Oxytocin modulates human chemosensory decoding of sex in a dose-dependent manner.

There has been accumulating evidence of human social chemo-signaling, but the underlying mechanisms remain poorly understood. Considering the evolutionarily conserved roles of oxytocin and vasopressin in reproductive and social behaviors, we examined whether the two neuropeptides are involved in the subconscious processing of androsta-4,16,-dien-3-one and estra-1,3,5 (10),16-tetraen-3-ol, two human chemosignals that convey masculinity and femininity to the targeted recipients, respectively. Psychophysical data collected from 216 heterosexual and homosexual men across five experiments totaling 1056 testing sessions consistently showed that such chemosensory communications of masculinity and femininity were blocked by a competitive antagonist of both oxytocin and vasopressin receptors called atosiban, administered nasally. On the other hand, intranasal oxytocin, but not vasopressin, modulated the decoding of androstadienone and estratetraenol in manners that were dose-dependent, nonmonotonic, and contingent upon the recipients' social proficiency. Taken together, these findings establish a causal link between neuroendocrine factors and subconscious chemosensory communications of sex-specific information in humans.

Isolation and functional characterization of a novel endogenous inverse agonist of estrogen related receptors (ERRs) from human pregnancy urine.

Estrogen-receptor related receptors (ERRs) which consists of ERRα, ERRß and ERRγ belong to the orphan nuclear receptor subfamily 3, group B (NR3B) subfamily, and are constitutively active. ERRs have been shown to actively modulate estrogenic responses, and to play an essential role in pregnancy, and are implicated in breast cancer progression. Despite intensive efforts, no endogenous ligand other than the ubiquitous sterol, cholesterol which binds ERRα, has been identified for ERRs so far. The discovery of ligands that bind these orphan receptors will allow the manipulation of this pathway and may lead to novel strategies for the treatment of cancer and other diseases. We previously reported the identification of a novel endogenous estradienolone-like steroid (ED) that is strongly bound to sex hormone binding globulin, in pregnant women. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. Furthermore, the anti-mitogenic effect of ED in breast cancer cells is ERRα-dependent. These data suggest that ED-ERR interaction may represent a novel physiologically relevant hormone response pathway in the human. The finding that ED inhibits both ER negative and ER positive breast cancer cell growth may have important implications in pathophysiology breast cancer.

3,17ß-Bis-sulfamoyloxy-2-methoxyestra-1,3,5(10)-triene and Nonsteroidal Sulfamate Derivatives Inhibit Carbonic Anhydrase IX: Structure-Activity Optimization for Isoform Selectivity.

3,17ß-Bis-sulfamoyloxy-2-methoxyestra-1,3,5(10)-triene (STX140), a bis-sulfamate derivative of the endogenous steroid 2-methoxyestradiol, has shown promising anticancer potency both in vitro and in vivo, with excellent bioavailability. Its activity against taxane-resistant xenografts makes it a potential drug candidate against triple-negative breast cancer (TNBC). These properties are linked to the ability of STX140 to act in a multitargeting fashion in vivo as a microtubule disruptor, leading to cell cycle arrest and with both proapoptotic and anti-angiogenic activities. Carbonic anhydrase IX (CA IX) is a well-established biomarker for aggressive cancers, including TNBC. This study reports, for the first time, the inhibitory activities of a series of steroidal and nonsteroidal sulfamate derivatives against CA IX in comparison to the ubiquitous CA II, with some compounds demonstrating 100-200-fold selectivity for CA IX over CA II. X-ray crystallographic studies of four of the most promising compounds reveal that isoform-specific residue interactions are responsible for the high specificity.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women II: Total estrogenicity calculations accounting for competitive protein and receptor binding and potency.

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4-99.2% (median: 97.3%), ER-ß, 82.7-97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women.

Discovery of Vilaprisan (BAY 1002670): A Highly Potent and Selective Progesterone Receptor Modulator Optimized for Gynecologic Therapies.

Progesterone plays an important role in the female reproductive system. However, there is also evidence that gynecologic disorders/diseases such as uterine fibroids and endometriosis are progesterone-dependent. Steroidal and non-steroidal selective progesterone receptor modulators (SPRMs) have shown potential for the treatment of such diseases. Steroidal SPRMs, including mifepristone and ulipristal acetate, have proven effective in clinical trials. However, several steroidal SPRMs containing a dimethylamino substituent have been associated with elevated liver enzymes in patients. An earlier drug discovery program identified lonaprisan as a highly selective SPRM that did not show drug-related change in liver enzyme activity. Building on data obtained from that work, here we describe the research program that culminated in the discovery of a novel steroidal SPRM, vilaprisan, which combines an extremely high potency with very favorable drug metabolism and pharmacokinetic properties. Vilaprisan has entered clinical development and is currently undergoing phase 3 clinical trials.

Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16ß-( m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16ß-( m-carbamoylbenzyl)-17ß-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17ß-HSD1-2-MeO-CC-156-NADP+ and 17ß-HSD1-PBRM-NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.

Dynamic changes in binding interaction networks of sex steroids establish their non-classical effects.

Non-classical signaling in the intracellular second messenger system plays a pivotal role in the cytoprotective effect of estradiol. Estrogen receptor is a common target of sex steroids and important in mediating estradiol-induced neuroprotection. Whereas the mechanism of genomic effects of sex steroids is fairly understood, their non-classical effects have not been elucidated completely. We use real time molecular dynamics calculations to uncover the interaction network of estradiol and activator estren. Besides steroid interactions, we also investigate the co-activation of the receptor. We show how steroid binding to the alternative binding site of the non-classical action is facilitated by the presence of a steroid in the classical binding site and the absence of the co-activator peptide. Uncovering such dynamic mechanisms behind steroid action will help the structure-based design of new drugs with non-classical responses and cytoprotective potential.

Sorption and desorption of 17α-trenbolone and trendione on five soils.

The metabolites 17α-trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, and its metabolite trendione in the environment. Predictions of the fate and environmental concentrations of these hormones after land application require accurate estimates of the sorption of these compounds in soils. The sorption and desorption of 17α-trenbolone and trendione were measured at 5 nominal concentrations in 5 soils from different geologic settings using a batch equilibrium technique following guideline 106 of the Organisation for Economic Co-operation and Development. Both the sorption and desorption of 17α-trenbolone and trendione to soils were adequately described by the Freundlich sorption model and by linear partition coefficients. The mean sorption coefficients were 9.04 mL/g and 32.2 mL/g for 17α-trenbolone and trendione, respectively. The corresponding mean Freundlich sorption exponents were 0.88 and 0.98, respectively. Sorption of 17α-trenbolone and trendione was correlated principally with soil organic carbon. Average sorption coefficients normalized to soil organic carbon content (KOC ) were 460 mL/g and 1804 mL/g for 17α-trenbolone and trendione, respectively. The mean desorption coefficients were 22.1 mL/g and 43.8 mL/g for 17α-trenbolone and trendione, respectively. Calculated hysteresis coefficients based on the difference in the area between sorption and desorption isotherms indicated that sorption equilibrium was not fully reversible and hysteresis of desorption isotherms occurred for both 17α-trenbolone and trendione. Environ Toxicol Chem 2017;36:613-620. © 2016 SETAC.

Degradation and transformation of 17α-trenbolone in aerobic water-sediment systems.

Synovex® ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers. Trenbolone acetate is rapidly hydrolyzed in cattle to form 17ß-trenbolone and its isomer, 17α-trenbolone, which are further transformed to a secondary metabolite, trendione. As part of the environmental assessment for the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14 C]-17α-trenbolone in 2 representative water-sediment systems under aerobic conditions. The same transformation products, 17ß-trenbolone and trendione, were formed, principally in the sediment phase, in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of 17ß-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17α-trenbolone + 17ß-trenbolone + trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17α-trenbolone + 17ß-trenbolone + trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2017;36:630-635. © 2016 SETAC.

Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17ß from water.

Six experiments were carried out to define the optimum conditions for investigating the dynamics of uptake and metabolism of tritiated E2 from water by adult blue mussels, Mytilus spp. Optimum uptake was achieved using 400mL aerated sea water animal-1 and an incubation period of no more than 24h. The pattern of disappearance conformed closest to an inverse hyperbolic curve with the percentage of radiolabel that could be measured in the water reaching an asymptote that was on average 50% of the original. This apparent inability of the animals to absorb all the radiolabel was investigated further. Solvent partition and chromatography revealed that, after 24h, c. 60% of the radiolabel still present in the water was composed of water soluble conjugates, c. 25% was composed of tritiated water and only 15% ran on and around the chromatographic position of E2. The major water soluble constituent was identified by chromatography and mass-spectrometry as 1,3,5(10)-estratriene-3,17ß-diol 3-sulfate (estradiol 3-S). The clearance rate of radiolabel was 46.9±1.8mLanimal-1h-1. This was not significantly affected by the addition of as much as 25µgL-1 cold E2 to the water, demonstrating that mussels have a large capacity for E2 uptake. A new procedure involving solvent partition was developed for separating the free, esterified and sulfated forms of E2 present in the flesh of mussels. This involved extracting the soft tissue with organic solvents and then treating a portion of dried extract with a combination of heptane (dissolved fatty acid esters of E2) and 80% ethanol (dissolved free and sulfated E2). The latter fraction was further partitioned between water (sulfate) and diethyl ether (free steroid). This procedure was much cheaper and less time-consuming than chromatography. Approximately 80% of the radioactivity that was taken up by the animals was present in the form of ester. Moreover, E2 was the only steroid identified after saponification of these esters. Of the remaining radioactivity, c. 10% was in the form of unidentified free steroids and c. 10% was estradiol 3-S. In order to determine how rapidly mussels were able to depurate tritiated E2 and its metabolites, two experiments were carried out. Animals from the first experiment purged up to 63% of radioactivity in 20days under flow-through conditions; whereas animals from the second experiment released only 16% of radioactivity in 10days under semi-static conditions. The ratios of the different forms of E2 did not change substantially during the course of depuration.

Transferred multipolar atom model for 10ß,17ß-dihydroxy-17α-methylestr-4-en-3-one dihydrate obtained from the biotransformation of methyloestrenolone.

Biotransformation is the structural modification of compounds using enzymes as the catalysts and it plays a key role in the synthesis of pharmaceutically important compounds. 10ß,17ß-Dihydroxy-17α-methylestr-4-en-3-one dihydrate, C19H28O3·2H2O, was obtained from the fungal biotransformation of methyloestrenolone. The structure was refined using the classical independent atom model (IAM) and a transferred multipolar atom model using the ELMAM2 database. The results from the two refinements have been compared. The ELMAM2 refinement has been found to be superior in terms of the refinement statistics. It has been shown that certain electron-density-derived properties can be calculated on the basis of the transferred parameters for crystals which diffract to ordinary resolution.

Relationship between the climbing up and climbing down stairs domain scores on the FES-DMD, the score on the Vignos Scale, age and timed performance of functional activities in boys with Duchenne muscular dystrophy / Relação entre escore FES-DMD-subir e descer escada com escore Escala Vignos, idade e tempo de realização das atividades em meninos com Distrofia Muscular de Duchenne

BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA) of going up and down stairs in boys with Duchenne muscular dystrophy (DMD). METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD); age in years; functional classification using the Vignos Scale (VS), and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004) and strong relationships with VS (r=0.72, p=0.001) and TA for this task (r=0.83, p<0.001). There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032), VS (r=0.65, p=0.002) and TA for this task (r=0.40, p=0.034). CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information. .

CONTEXTUALIZAÇÃO: Conhecer as potencialidades e limitações das informações geradas por diferentes instrumentos de avaliação favorece o desenvolvimento mais preciso do diagnóstico funcional e da tomada de decisão terapêutica. OBJETIVO : Investigar a relação entre o número de movimentos compensatórios ao subir e descer escadas, idade, classificação funcional e tempo de realização de atividade (TA) em meninos com Distrofia Muscular de Duchenne (DMD). MÉTODO : Foi utilizado banco de filmes de 30 meninos com DMD realizando atividades funcionais. Os movimentos compensatórios foram avaliados pela Escala de Avaliação Funcional para Distrofia Muscular de Duchenne (FES-DMD), domínio subir e descer escada; a idade, mensurada em anos; a classificação funcional foi pesquisada pela Escala de Vignos (EV), e o TA foi cronometrado. Foi utilizado o teste de correlação de Spearman. RESULTADOS : Existe moderada relação entre a FES-DMD-subir escada e a idade (r=0,53, p=0,004) e forte relação com a EV (r=0,72, p=0,001) e TA dessa tarefa (r=0,83, p<0,001). Houve fraca relação entre a FES-DMD-descer escada e a idade (r=0,40, p=0,032), EV (r=0,65, p=0,002) e o TA dessa tarefa (r=0,40, p=0,034). CONCLUSÃO : Esses achados indicam que a avaliação da tarefa de subir escada pode trazer informações mais relevantes sobre a evolução da doença, embora a atividade de descer escada deva ser pesquisada visando à orientação e prevenção de acidentes. A utilização conjunta de dados provenientes da FES-DMD, da idade e do TA pode se complementar para formulação do diagnóstico funcional. Estudos longitudinais e com outras faixas etárias mais amplas podem complementar tal informação. .

Chemosensory communication of gender through two human steroids in a sexually dimorphic manner.

Recent studies have suggested the existence of human sex pheromones, with particular interest in two human steroids: androstadienone (androsta-4,16,-dien-3-one) and estratetraenol (estra-1,3,5(10),16-tetraen-3-ol). The current study takes a critical step to test the qualification of the two steroids as sex pheromones by examining whether they communicate gender information in a sex-specific manner. By using dynamic point-light displays that portray the gaits of walkers whose gender is digitally morphed from male to female [1, 2], we show that smelling androstadienone systematically biases heterosexual females, but not males, toward perceiving the walkers as more masculine. By contrast, smelling estratetraenol systematically biases heterosexual males, but not females, toward perceiving the walkers as more feminine. Homosexual males exhibit a response pattern akin to that of heterosexual females, whereas bisexual or homosexual females fall in between heterosexual males and females. These effects are obtained despite that the olfactory stimuli are not explicitly discriminable. The results provide the first direct evidence that the two human steroids communicate opposite gender information that is differentially effective to the two sex groups based on their sexual orientation. Moreover, they demonstrate that human visual gender perception draws on subconscious chemosensory biological cues, an effect that has been hitherto unsuspected.

Mass balance approaches to characterizing the leaching potential of trenbolone acetate metabolites in agro-ecosystems.

Several studies have documented the occurrence and fate of trenbolone acetate (TBA) metabolites in soil and water. However, considerable uncertainty still exists with respect to TBA risk in agro-ecosystems because limited data are available to quantify excretion, transformation, and leaching processes. To address these uncertainties, we used experimental mesocosms and a mass balance approach to estimate the TBA metabolite leaching potential from manure excreted by implanted (40 mg TBA, 8 mg 17ß-estradiol) beef cattle. Manure sample analysis indicates that over 113 days, a maximum of 9.3% (3,200 µg/animal unit [AU]) of the implant dose was excreted as 17α-trenbolone (17α-TBOH), and <1% was excreted as 17ß-trenbolone (65 µg/AU) or trendione (3 µg/AU). While most (>97%) of the total excreted mass of 17α-TBOH transforms to uncharacterized products, 0.3-0.6% (100-220 µg/AU) of the implant dose accumulates on land surfaces and is available for subsequent transport. During rainfall or irrigation events, a maximum of 0.005-0.06% (1.6-22 µg/AU 17α-TBOH) or 0.005-0.012% (1.8-4 µg/AU 17α-TBOH) of the dose leached into runoff, respectively. Leaching potentials peak at 5-30 days postimplantation, suggesting that targeted timing of implantation and irrigation could minimize steroid leaching during rainfall and irrigation events.

Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures: a review.

Structural modification of steroids through whole-cell biocatalysis is an invaluable procedure for the production of active pharmaceutical ingredients (APIs) and key intermediates. Modifications could be carried out with regio- and stereospecificity at positions hardly available for chemical agents. Much attention has been focused recently on the biotransformation of 17α-ethynyl substituted steroidal drugs using fungi, bacteria and plant cell cultures in order to obtained novel biologically active compounds with diverse structure features. Present article includes studies on biotransformation on 17α-ethynyl substituted steroidal drugs using microorganisms and plant cell cultures. Various experimental and structural elucidation methods used in biotransformational processes are also highlighted.

New metabolites from fungal biotransformation of an oral contraceptive agent: methyloestrenolone.

Fungal cell cultures were used for the first time for the biotransformation of methyloestrenolone (1), an oral contraceptive. Fermentation of 1 with Macrophomina phaseolina, Aspergillus niger, Gibberella fujikuroi, and Cunninghamella echinulata produced eleven metabolites 2-12, six of which 2-5, 11 and 12 were found to be new. These metabolites were resulted from the hydroxylation at C-1, C-2, C-6, C-10, C-11, and C-17α-CH3, as well as aromatization of ring A of the steroidal skeleton of substrate 1. The transformed products were identified as 17α-methyl-6ß,17ß-dihydroxyestr-4-en-3-one (2), 17α-(hydroxymethyl)-11ß,17ß-dihydroxyestr-4-en-3-one (3), 17α-methyl-2α,11ß,17ß-trihydroxyestr-4-en-3-one (4), 17α-methyl-1ß,17ß-dihydroxyestr-4-en-3-one (5), 17α-methyl-11α,17ß-dihydroxyestr-4-en-3-one (6), 17α-methyl-11ß,17ß-dihydroxyestr-4-en-3-one (7), 17α-methyl-10ß,17ß-dihydroxyestr-4-en-3-one (8), 17α-(hydroxymethyl)-17ß-hydroxyestr-4-en-3-one (9), 17α-methylestr-1,3,5(10)-trien-3,17ß-diol (10), 17α-methyl-3,17ß-dihydroxyestr-1,3,5(10)-trien-6-one (11), and 17α-methyl-6ß,10ß,17ß-trihydroxyestr-4-en-3-one (12).

Uptake of 17ß-trenbolone and subsequent metabolite trendione by the pinto bean plant (Phaseolus vulgaris).

Manure from livestock feeding operations is commonly applied to agricultural fields as an alternative to commercial fertilizers. Trenbolone acetate (TbA) is a synthetic growth promoter frequently utilized in beef cattle feeding operations. Metabolites of TbA can be present in manure and subsequently applied to fields. Fate ofTbA metabolites 17ß-trenbolone (17ßTb), 17α-trenbolone (17αTb), and trendione (TbO) have been assessed in manure and soils, but plant uptake in agricultural fields is not fully understood. The objective of this study was to investigate potential plant uptake and biotransformation of 17ßTb using the pinto bean plant (Phaseolus vulgaris). Vegetated (n=20) and control sands (n=16) were amended with 17ßTb at a level of 1µg/g once per week for a total of four weeks. Sand, above-ground plant portion and below-ground plant portion were collected each week and then analyzed for 17ßTb, 17αTb, and TbO. By week four, low concentrations of 17ßTb (10±4.9µg/g fresh weight) were taken up into the roots of plants and, to a much lesser extent, translocated throughout the plant (0.04±0.02µg/g fresh weight). Extensive transformation of 17ßTb to the metabolite trendione (TbO) occurred in vegetated sand, while minimal TbO was detected in control sand. These results suggest the biotransformation of 17ßTb to TbO is predominantly through microbial degradation. Trenbolone (Tb) metabolites can then be taken up into plants but remain concentrated in the roots with only slight translocation to above ground portions of the plant. After four weeks, maximum observed concentrations of total Tb (parent+metabolites) in fresh plant tissues were 33.0µg/g in roots and 0.25µg/g in leaves. No phytotoxicity was observed to pinto bean plants throughout the four week study.

The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea.

Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

The role of Ala231 and Trp227 in the substrate specificities of fungal 17ß-hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase: Steroids versus smaller substrates.

17ß-Hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus; 17ß-HSDcl and 3HNR, respectively) are two homologous short-chain dehydrogenase/reductase proteins that are 58% identical and have 86% similar amino acids. The minor differences in their substrate-binding regions are believed to be crucial for their substrate specificities. 3HNR shows high affinity for substrates with two rings, like trihydroxynaphthalene and 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), while 17ß-HSDcl can accommodate ligands with four rings, like steroids. In the present study, we examined the role of Ala231 in 17ß-HSDcl and Trp227 in 3HNR, as the potential key amino acids in the determination of substrate recognition based on size. We constructed Ala231Trp 17ß-HSDcl and Trp227Ala 3HNR mutant proteins and used spectrophotometric analyses to compare their catalytic activities with those of the wild-type enzymes, for oxidation of 4-estrene-17ß-ol-3-one and DDBO and for reduction of 4-estrene-3,17-dione and 9,10-phenanthrenequinone (PQ). The Ala231Trp side-chain substitution in 17ß-HSDcl abolished and decreased (by 14.6-fold) the initial rates for steroid oxidation and reduction, respectively, while the initial rate for PQ reduction was increased 5.6-fold. The bulky Trp227Ala side-chain substitution in 3HNR enabled oxidation of 4-estrene-17ß-ol-3-one, increased the initial rates for reduction of 4-estrene-3,17-dione and PQ by 4.5-fold and 1.5-fold, respectively, while the initial rate for DDBO oxidation was decreased 4.1-fold. Our TLC analysis and docking simulations also support these findings. Our study thus confirms the important roles of Ala231 in 17ß-HSDcl and Trp227 in 3HNR, for the selection between larger and smaller substrates. Article from a special issue on steroids and microorganisms.

Stimulation of Ca2+-sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney.

We used the patch-clamp technique to study the effect of changing the external Ca2+ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2+ concentration from 1 mM to 2 or 3 mM inhibited the basolateral 50-pS K channels while decreasing external Ca2+ to 10 µM increased the 50-pS K channel activity. The effect of the external Ca2+ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2+ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2+-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2+ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analog increased the 50-pS K channel activity but did not block the effect of raising the external Ca2+ on the K channels. Neither inhibition of phospholipase A2 (PLA2) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2+ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2+. We conclude that the external Ca2+ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2+ is mediated by the CaSR which stimulates PLC-PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2+ level modulates the reabsorption of divalent cations.