Activity-guided screening of bioactive natural compounds implementing a new glucocorticoid-receptor-translocation assay and detection of new anti-inflammatory steroids from bacteria.

Using an in vitro cell-based assay in a flow-design, we have applied activity-guided screening to search for new bioactive compounds isolated from microorganisms. A first assay employs the stable expression of nuclear factor kappa B (NF-κB) while a second assay utilizes the glucocorticoid receptor (GR) coupled to green fluorescent protein. A specialized assay was implemented for both the translocation of NF-κB and to inhibit the translocation of cytokine-mediated NF-κB. In addition, we developed in a wide palette of cell lines used for a highly specialized GR-translocation assay to detect anti-inflammatory effects. This approach demonstrates the straight-forward combination of cell-based assays arranged with an automated fluorescence microscope. This allows for the direct sorting of extracts which are acting in a pharmaceutically interesting way. Initial results using this technique have led to the detection of new anti-inflammatory steroids from bacterial crude extracts.

Marine antifungal theonellamides target 3beta-hydroxysterol to activate Rho1 signaling.

Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-beta-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-beta-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3beta-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis.

Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: cloning, expression, characterization, and developmental expression.

By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at approximately 6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.

The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors.

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10(-10)-10(-5) M) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl beta-carboline-3-carboxylate (beta-CCM) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, beta-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3 beta-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.

cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-hydroxysteroids.

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

Isolation and characterization of a stereospecific 3beta-hydroxysteriod sulfotransferase (pregnenolone sulfotransferase) cDNA.

In contrast to humans, who possess a hydroxysteroid sulfotransferase (HSST), namely, DHEA sulfotransferase (DHEA-ST), that displays broad substrate specificities, HSSTs of the guinea pig show a high substrate stereoselectivity, as shown by the recent cloning of a chiral-specific 3alpha-hydroxysteroid sulfotransferase. Herein, we report the cloning and expression of the substrate and chiral-specific pregnenolone sulfotransferase (PREG-ST). Transfection of the pCMV expression vector containing PREG-ST cDNA in transformed human embryonal kidney (293) cells showed that the expressed enzyme selectively catalyzes the 3beta-hydroxysteroid substrate. It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were transformed at a much lower rate, and androsterone, a 3alpha-hydroxysteroid, was not significantly metabolized (30-fold lower). Thus, the enzyme was identified as pregnenolone sulfotransferase. DNA analysis predicts a protein of 287 amino acids with a calculated molecular mass of 34,199 daltons. Alignment of the amino acid sequence with other sulfotransferases indicated that guinea pig pregnenolone sulfotransferase shares 75 and 80% homology with human DHEA sulfotransferase and rat hydroxysteroid dehydrogenase, respectively. RNA blot analysis using guinea pig liver, intestine, adrenal, kidney, epididymis, testis, and lung showed a single RNA species at 1.3 kb is expressed in liver, intestine, and kidney. Guinea pig 3beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.

The corticotropin-releasing factor test in the differential diagnosis of Cushing's syndrome: a comparison with the lysine-vasopressin test.

The diagnostic accuracy of the CRH test was compared with that of the LVP test in 28 consecutive patients with ACTH-dependent Cushing's syndrome. A false negative response to CRH was found in 3 of 21 patients with pituitary-dependent Cushing's disease and to LVP in 4. The 7 patients with ectopic ACTH secretion were unresponsive to CRH, whereas 2 did respond to LVP. CRH and high-dose dexamethasone tests combination led to concordant results in 79% of patients. In all cases the etiological diagnosis suggested was correct. LVP and high-dose dexamethasone tests combination led to concordant results in only 71% of patients and the etiological diagnosis suggested was erroneous in one. Individual tolerance to the CRH test was also clearly better than that to the LVP test. It is concluded that the CRH test, alone or in combination with the high-dose dexamethasone test must be preferentially used to the LVP test in the differential diagnosis of ACTH-dependent Cushing's disease.