Genetic Disruption of cyp21a2 Leads to Systemic Glucocorticoid Deficiency and Tissues Hyperplasia in the Teleost Fish Medaka (Oryzias latipes).

cytochrome P-450, 21-hydroxylase (cyp21a2), encodes an enzyme required for cortisol biosynthesis, and its mutations are the major genetic cause of congenital adrenal hyperplasia (CAH) in humans. Here, we have generated a null allele for the medaka cyp21a2 with a nine base-pair insertion which led to a truncated protein. We have observed a delay in hatching and a low survival rate in homozygous mutants. The interrenal gland (adrenal counterpart in teleosts) exhibits hyperplasia and the number of pomca-expressing cells in the pituitary increases in the homozygous mutant. A mass spectrometry-based analysis of whole larvae confirmed a lack of cortisol biosynthesis, while its corresponding precursors were significantly increased, indicating a systemic glucocorticoid deficiency in our mutant model. Furthermore, these phenotypes at the larval stage are rescued by cortisol. In addition, females showed complete sterility with accumulated follicles in the ovary while male homozygous mutants were fully fertile in the adult mutants. These results demonstrate that the mutant medaka recapitulates several aspects of cyp21a2-deficiency observed in humans, making it a valuable model for studying steroidogenesis in CAH.

Multiplexed Serum Steroid Profiling Reveals Metabolic Signatures of Subtypes in Congenital Adrenal Hyperplasia.

Context: Altered metabolic signatures on steroidogenesis may characterize individual subtypes of congenital adrenal hyperplasia (CAH), but conventional diagnostic approaches are limited to differentiate subtypes. Objective: We explored metabolic characterizations and identified multiple diagnostic biomarkers specific to individual subtypes of CAH. Methods: Liquid chromatography-mass spectrometry-based profiling of 33 adrenal steroids was developed and applied to serum samples obtained from 67 CAH patients and 38 healthy volunteers. Results: Within- and between-run precisions were 95.4% to 108.3% and 94.1% to 110.0%, respectively, while all accuracies were <12% and the correlation coefficients (r2) were > 0.910. Metabolic ratios corresponding to 21-hydroxylase characterized 21-hydroxylase deficiency (21-OHD; n = 63) from healthy controls (area under the curve = 1.0, P < 1 × 10-18 for all) and other patients with CAH in addition to significantly increased serum 17α-hydroxyprogesterone (P < 1 × 10-16) and 21-deoxycortisol (P < 1 × 10-15) levels. Higher levels of mineralocorticoids, such as corticosterone (B) and 18-hydroxyB, were observed in 17α-hydroxylase deficiency (17α-OHD; N = 3), while metabolic ratio of dehydroepiandrosterone sulfate to pregnenolone sulfate was remarkably decreased against all subjects. A patient with 11ß-hydroxylase deficiency (11ß-OHD) demonstrated significantly elevated 11-deoxycortisol and its metabolite tetrahydroxy-11-deoxyF, with reduced metabolic ratios of 11ß-hydroxytestosterone/testosterone and 11ß-hydroxyandrostenedione/androstenedione. The steroid profiles resulted in significantly decreased cortisol metabolism in both 21-OHD and 17α-OHD but not in 11ß-OHD. Conclusion: The metabolic signatures with specific steroids and their corresponding metabolic ratios may reveal individual CAH subtypes. Further investigations with more substantial sample sizes should be explored to enhance the clinical validity.

LC-MS based simultaneous profiling of adrenal hormones of steroids, catecholamines, and metanephrines.

Metabolic changes in adrenocortical steroids and medullary catecholamines characterize adrenal tumors, but they are measured using different analytical protocols. To increase bioanalytical validity while maintaining sample homogeneity, LC-MS-based profiling of 29 cortical steroids and 6 medullary amines, including catecholamines and metanephrines, in a single run was developed. Alkyloxycarbonylation with isobutyl chloroformate was employed together with our comprehensive steroid assay, and all adrenal hormones were separated on a reversed-phase C18 column (50 × 2.1 mm, 1.9 µm) at a flow rate of 0.3 ml/min. The lower limits of quantification for all analytes ranged from 0.1 to 2.0 ng/ml, with extraction recoveries of 58.5%-109.5%, while the imprecision and accuracy were 1.6%-14.8% and 89.2%-114.9%, respectively. The validated LC-MS assay was applied to serum samples obtained from 60 patients with adrenal Cushing syndrome, primary aldosteronism, and pheochromocytoma/paraganglioma (PPGL). In addition to the characteristic metabolic changes in glucocorticoids, mineralocorticoids, catecholamines, and metanephrine, the molecular ratios of dehydroepiandrosterone sulfate and 20α-dihydrocortisol indicated Cushing syndrome and primary aldosteronism (P < 0.01 for all compounds), respectively. Moreover, the interactive molecular ratios of 11-deoxycortisol with normetanephrine, metanephrine, norepinephrine, and epinephrine (P < 0.01 all compounds) were proposed to characterize the metabolic features of PPGL. Novel LC-MS-based quantitative profiling of steroids, catecholamines, and metanephrines in human serum was successfully established and characterized metabolic features of individual adrenal tumors that could be used for clinical purposes.

Interactive metabolic signatures of testicular testosterone with bilateral adrenalectomy in mice.

The hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes have reciprocal relationships with steroidogenesis regulation. However, the relationship between testicular steroids and defective glucocorticoid production under chronic stress remains unclear. Metabolic changes of testicular steroids in bilateral adrenalectomized (bADX) 8-week-old C57BL/6 male mice were measured using gas chromatography-mass spectrometry. Twelve weeks after surgery, testis samples were obtained from the model mice, which were divided into tap-water (n = 12) and 1 % saline (n = 24) supplementation groups, and their testicular steroid levels were compared with those of sham controls (n = 11). An increased survival rate with lower testicular levels of tetrahydro-11-deoxycorticosterone was observed in the 1 % saline group compared to both the tap-water (p = 0.029) and sham (p = 0.062) groups. Testicular corticosterone levels were significantly decreased in both tap-water (4.22 ± 2.73 ng/g, p = 0.015) and 1 % saline (3.70 ± 1.69, p = 0.002) groups compared to those in sham controls (7.41 ± 7.39). Testicular testosterone levels tended to increase in both bADX groups compared to those in the sham controls. In addition, increased metabolic ratios of testosterone to androstenedione in tap-water (2.24 ± 0.44, p < 0.05) and 1 % saline (2.18 ± 0.60, p < 0.05) mice compared to sham controls (1.87 ± 0.55) suggested increased production of testicular testosterone. No significant differences in serum steroid levels were observed. Defective adrenal corticosterone secretion and increased testicular production in bADX models revealed an interactive mechanism underlying chronic stress. The present experimental evidence suggests the crosstalk between the HPA and HPG axes in homeostatic steroidogenesis.

The genome insights of Streptomyces lannensis T1317-0309 reveals actinomycin D production.

The members of Streptomyces have been identified as a major source of antimicrobial agents with broad spectrum. This study is mainly focused on bioactivity-guided isolation and characterization of bioactive molecule from strain Streptomyces sp. T1317-0309 and its whole-genome sequence analysis for possible isolation of novel natural products. Strain Streptomyces sp. T1317-0309 showed 100% sequence similarity with strain Streptomyces lannensis TA4-8T consisting 10, 453,255 bp of genome with 5 scaffolds and 69.9 mol% G + C content. The genome analyses revealed a total of 17 putative biosynthetic gene clusters (BGCs) responsible for various secondary metabolites including actinomycin, bacteriocin, ectoine, melanin, terpene, siderophore, betalactone, NRPS, T2PKS, and T3PKS. The BGC and bioactivity-guided purification of ethyl acetate extract of strain T1317-0309 showed the great potency of antimicrobial activities against various gram-positive multi-drug resistant human pathogens including MRSA. The BGC-predicted bioactive secondary metabolite was identified by various NMR analyses and confirmed as actinomycin D. In addition, this study reveals the first genome study of Streptomyces lannensis as a novel source for actinomycin D.