anticarcinogenic properties, among others have been summarized. Furthermore, this review revealed some commercial applications of Iturins and their relevance in research works. For example, in food packaging, clean water has good development potential.This can promote the commercial application of Iturins instead of other chemical and biological control agents that are environmentally friendly, pollution-free and have no side effects on humans. Furthermore, work documented anticancer, hemolytic and other biological activities of Iturin. |
W3176891009 | Bacillus subtilis Z-14 can inhibit phytopathogenic fungi, and is used as a biocontrol agent for wheat take-all disease. The present study used the soil-borne fungus Gaeumannomyces graminis var. tritici ( Ggt ), which causes wheat take-all disease, and the soil microbial community as indicators, and investigated the antifungal effects of fengycin and iturin A purified from strain Z-14 using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, respectively. The results showed that fengycin destroyed the internal structure of Ggt cells by digesting the cytoplasm and organelles, forming vacuoles, and inducing hyphal shrinkage and distortion. Iturin A induced cell wall disappearance, membrane degeneration, intracellular material shrinkage, and hyphal fragmentation. A biocontrol test demonstrated a 100% control effect on wheat take-all when wheat seedlings were treated with fengycin at 100 μg/ml or iturin A at 500 μg/ml. Iturin A and fengycin both reduced the relative abundance of Aspergillus and Gibberella . At the genus level, iturin A reduced the relative abundance of Mortierella and Myrothecium , while fengycin reduced that of Fusarium. Only fengycin treatment for 7 days had a significant effect on soil bacterial diversity. |
W3145434124 | In comparison to other natural and synthetic emulsifiers, the results showed that surfactin is a strong candidate to form and stabilize O/W emulsions under the reported conditions. |
W3034283818 | A polar head and an apolar tail chemically characterize surfactants, they show different properties and are categorized by different factors such as head charge and molecular weight. They work by reducing the surface tension between oil and water phases to facilitate the formation of one homogeneous mixture. In this respect, they represent unavoidable ingredients, their main application is in the production of detergents, one of if not the most important categories of cosmetics. Their role is very important, it should be remembered that it was precisely soaps and hygiene that defeated the main infectious diseases at the beginning of the last century. Due to their positive environmental impact, the potential uses of microbial sourced surfactants are actively investigated. These compounds are produced with different mechanisms by microorganisms in the aims to defend themselves from external threats, to improve the mobility in the environment, etc. In the cosmetic field, biosurfactants, restricted in the present work to those described above, can carry high advantages, in comparison to traditional surfactants, especially in the field of sustainable and safer approaches. Besiede this, costs still remain an obsatcle to their diffusion; in this regard, exploration of possible multifunctional actions could help to contain application costs. To highlight their features and possible multifunctional role, on the light of specific biological profiles yet underestimated, we have approached the present review work. |
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W3163181598 | Abstract Background Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. Results In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks . Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC–MS showed that the proportion of n C 14 -surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more n C 14 -surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more n C 14 -surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. Conclusion The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of n C 14 -surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin. Graphic abstract |
W3088135731 | Glycogen is a highly branched polysaccharide that is widely present in all life domains. It has been identified in many bacterial species and functions as an important energy storage compound. In addition, it plays important roles in bacterial transmission, pathogenicity, and environmental viability. There are five essential enzymes (coding genes) directly involved in bacterial glycogen metabolism, which forms a single operon <i>glgBXCAP</i> with a suboperonic promoter in <i>glgC</i> gene in <i>Escherichia coli</i>. Currently, there is no comparative study of how the disruptions of the five glycogen metabolism genes influence bacterial phenotypes, such as growth rate, biofilm formation, and environmental survival, etc. In this study, we systematically and comparatively studied five <i>E. coli</i> single-gene mutants (Δ<i>glgC</i>, Δ<i>glgA</i>, Δ<i>glgB</i>, Δ<i>glgP</i>, Δ<i>glgX</i>) in terms of glycogen metabolism and explored their phenotype changes with a focus on environmental stress endurance, such as nutrient deprivation, low temperature, desiccation, and oxidation, etc. Biofilm formation in wild-type and mutant strains was also compared. <i>E. coli</i> wild-type stores the highest glycogen content after around 20-h culture while disruption of degradation genes (<i>glgP</i>, <i>glgX</i>) leads to continuous accumulation of glycogen. However, glycogen primary structure was abnormally changed in Δ<i>glgP</i> and Δ<i>glgX</i>. Meanwhile, increased accumulation of glycogen facilitates the growth of <i>E. coli</i> mutants but reduces glucose consumption in liquid culture and <i>vice versa</i>. Glycogen metabolism disruption also significantly and consistently increases biofilm formation in all the mutants. As for environmental stress endurance, glycogen over-accumulating mutants have enhanced starvation viability and reduced desiccation viability while all mutants showed decreased survival rate at low temperature. No consistent results were found for oxidative stress resistance in terms of glycogen metabolism disruptions, though Δ<i>glgA</i> shows highest resistance toward oxidation with unknown mechanisms. In sum, single gene disruptions in <i>glgBXCAP</i> operon significantly influence bacterial growth and glucose consumption during culture. Accumulation and structure of intracellular glycogen were also significantly altered. In addition, we observed significant changes in <i>E. coli</i> environmental viabilities due to the deletions of certain genes in the operon. Further investigations shall be focused on the molecular mechanisms behind these phenotype changes. |
W3091847020 | The developing field of bio-nanotechnology aims to advance colloidal research via the introduction of multifunctional nanoparticles to augment the dermal effectiveness of active substances. Self-emulsifying drug delivery systems (SEDDS)—isotropic mixtures of oils, surfactants, solvents and co-solvents or surfactants—are attracting interest in the cosmeceutical field. As part of this study, SEDDS systems containing vitamin C or vitamin E and curcumin were developed, whereby the bioavailability of the active compounds increased by enhancing their permeability to deeper layers of the skin. A composition consisting of 50% surfactin from Bacillus subtilis, 30% Transcutol and 20% oil phase was designed to encapsulate the active substances, i.e., vitamin C or vitamin E and curcumin, contained in the oil phase. The developed carriers were characterized by average particle sizes of 69–183 nm. The formulations with the vitamins were found to be physically and chemically stable for 6 months. Transdermal tests were carried out, showing that the carriers enable the transport of active substances deep into the skin, stopping at the dermis border. The formulations with vitamin C and vitamin E reduced the discoloration, the vascular lesions, and the depth of the wrinkles on the tested skin, which can be useful in cosmetics in the treatment of problem skin, including capillary and sensitive skin. |
W4283310922 | Microbial biosurfactants have attracted the attention of researchers and companies for the last decades, as they are considered promising candidates to replace chemical surfactants in numerous applications. Although in the last years, considerable advances were performed regarding strain engineering and the use of low-cost substrates in order to reduce their production costs, one of the main bottlenecks is their production at industrial scale. Conventional aerobic biosurfactant production processes result in excessive foaming, due to the use of high agitation and aeration rates necessary to increase dissolved oxygen concentration to allow microbial growth and biosurfactant production. Different approaches have been studied to overcome this problem, although with limited success. A not widely explored alternative is the development of foam-free processes through the anaerobic growth of biosurfactant-producing microorganisms. Surfactin, produced by Bacillus subtilis, is the most widely studied lipopeptide biosurfactant, and the most powerful biosurfactant known so far. Bacillus licheniformis strains produce lichenysin, a lipopeptide biosurfactant which structure is similar to surfactin. However, despite its extraordinary surface-active properties and potential applications, lichenysin has been scarcely studied. According to previous studies, B. licheniformis is better adapted to anaerobic growth than B. subtilis, and could be a good alternative for the anaerobic production of lipopeptide biosurfactants. In this review, the potential and limitations of surfactin and lichenysin production under anaerobic conditions will be analyzed, and the possibility of implementing foam-free processes for lichenysin production, in order to expand the market and applications of biosurfactants in different fields, will be discussed. |
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W4292953972 | Surfactin is an excellent biosurfactant with a wide range of application prospects in many industrial fields. However, its low productivity and high cost have largely limited its commercial applications. In this review, the pathways for surfactin synthesis in <i>Bacillus</i> strains are summarized and discussed. Further, the latest strategies for improving surfactin production, including: medium optimization, genome engineering methods (rational genetic engineering, genome reduction, and genome shuffling), heterologous synthesis, and the use of synthetic biology combined with metabolic engineering approaches to construct high-quality artificial cells for surfactin production using xylose, are described. Finally, the prospects for improving surfactin synthesis are discussed in detail. |
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W4327804028 | A new member of the surfactin family, surfactin-C 17 , with the longest fatty acid chain showed strong surface and biological activities. |
W4213317657 | This study aims to find the targets that may influence the production of bacitracin based on RNA sequencing in <i>Bacillus licheniformis</i>. Transcriptional profiling revealed that (i) the expression of the <i>bacT</i> gene, encoding a type II thioesterase (TEII<sub>bac</sub>), was positively correlated with bacitracin production and (ii) the oxygen uptake exhibited significant influence on precursor synthesis. The verified experiments showed that the overexpression of TEII<sub>bac</sub> with an endogenous promoter increased the bacitracin A titer by 37.50%. Furthermore, the increase of oxygen availability through <i>Vitreoscilla</i> hemoglobin (VHb) expression increased the bacitracin A titer by 126.67% under oxygen-restricted conditions. From the transcriptome perspective, the results of this paper demonstrate that TEII<sub>bac</sub> and oxygen supply are crucial to bacitracin production. This study also provides insights into the construction of chassis cells for the industrial production of secondary metabolites with a preference for aerobic conditions. |
W4224036818 | The current work aims to formulate a stable cosmetic nanoemulsion using nature-derived ingredients such as surfactin (a lipopeptide-type biosurfactant produced by Bacillus sp) as an emulsifier, plant-based essential oil as a bioactive ingredient, and coconut oil as the base oil using high-energy ultrasonication method. To accomplish a stable nanoemulsion formulation, experiments with varying combinations of surfactin concentration, essential oil concentration, ultrasonication time, and amplitude were performed. Statistical analysis of samples, based on the responses such as visual observation, droplet size, and polydispersity index, facilitated proper segregation of best-performing samples from those of poorly performing. Nanoemulsions formulated using optimal conditions of oil-to-surfactant ratio (O/S) of 7.4:1 (%w/w), ultrasonication amplitude of 40%, and ultrasonication time of 4.5 min showed greater stability with average droplets size of 170 ± 12 nm, polydispersity index (PDI) of 0.17 ± 0.01 and zeta potential of −56 ± 0.5 mV. The nanoemulsion remained completely stable with no sign of phase separation during the test period of 200 days. Further, the performance of samples toward different stability tests such as heat treatment, centrifugation test, NaCl treatment was satisfactory. In addition, nanoemulsion exhibited significant antioxidant and antimicrobial activity toward Staphylococcus aureus. Thus, the current study demonstrates the imminent potential of surfactin as a bio-emulsifier in skin-care cosmetics. |
W4210817322 | Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains. |
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W1523693806 | Autophagy is an important intracellular degradative process that delivers cytoplasmic proteins to lysosome for degradation. Dysfunction of autophagy is implicated in several human diseases, such as neurodegenerative diseases, infectious diseases, and cancers. Autophagy-related proteins are constitutively expressed in the eye. Increasing studies have revealed that abnormal autophagy is an important pathological feature of several ocular diseases. Pharmacological manipulation of autophagy may provide an alternative therapeutic target for some ocular diseases. In this manuscript, we reviewed the relevant progress about the role of autophagy in the pathogenesis of ocular diseases. |
W1788032165 | We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17β-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17β-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 μM; its uptake was Na + independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver. |
W1948123184 | The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action. |
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W1126765015 | We previously showed that anthraquinones (including rhein, emodin, aloe-emodin, chrysophanol and physcion) were inhibitors of human organic anion transporter 1 (hOAT1) and hOAT3, causing transporter-mediated drug-drug interactions in rats. In this study, the time-dependent inhibition (TDI) of hOAT1 and hOAT3 by anthraquinones was investigated. Madin-Darby canine kidney (MDCK)-hOAT1, HEK293-hOAT3 and their parental cells were used. Preincubation with chrysophanol or physcion for 30 min significantly increased the inhibition of hOAT1, but preincubation with rhein, emodin, aloe-emodin or probenecid had no effect on hOAT1 activity. By contrast, preincubation of hOAT3 with emodin, aloe-emodin, chrysophanol or physcion for 30 min significantly increased its inhibition, but preincubation with rhein or probenecid had no effect on activity. As the incubating time lengthened, from 0 to 60 min, both the inhibition of hOAT1 by chrysophanol and physcion and the inhibition of hOAT3 by emodin, aloe-emodin, chrysophanol and physcion were observed to increase in a time-dependent manner. In conclusion, our results suggest that some anthraquinones contribute to the TDI of hOAT1 and hOAT3. An inhibition study without the preincubation procedure may underestimate the inhibitory potential of anthraquinones against hOAT1 and hOAT3. The underlying mechanisms of TDI of hOAT1 and hOAT3 need to be further investigated. |
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W1583737843 | Protein phosphatase 2B (PP2B) is one of the major brain phosphatases and can dephosphorylate tau at several phosphorylation sites in vitro. Previous studies that measured PP2B activity in human brain crude extracts showed that PP2B activity was either unchanged or decreased in Alzheimer's disease (AD) brain. These results led to the speculation that PP2B might regulate tau phosphorylation and that a down-regulation of PP2B might contribute to abnormal hyperphosphorylation of tau. In this study, we immunoprecipitated PP2B from brains of six AD subjects and seven postmortem- and age-matched controls and then measured the phosphatase activity. We found a three-fold increase in PP2B activity in AD brain as compared with control brains. The activation was due to the partial cleavage of PP2B by calpain I that was activated in AD brain. The truncation of PP2B appeared to alter its intracellular distribution in the brain. In human brains, PP2B activity correlated positively, rather than negatively, to the levels of tau phosphorylation at several sites that can be dephosphorylated by PP2B in vitro. Truncation of PP2B in the frontal cortex was more than in the temporal cortex, and tau phosphorylation was also more in the frontal cortex. Taken together, these results indicate that truncation of PP2B by calpain I elevates its activity but does not counteract the abnormal hyperphosphorylation tau in AD brain. |
W1497188274 | The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (Ki) from 6 nM for wild-type FKBP12 to 3.7 microM for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-A2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site. |
W1875385555 | Aims In contrast to cyclosporin, only limited information exists on the interaction potential between the immunosuppressive agent tacrolimus and HMG‐CoA reductase inhibitors, which are metabolized via the cytochrome P450 system. The aim of this study was to investigate the pharmacokinetics, and adverse effects of cerivastatin combined with tacrolimus in renal transplant patients. Methods Ten patients with stable kidney graft functions and LDL‐cholesterol serum concentrations > 110 mg dl −1 were included in the study. After an observation period of 3 months, cerivastatin (0.2 mg daily) was administered for an additional 3 months. Tacrolimus steady‐state pharmacokinetics and cerivastatin single‐ and multiple‐dose pharmacokinetics were determined. Lipid concentrations, routine laboratory parameters and adverse events were obtained and analysed throughout the study period of 6 months. Results Blood tacrolimus trough concentrations were not affected by cerivastatin (mean ± SD 8.6 ± 2.1 ng ml −1 before, and 8.7 ± 2.4 ng ml −1 at day 90 of cerivastatin dosing, with a 95% confidence interval on the difference = 0.97, 1.08). The mean area under the blood concentration–time curve to 24 h (AUC (0,24 h) ) for cerivastatin was 14.5 ± 2.53 µg l −1 h −1 at day 1 after starting treatment and 19.02 ± 3.55 µg l −1 h −1 (3 months later), resulting in a 35% higher (AUC (0,24 h) ) compared with the first dose. Total cholesterol, LDL‐cholesterol and triglyceride concentrations were significantly lowered by cerivastatin whereas no significant effect of cerivastatin on serum creatininkinase concentrations was observed and no adverse effects were documented. Conclusions Tacrolimus increased the AUC (0, 24 h) of cerivastatin by a mean of 35% in renal transplant patients. Cerivastatin had no detectable effect on the pharmacokinetics of tacrolimus. |
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W1967676836 | The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity‐based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease‐relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K m for ATP . In addition, profiling at a physiological ATP concentration (1 m m ) was carried out, and the IC 50 values of the inhibitors against each kinase were compared with the estimated plasma‐free concentration (calculated from published pharmacokinetic parameters of plasma C trough and C max values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off‐target kinases in drug side effects. Thus, large‐scale kinase profiling at both K m and physiological ATP concentrations could be useful in characterizing the targets and off‐targets of kinase inhibitors. |
W1977839873 | null |
W1969352301 | The nuclear translocation of peptide hormones, such as the somatolactogenic hormone prolactin, after receptor internalization has been widely reported. Prolactin has been demonstrated to interact with cyclophilin B, a member of the immunophilin family of proteins. Cyclophilin B interaction with prolactin potentiated prolactin-induced proliferation, cell growth, and the nuclear retrotransport of prolactin. These effects could be abrogated by the removal of the peptidyl-prolyl isomerase activity of cyclophilin B. Our findings indicate that the intranuclear prolactin/cyclophilin B complex acts as a transcriptional inducer by interacting directly with Stat5, resulting in the removal of the Stat-repressor protein inhibitor of activated Stat 3 (PIAS3), thereby enhancing Stat5 DNA-binding activity and prolactin-induced, Stat5-mediated gene expression. |
W1967808726 | Atorvastatin is increasingly used as a cholesterol-lowering agent in solid organ transplant recipients receiving cyclosporine A (CsA). However, the potential bilateral pharmacokinetic interaction between atorvastatin and CsA in renal transplant recipients has not previously been examined. Baseline 12-h CsA pharmacokinetic investigation was performed in 21 renal transplant recipients and repeated after 4 weeks of atorvastatin treatment (10 mg/ d). At week 4, 24-h pharmacokinetics of atorvastatin was also performed. All patients received basiliximab induction followed by CsA and prednisolone immunosuppression. Compared with historic controls, CsA-treated patients showed, on average, sixfold higher plasma HMG-CoA reductase inhibitory activity after 4 weeks of atorvastatin treatment (p < 0.05). Atorvastatin had a moderate effect on the pharmacokinetics of CsA and reduced the AUC0-12 (area under curve, 0-12h) by 9.5 +/- 18% (p = 0.013) and Cmax (maximal concentration) by 13.5 +/- 24% (p =0.009), while C12 (trough level) was unchanged (p =0.42). Total and LDL cholesterol decreased by 26.8 +/- 8.4% (p < 0.0001) and 41.5 +/- 11.0% (p < 0.0001), respectively. Bilateral pharmacokinetic interaction between atorvastatin and CsA resulted in sixfold higher plasma HMG-CoA reductase inhibitory activity, but only a moderate decrease in systemic exposure of CsA. |
W1968824596 | null |
W2012034410 | Protein expression across human tissues Sequencing the human genome gave new insights into human biology and disease. However, the ultimate goal is to understand the dynamic expression of each of the approximately 20,000 protein-coding genes and the function of each protein. Uhlén et al. now present a map of protein expression across 32 human tissues. They not only measured expression at an RNA level, but also used antibody profiling to precisely localize the corresponding proteins. An interactive website allows exploration of expression patterns across the human body. Science , this issue 10.1126/science.1260419 |
W2022687771 | Hepatitis C virus (HCV) is a highly variable plus-strand RNA virus of the family <i>Flaviviridae</i>. Viral strains are grouped into six epidemiologically relevant genotypes that differ from each other by more than 30% at the nucleotide level. The variability of HCV allows immune evasion and facilitates persistence. It is also a substantial challenge for the development of specific antiviral therapies effective across all HCV genotypes and for prevention of drug resistance. Novel HCV cell culture models were instrumental for identification and profiling of therapeutic strategies. Concurrently, these models revealed numerous host factors critical for HCV propagation, some of which have emerged as targets for antiviral therapy. It is generally assumed that the use of host factors is conserved among HCV isolates and genotypes. Additionally, the barrier to viral resistance is thought to be high when interfering with host factors. Therefore, current drug development includes both targeting of viral factors but also of host factors essential for virus replication. In fact, some of these host-targeting agents, for instance inhibitors of cyclophilin A, have advanced to late stage clinical trials. Here, we highlight currently available cell culture systems for HCV, review the most prominent host-targeting strategies against hepatitis C and critically discuss opportunities and risks associated with host-targeting antiviral strategies. |
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W2017265065 | Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1alpha and HIF-1beta subunits. O2-dependent degradation of the HIF-1alpha subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1alpha is regulated by the competition of RACK1 and HSP90 for binding to HIF-1alpha. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1alpha. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1alpha. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1alpha. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1alpha degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1alpha each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1alpha. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1alpha degradation. These results indicate that intracellular calcium levels can regulate HIF-1alpha expression by modulating calcineurin activity and RACK1 dimerization. |
W2016394900 | null |
W2005518854 | Objective The mutual drug-drug interaction potential of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin and cyclosporine (INN, ciclosporin) in kidney transplant recipients receiving individual immunosuppressive treatment was evaluated with respect to pharmacokinetic behavior of either drug and tolerability of concomitant use. Methods Plasma and urine concentrations of cerivastatin and its major metabolites were determined after administration of 0.2 mg single-dose cerivastatin to 12 kidney transplant recipients (9 men and 3 women) who were receiving stable individual cyclosporine treatment (mainly 200 mg twice a day). These results were compared with the single-dose pharmacokinetic results obtained from a healthy control group (n = 12, age-comparable men). Cerivastatin steady-state pharmacokinetics were evaluated in the same patients during continued immunosuppressive treatment 4 to 6 weeks later, after a 7-day treatment of 0.2 mg cerivastatin once a day. Cyclosporine steady-state concentration–time profiles were determined in blood with monoclonal (EMIT [enzyme multiplied immunoassay technique] assay, parent drug specific) and polyclonal antibodies (FPIA [fluorescence polarization immunoassay] assay, cyclosporine plus metabolites) during cerivastatin cotreatment and compared with predosing data. Results Coadministration of 0.2 mg cerivastatin once a day to the kidney transplant recipients treated with individual doses of cyclosporine and other immunosuppressive agents resulted in a 3- to 5-fold increase in cerivastatin and metabolites plasma concentrations. Cerivastatin and metabolites elimination half-lives were unaffected, and no accumulation occurred during multiple-dosing conditions. Cerivastatin had no influence on steady-state blood concentrations of cyclosporine or cyclosporine metabolites in these patients. The concomitant use of both drugs was well tolerated. Conclusions Cerivastatin and metabolites plasma concentrations were significantly increased in kidney transplant recipients treated with cyclosporine and other immunosuppressive agents. Displacement from the main site for cerivastatin distribution—the liver—by cyclosporine-inhibited liver transport processes may explain the decrease in both metabolic clearance and volume of distribution for cerivastatin and metabolites. Clinical Pharmacology & Therapeutics (1999) 65, 251–261; doi: |
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W2019122675 | Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding. |
W2015591423 | Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported. |
W2004287689 | Abstract Statins are relatively safe first-line agents to use in the setting of dyslipidemia associated with immunosuppressive therapy in subjects undergoing liver transplantation, and also in HIV-infected patients with dyslipidemia due to antiretroviral drugs, especially ritonavir-boosted protease inhibitors. Rosuvastatin, a new statin, has demonstrated higher potency than previously released statins and is not extensively metabolized by the liver P450 system; therefore, the probability of deleterious pharmacokinetic interactions with commonly used immunosuppressants and antiretroviral drugs is reduced. We present the first case of severe rhabdomyolysis in a liver transplant patient receiving rosuvastatin for the treatment of immunosuppressive therapy-related grade IV dyslipidemia, an HIV-infected subject on protease inhibitor-sparing HAART, that resolved after rosuvastatin withdrawal, probably related to interactions between calcineurin inhibitors and hepatic rosuvastatin uptake transporters such as Organic Anion Transporting Polypeptides (OATPs). Liver Transpl, 2011. © 2011 AASLD. |
W2053519812 | Prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP) and cyclophilin B (CyPB) form a complex in the endoplasmic reticulum which is responsible for 3-hydroxylation of a limited number of proline residues in types I, II and V collagens. In this complex, CRTAP serves the role of helper protein, while P3H1 provides the enzymatic activity for the modification. In type I collagen, the major protein of the extracellular matrix of bone, the complex 3-hydroxylates only the a1(I)Pro986 residue. P3H1 and CRTAP each also have independent roles as components of matrix. Furthermore, the two proteins have significant homology with each other. The critical importance of the components of the complex for normal bone development has been revealed by a Crtap knock-out mouse and by infants and children with null mutations of CRTAP and LEPRE1, the gene that encodes P3H1. On a clinical level, defects in the components of the prolyl 3-hydroxylation complex have been shown to be the long-sought cause of severe and lethal recessive osteogenesis imperfecta. |
W2050932098 | Two classes of hepatitis C antiviral agents currently exist, i.e., direct-acting antivirals and host-targeting antivirals. Direct-acting antivirals target viral proteins including NS3/NS4A protease, NS5B polymerase and NS5A protein, while host-targeting antivirals target various host proteins critical for replication of the hepatitis C virus (HCV). Alisporivir is the most advanced host-targeting antiviral in clinical development. Alisporivir blocks HCV replication by neutralizing the peptidyl-prolyl isomerase activity of the abundant host cytosolic protein, cyclophilin A. Due to its unique mechanism of antiviral action, alisporivir is pangenotypic, provides a high barrier for development of viral resistance, and does not permit cross-resistance to direct-acting antivirals. Alisporivir has an excellent pharmacokinetic and safety profile. Phase I and II clinical studies have demonstrated that alisporivir causes a dramatic reduction in viral loads in HCV-infected patients. Alisporivir was shown to be highly potent in treatment-naïve and treatment-experienced patients with genotype 1 as well as in those with genotypes 2 or 3. Low viral breakthrough rates were observed and the most frequent clinical and laboratory adverse events associated with alisporivir in combination with pegylated interferon-alpha and ribavirin were similar to those associated with pegylated interferon-alpha and ribavirin used alone. A laboratory abnormality observed in some patients receiving alisporivir is hyperbilirubinemia, which is related to transporter inhibition and not to liver toxicity. The most recent clinical results suggest that alisporivir plus other direct-acting antivirals should provide a successful treatment option for difficult-to-treat populations, such as nonresponders to prior interferon-alpha therapy and patients with cirrhosis. In conclusion, alisporivir represents an attractive candidate component of future interferon-free regimens. |
W2036741913 | Cyclosporin A (CsA) causes a number of clinically relevant drug-drug interactions (DDIs) by inhibiting OATP1B1 and OATP1B3. In the present study, long-lasting inhibitory effects of CsA on these transporters were examined in comparison to tacrolimus (TCR). OATP1B1- and OATP1B3-expressing HEK293T cells, OATP1B1-expressing MDCK II cells, and human hepatocytes were preincubated with CsA or TCR, and uptake studies were carried out in their presence or absence. Western blot or immunohistochemical studies were done in OATP1B1-expressing HEK293T cells. The pretreatment of OATP1B1- and OATP1B3-expressing cells with 0.5-10 µM CsA, but not TCR, resulted in a reduction in their activity, even after washing out CsA from the incubation media. Preincubating the cells with CsA significantly enhanced its inhibitory effects on OATP1B1 and OATP1B3 by coincubation at 0.1-1 µM. Preincubation with 1 µM CsA caused a reduction in OATP1B1 activity for at least 18 h after its removal. The expression of OATP1B1 was not affected by incubation with CsA and no obvious change in its intracellular localization was observed. The long-lasting inhibition by CsA was also observed in human hepatocytes. Thus, CsA has a long-lasting inhibitory effect on OATP1B1 and OATP1B3. It may attribute to the clinically relevant DDIs between OATP substrates and CsA. |
W2041587518 | The mitochondrial permeability transition (mPT) involves the opening of a non-specific pore in the inner membrane of mitochondria, converting them from organelles whose production of ATP sustains the cell, to instruments of death. Here, I first summarize the evidence in favour of our model for the molecular mechanism of the mPT. It is proposed that the adenine nucleotide translocase (ANT) is converted into a non-specific pore through a calcium-mediated conformational change. This requires the binding of a unique cyclophilin (cyclophilin-D, CyP-D) to the ANT, except when matrix [Ca2+] is very high. Binding of CyP-D is increased in response to oxidative stress and some thiol reagents which sensitize the mPT to [Ca2+]. Matrix adenine nucleotides decrease the sensitivity of the mPT to [Ca2+] by binding to the ANT. This is antagonized by carboxyatractyloside (an inhibitor of the ANT) and by modification of specific thiol groups on the ANT by oxidative stress or thiol reagents; such treatments thus enhance the mPT. In contrast, decreasing intracellular pH below 7.0 greatly desensitizes the mPT to [Ca2+]. Conditions which sensitize the mPT towards [Ca2+] are found in hearts reperfused after a period of ischaemia, a process that may irreversibly damage the heart (reperfusion injury). We have demonstrated directly that mPT pores open during reperfusion (but not ischaemia) using a technique that involves entrapment of [3H]deoxyglucose in mitochondria that have undergone the mPT. The mPT may subsequently reverse in hearts that recover from ischaemia/reperfusion, the extent of resealing correlating with recovery of heart function. A variety of agents that antagonize the mPT protect the heart from reperfusion injury, including cyclosporin A, pyruvate and propofol. Mitochondria that undergo the mPT and then reseal may cause cytochrome c release and thus initiate apoptosis in cells subjected to stresses less severe than those causing necrosis. An example is the apoptotic cell death in the hippocampus that occurs several days after insulin-induced hypoglycaemia, and can be prevented by prior treatment with cyclosporin A. |
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W2046996355 | The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment. |
W2049347116 | The plasma concentrations of pravastatin in pediatric cardiac recipients receiving triple immunosuppressive medication are nearly 10-fold higher than in hypercholesterolemic children after the same pravastatin dose. However, the short-term therapy of pravastatin was well tolerated and effective in lowering serum cholesterol levels in cardiac recipients. |
W2061730574 | Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo. |
W2043034268 | Reduced function of OATP1B1 related to genetic variation and drug-drug interactions likely contributed to cerivastatin-induced rhabdomyolysis. Although cerivastatin is no longer in clinical use, these findings may translate to related statins and other substrates of OATP1B1. |
W2055013289 | A protein has been identified that interacts specifically with both the Src homologous 3 (SH3) domain and carboxy-terminal sequences of the c-Abl tyrosine kinase. The cDNA encoding the Abl interactor protein (Abi-2), was isolated from a human lymphocyte library using the yeast two-hybrid system with the Abl SH3 domain as bait. Abi-2 binds to c-Abl in vitro and in vivo. Abi-2 is a novel protein that contains an SH3 domain and proline-rich sequences critical for binding to c-Abl. A basic region in the amino terminus of Abi-2 is homologous to the DNA-binding sequence of homeo-domain proteins. We show that Abi-2 is a substrate for the c-Abl tyrosine kinase. Expression of an Abi-2 mutant protein that lacks sequences required for binding to the Abl SH3 domain but retains binding to the Abl carboxyl terminus activates the transforming capacity of c-Abl. The properties of Abi-2 are consistent with a dual role as regulator and potential effector of the c-Abl protein and suggest that Abi-2 may function as a tumor suppressor in mammalian cells. |
W2062120260 | null |
W2084977836 | Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals. |
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W2085448380 | Rosuvastatin exposure was significantly increased in transplant recipients on an antirejection regimen including cyclosporine. Cyclosporine inhibition of OATP-C-mediated rosuvastatin hepatic uptake may be the mechanism of the drug-drug interaction. Coadministration of rosuvastatin with cyclosporine needs to be undertaken with caution. |
W2077798795 | Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities. |
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W2112078820 | We present a statistical model to estimate the accuracy of peptide assignments to tandem mass (MS/MS) spectra made by database search applications such as SEQUEST. Employing the expectation maximization algorithm, the analysis learns to distinguish correct from incorrect database search results, computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. Using SEQUEST search results for spectra generated from a sample of known protein components, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptides. This analysis makes it possible to filter large volumes of MS/MS database search results with predictable false identification error rates and can serve as a common standard by which the results of different research groups are compared. |
W2089850443 | Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 × 10 -7 moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH 2 -terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56°C and at p H 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism. |
W2145804243 | null |
W2091423586 | Abstract Dasatinib is a BCR-ABL inhibitor with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Imatinib failure is commonly caused by BCR-ABL mutations. Here, dasatinib efficacy was analyzed in patients recruited to phase 2/3 trials with chronic-phase chronic myeloid leukemia with or without BCR-ABL mutations after prior imatinib. Among 1043 patients, 39% had a preexisting BCR-ABL mutation, including 48% of 805 patients with imatinib resistance or suboptimal response. Sixty-threedifferent BCR-ABL mutations affecting 49 amino acids were detected at baseline, with G250, M351, M244, and F359 most frequently affected. After 2 years of follow-up, dasatinib treatment of imatinib-resistant patients with or without a mutation resulted in notable response rates (complete cytogenetic response: 43% vs 47%) and durable progression-free survival (70% vs 80%). High response rates were achieved with different mutations except T315I, including highly imatinib-resistant mutations in the P-loop region. Impaired responses were observed with some mutations with a dasatinib median inhibitory concentration (IC50) greater than 3nM; among patients with mutations with lower or unknown IC50, efficacy was comparable with those with no mutation. Overall, dasatinib has durable efficacy in patients with or without BCR-ABL mutations. All trials were registered at http://www.clinicaltrials.gov as NCT00123474, NCT00101660, and NCT00103844. |
W2099236176 | Cyclosporine raised the plasma concentrations of repaglinide, probably by inhibiting its CYP3A4-catalyzed biotransformation and OATP1B1-mediated hepatic uptake. Coadministration of cyclosporine may enhance the blood glucose-lowering effect of repaglinide and increase the risk of hypoglycemia. |
W2131983256 | Aims Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion‐transporting polypeptide ( OATP ) 1 B , but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral ( PO ) and intravenous ( IV ) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. Methods The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV . The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. Results Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7‐ and 7.6‐fold for pitavastatin and 4.4‐ and 3.3‐fold for rosuvastatin, after PO and IV rifampicin, respectively. I n vitro studies showed that rifampicin was an inhibitor of OATP1B 1 and OATP1B 3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. Conclusions The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose. |
W2139413142 | null |
W2152561379 | ABSTRACT SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro . SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 μM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro . SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV. |
W2102417187 | null |
W2111809733 | The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus. |
W2153607816 | null |
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