Identification and expression of the 11β-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11β-hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β-hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β-hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes.     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Bioactive Steroid Derivatives and Butyrolactone Derivatives from a Gorgonian‐Derived Aspergillus sp. Fungus

Six steroid derivatives, 1 – 6 , and five butyrolactone derivatives, 7 – 11 , were isolated from the fermentation broth of a gorgonian‐derived Aspergillus sp. fungus. Their structures were elucidated on the basis of NMR and MS spectral data. Compound 1 is a new, highly conjugated steroid. The NMR and MS data of 7 and 8 are reported for the first time, as their structures were listed in SciFinder Scholar with no associated reference. Compounds 1, 4, 5 , and 8 – 11 inhibited the larval settlement of barnacle Balanus amphitrite with EC₅₀ values ranging from 0.63 to 18.4 μg ml^?1. Butyrolactone derivatives 7 and 8 showed pronounced antibacterial activities against Staphylococcus aureus with the same MIC values as the positive control ciprofloxacin (MIC 1.56 μM for all three compounds).     

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15α-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein β subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Δfgb1 strain, suggesting a role for the G-protein β subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Δfgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.     

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedK _d=13±3 nM andB _max=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.     

Complex combined steroid mix of the female tract modulates human sperm

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca²⁺]_i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, ‘physiological standard’ steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca²⁺]_i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca²⁺]_i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10^-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.     

The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas

In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10⁻¹ mg/L), low (10⁻² mg/L), very low (10⁻⁶ mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).     

Laboratory-scale biosynthesis ofTrichoderma mycolytic enzymes for protoplast release fromCochliobolus lunatus

Summary Microorganism useful for the induction of enzymes lytic towards walls of filamentous fungusCochliobolus lunatus were studies. Production of specificTrichoderma viride mycolytic enzymes was studied in a laboratory fermentor. The product with high chitinase and relatively low protease activity gave better yields ofC. lunatus protoplasts than commercial Novozym 234.     

The involvement of cAMP in the growth inhibition of filamentous fungus Rhizopus nigricans by steroids

Several steroids, in particular progesterone, are toxic for the filamentous fungus Rhizopus nigricans and, at high concentrations, inhibit its growth. Previous studies on this microorganism revealed progesterone specific receptors coupled to G proteins at the plasma membrane. In this study, the next step of steroid signalling in R. nigricans following G protein activation is investigated, together with the possible impact of this pathway on fungal growth inhibition. The intracellular level of cAMP decreased in the presence of steroids, demonstrating the probable involvement of cAMP signalling in the response of R. nigricans to steroids. Results of the growth analysis in the presence of cAMP increasing agents suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.     

Fungal–plant signalling in the Ustilago maydis–maize pathosystem

The smut fungus Ustilago maydis needs the host plant maize for completion of its sexual life cycle. Recent experiments highlight the importance of cAMP and mitogen-activated protein (MAP) kinase signalling for cell fusion as well as for subsequent stages of plant colonisation and induction of disease symptoms. During these distinct developmental stages the same signalling cascades must be differentially regulated and accommodate multiple inputs and outputs.     

Phylogenetic study on wild allies of Lima bean,Phaseolus lunatus (Fabaceae), and implications on its origin

An investigation was made of the phylogenetic relationships among wild accessions of Lima bean (Phaseolus lunatus) and wild allies of Mesoamerican and Andean origins, using electrophoresis of seed storage proteins and isozymes. Mesoamerican wild species are phylogenetically more distant fromP. lunatus than Andean species, and apparently belong to the tertiary gene pool of Lima bean. The Andean wild species, which are investigated for the first time, reveal a high similarity to the Lima bean, and particularly with its Mesoamerican gene pool. These Andean species probably constitute a secondary gene pool of Lima bean, and are thus of considerable interest in the context of genetic improvement of the crop. Based on these observations, an Andean origin is suggested for the Andean wild species and forP. lunatus. These results point out the importance of collecting and conserving AndeanPhaseolus germplasm.     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type     

Steroid hormone activation of wandering in the isolated nervous system of Manduca sexta

Steroid hormones modulate motor circuits in both vertebrates and invertebrates. The insect Manduca sexta, with its well-characterized developmental and endocrinological history, is a useful model system in which to study these effects. Wandering is a stage-specific locomotor behavior triggered by the steroid hormone 20-hydroxyecdysone (20E), consisting of crawling and burrowing movements as the animal searches for a pupation site. This study was undertaken to determine whether the wandering motor pattern is activated by direct action of 20E on the CNS. 20E acts on the isolated larval nervous system to induce a fictive motor pattern showing features of crawling and burrowing. The latency of the response to 20E is long, suggestive of a genomic mechanism of action. The abdominal motoneurons or segmental pattern generating circuits are unlikely to be the primary targets of 20E action in inducing fictive wandering. Exposure of the segmental ganglia alone to hormone did not evoke fictive wandering. Therefore, as suggested by an earlier study, the likely site of 20E action is within the brain.     

Interspecific hybridization of phaseolus vulgaris with P. lunatus and P. acutifolius

Summary The influence of genotypic combinations on the growth of hybrid embryos between Phaseolus vulgaris and P. lunatus, and between P. vulgaris and P. acutifolius was examined. All embryos obtained from P. vulgaris × P. lunatus crosses developed only to a stage which appears to be comparable to the pre-heart-shape stage of selfed embryos. Reciprocal crosses were attempted, but pods abscised at a very early stage. Embryos derived from P. vulgaris × P. acutifolius and reciprocal crosses attained the cotyledon stage although no mature seeds were formed. A distinct characteristic of these embryos was the uneven development of the two cotyledons. The rate of growth and final size of these hybrid embryos seemed to be influenced by the genotypes of both parents.Immature embryos were cultured on defined medium and the effects of glutamine and gibberellin (GA₃) were examined. Glutamine was effective in increasing the survival rate; gibberellin had no apparent effect. Plants derived from cultured embryos of P. vulgaris × P. lunatus, P. vulgaris × P. acutifolius and P. acutifolius × P. vulgaris were obtained.Technical paper No. 00 of the Oregon Agricultural Experiment Station. Research was supported by the Oregon Agricultural Experiment Station, the Research Council of Oregon State University (National Institute of Health Biomedical Research Support Grant RR07079) and the Processor Research Council of Oregon. A.R. is supported by an African Graduate Fellowship from the African-American Institute     

Molecular mechanism(s) of endocrine‐disrupting chemicals and their potent oestrogenicity in diverse cells and tissues that express oestrogen receptors

Endocrine‐disrupting chemicals (EDCs) are natural or synthetic compounds present in the environment which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. Most EDCs tend to bind to steroid hormone receptors including the oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). As EDCs disrupt the actions of endogenous hormones, they may induce abnormal reproduction, stimulation of cancer growth, dysfunction of neuronal and immune system. Although EDCs represent a significant public health concern, there are no standard methods to determine effect of EDCs on human beings. The mechanisms underlying adverse actions of EDC exposure are not clearly understood. In this review, we highlighted the toxicology of EDCs and its effect on human health, including reproductive development in males and females as shown in in vitro and in vivo models. In addition, this review brings attention to the toxicity of EDCs via interaction of genomic and non‐genomic signalling pathways through hormone receptors.