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Ling-Xing Shan,1 Ping-Hua Sun,1,2 Bao-Qin Guo,1 Xing-Jun Xu,1 Zhi-Qiang Li,1 Jia-Zhi Sun,2 Shu-Feng Zhou,2 Wei-Min Chen1 1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, People's Republic of China; 2College of Pharmacy, University of South Florida, Tampa, FL, USA Abstract: Seventeen acylides bearing an aryl-tetrazolyl alkyl-substituted side chain were synthesized, starting from clarithromycin, via several reactions including hydrolysis, acetylating, esterification, carbamylation, and Michael addition. The structures of all new compounds were confirmed by 1H nuclear magnetic resonance spectroscopy, 13C nuclear magnetic resonance spectroscopy, and mass spectrometry. All these synthesized acylides were evaluated for in vitro antimicrobial activities against gram-positive pathogens (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative pathogens (Pseudomonas aeruginosa, Escherichia coli), using the broth microdilution method. Results showed that compounds 10e, 10f, 10g, 10 h, 10o have good antibacterial activities. Keywords: acylide, clarithromycin, synthesis, antibacterial activity

Although copper-nitrene has been extensively studied as a versatile active species in various transformations, asymmetric reactions involving copper-nitrene have been limited to the aziridination of olefins. Herein, we report the novel copper-nitrene-catalyzed desymmetric oxaziridination reaction of cyclic diketones with alkyl azides and the subsequent rearrangement of the resulting highly active intermediate, which produces a synthetically challenging chiral bicyclic lactam containing a quaternary carbon center. This procedure not only enriches the copper-nitrene-catalyzed asymmetric reactions, but also provides an alternative strategy to address the inherent challenges of catalytic asymmetric Schmidt reactions. This unique reaction could inspire the investigation of novel copper-nitrene-catalyzed asymmetric transformations and their reaction mechanisms., (© 2021 Wiley-VCH GmbH.)

Background and Purpose: GPR119 is a G protein-coupled receptor that is preferentially expressed in islet cells and mediates insulin secretion. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) act as endogenous ligands for this receptor, whereas PSN375963 and PSN632408 are two recently reported synthetic agonists. In this study, we explored mechanisms underlying GPR119-induced insulin secretion. In addition, we assessed the potential utility of the synthetic agonists as tools for exploring GPR119 biology., Experimental Approach: We examined natural and synthetic GPR119 agonist activity at GPR119 in MIN6c4 and RINm5f insulinoma cells. We evaluated insulin secretion, intracellular calcium [Ca(2+)](i), ion channel involvement and levels of cAMP., Key Results: We report that increases in insulin secretion induced by OEA were associated with increased cAMP and a potentiation of glucose-stimulated increases in [Ca(2+)](i). We also demonstrate that ATP-sensitive K(+) and voltage-dependent calcium channels were required for GPR119-mediated increases in glucose-stimulated insulin secretion. In contrast to OEA, the synthetic GPR119 agonist PSN375963 and PSN632408 have divergent effects on insulin secretion, cAMP and intracellular calcium in MIN6c4 cells., Conclusions and Implications: The endogenous ligand OEA signals through GPR119 in a manner similar to glucagon-like peptide-1 (GLP-1) and its receptor with respect to insulin secretion, [Ca(2+)](i) and cAMP. In addition, PSN375963 and PSN632408 substantially differ from OEA and from one another. These studies suggest that the commercially available synthetic agonists, although they do activate GPR119, may also activate GPR119-independent pathways and are thus unsuitable as GPR119-specific pharmacological tools.

Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P < 0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced to 32% of control (P < 0.01). Moreover, in Sertoli cells from 90-day-old rats, AR levels were partially maintained by LH and completely maintained by androgens. A similar trend was observed on day 35. On day 21, however, AR levels in immature Sertoli cells were unaffected in all treatment groups. These results indicate that androgen maximally stimulates AR levels in immature Leydig cells but is without significant effect in adult Leydig cells. In contrast, AR levels in Sertoli cells are more sensitive to androgen regulation in adult compared with immature animals. These findings indicate that there are distinct mechanisms controlling AR concentrations in Leydig and Sertoli cells during the development of the testis.

In vitro studies have shown that corticosterone (B) directly inhibits testosterone (T) production by purified Leydig cells but does so only at high concentrations. 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) in Leydig cells oxidatively inactivates B, lowering its effective concentration, thus protecting against the suppressive effect of glucocorticoid on T production. The aim of the present study was to assess the significance of B at physiological levels in modulating T production and 11 beta-HSd activity in Leydig cells. To determine the effects of endogenous B on Leydig cell steroidogenesis, male rats (200-250 g body wt) were adrenalectomized (ADX), while control rats were subjected to sham surgery (SHAM). Seven days after surgery: T and LH were measured in serum; T production was measured in aliquots of spent culture media from 3-h incubations of purified Leydig cells; 11 beta-HSD activity and messenger RNA was measured in purified Leydig cells. ADX rats had elevated serum T (P < 0.05) in contrast to SHAM control or ADX rats that received B replacement (1 mg/100 g body wt per day, i.p., on the final 3 days). Serum LH levels were uninfluenced by ADX, with or without B replacement (SHAM), 0.45 +/- 0.16 ng/ml; ADX, 0.35 +/- 0.13 ng/ml; ADX + B, 0.61 +/- 0.09 ng/ml, NS, P > 0.05). This indicated that the alteration of T production was induced by a mechanism that is independent of LH. ADX nearly doubled LH-stimulated T production by purified Leydig cells, from 106.3 +/- 9.3 (SHAM) to 183.2 +/- 16.7 (ADX) ng/10(6) cells.3 h (mean +/- SEM for three replications of the experiment, P < or = 0.02). T production by Leydig cells from the ADX + B treatment group was suppressed to 53% of SHAM values, indicating that B inhibits T production after ADX. The oxidative activity of 11 beta-HSD in Leydig cells exceeded its reductive activity, and both activities declined after ADX. The decline in 11 beta-HSD activities after ADX was prevented by B replacement. Similarly, the steady state levels of 11 beta-HSD messenger RNA declined in Leydig cells after ADX, and this decline was prevented by B replacement. We conclude that physiological levels of B exert a tonic, negative control directly on Leydig cell steroidogenesis and also induce intracellular 11 beta-HSD activity, thereby protecting against B-mediated inhibition of T production. By modulating the level of active glucocorticoid in Leydig cells, 11 beta-HSD is thus a significant determinant of their steroidogenic capacity.

To investigate the mechanism of androgen-induced thymolysis, the effects of various androgens, including testosterone (T), 19-nortestosterone, and 7 alpha-methyl-19-nortestosterone (MENT), were compared with those of estradiol and dexamethasone (DEX) in intact, castrated, and adrenalectomized male rats. The potency comparisons on thymus regression, based on mass of steroids, showed DEX to be the most potent, followed by estradiol and the androgens. Among the androgens, MENT was the most potent, followed by nortestosterone and T, an order similar to their anabolic potency on muscle. As the thymolytic effects of T and MENT were not altered by the concomitant administration of an aromatase inhibitor or a 5-reductase inhibitor, it was concluded that the effects of androgens were not mediated by their conversion to estrogens or 5 alpha-reduced metabolites. Involvement of glucocorticoid receptors in androgen action was excluded because mifepristone (an antiglucocorticoid) blocked DEX-induced, but not T- or MENT-induced, thymus regression. Flutamide, an antiandrogen, significantly blocked the thymolytic effect of T and MENT, providing further support for this conclusion. This suggested that the thymolytic action of androgens is an intrinsic property mediated via androgen receptors (AR). The occurrence of AR in the thymus was demonstrated by binding assays and the presence of AR messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction. Quantitative reverse transcriptase-polymerase chain reaction for AR mRNA in the thymus showed 6-fold more AR mRNA in the thymic epithelial cells than in the thymocytes. However, epithelial cells represent only a small fraction of the thymus. Hence, it is hypothesized that the androgens produce their thymolytic effects by stimulating the secretion of a factor(s) by the thymic epithelial cells that, in turn, causes regression of the thymus.

Testosterone produced by Leydig cells is critical for the maintenance of spermatogenesis by Sertoli cells throughout adulthood in the rat. However, the presence of androgen receptors (AR) in Leydig cells in prepubertal rats suggests additional roles for androgen in early Leydig cell function and differentiation. In the present study, AR messenger RNA (mRNA) was directly measured by in situ hybridization in sections of rat testes at three developmental stages: on day 21 postpartum, when Leydig cells exist as mesenchymal-like progenitors; on day 35, when they are still immature, producing low amounts of testosterone; and on day 90, when they are fully functional in the sexually mature animal. Testicular AR mRNA was detected in Leydig cells, pericytes, peritubular myoid cells, and Sertoli cells. On day 90, AR mRNA levels in Sertoli cells varied with the cycle of the seminiferous epithelium, achieving peak intensity at stages VII-VIII. Measurements were made by image analysis and expressed as integrated signal intensities per unit labeled area (mean +/- SEM; n = 3 rats at each age). The results showed that levels of Leydig cell and Sertoli cell AR mRNA change significantly during development (P < 0.05). Leydig cell AR mRNA was intermediate on day 21 (at 17.3 +/- 0.7), highest on day 35 (at 26.9 +/- 1.6), and lowest on day 90 (at 11.8 +/- 1.1). The trend for isolated Leydig cells from these three ages was identical. In contrast, Sertoli cell AR mRNA was lowest on day 21 (at 19.3 +/- 1.0), intermediate on day 35 (at 24.5 +/- 1.4), and highest on day 90 (at 36.9 +/- 0.5). In Leydig cells, the highest level of AR mRNA was present during puberty, whereas the greatest amount of AR mRNA in Sertoli cells was present on day 90. This indicates that Leydig cells and Sertoli cells use different mechanisms to maintain AR levels. We infer from these data that Leydig cells are maximally sensitive to androgen during puberty, which is consistent with our hypothesis that androgens facilitate their differentiation.

Adult Leydig cells originate within the testis postnatally. Their formation is a continuous process involving gradual transformation of progenitors into the mature cell type. Despite the gradual nature of these changes, studies of proliferation, differentiation and steroidogenic function in the rat Leydig cell led to the recognition of three distinct developmental stages in the adult Leydig cell lineage: Leydig cell progenitors, immature Leydig cells and adult Leydig cells. In the first stage, Leydig cell progenitors arise from active proliferation of mesenchymal-like stem cells in the testicular interstitium during the third week of postnatal life and are recognizable by the presence of Leydig cell markers such as histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and the present of luteinizing hormone (LH) receptors. They proliferate actively and by day 28 postpartum differentiate into immature Leydig cells. In the second stage, immature Leydig cells are morphologically recognizable as Leydig cells. They have an abundant smooth endoplasmic reticulum and are steroidogenically active, but primarily produce 5 alpha-reduced androgens rather than testosterone. Immature Leydig cells divide only once, giving rise to the total adult Leydig cell population. In the third and final stage, adult Leydig cells are fully differentiated, primarily produce testosterone and rarely divide. LH and androgen act together to stimulate differentiation of Leydig cell progenitors into immature Leydig cells. Preliminary data indicate that insulin like growth factor-1 (IGF-1) acts subsequently in the transformation of immature Leydig cells into adult Leydig cells.

The postnatal differentiation of rat Leydig cells may be subdivided into three stages based on morphology and steroid production. The purpose of this study was to clarify the developmental mechanisms underlying increased testosterone production by measuring steady state levels of the mRNAs for three steroidogenic enzymes in isolated Leydig cells at each stage of differentiation. These include Leydig cell progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90. The steroidogenic enzymes were 1) cholesterol side-chain cleavage enzyme (CSCC), 2) 17 alpha-hydroxylase (P450-17 alpha), and 3) 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD). We report that levels of CSCC and P450-17 alpha mRNAs increase, whereas 3 alpha HSD mRNA levels decline during the course of Leydig cell differentiation. The levels of 3 alpha HSD mRNA were high in progenitor Leydig cells that appeared to contain little smooth endoplasmic reticulum and decreased in cells as smooth endoplasmic reticulum developed and other enzyme mRNAs increased. These observations suggest that the factors that regulate 3 alpha HSD mRNA levels are startlingly different from those that regulate the mRNA levels of CSCC and P450-17 alpha. We conclude that the progressive increase in the capacity of differentiating Leydig cells to produce testosterone can be explained in part by an increase in the activity of enzymes that synthesize testosterone (CSCC and P450-17 alpha) and a decrease in the activity of an enzyme that metabolizes testosterone and its precursors (3 alpha HSD).

To further assess the hormonal response capabilities of Leydig cell progenitors (PLC) from 21-day-old rats, their levels of LH and androgen receptors (LH-R and AR) were measured and compared to those of isolated immature (ILC) and adult Leydig cells (ALC) from 35- and 90-day-old rats, respectively. Levels of LH receptor were estimated by Scatchard analysis of binding to [125I]hCG, and levels of LH receptor mRNA were determined by Northern blot analysis using a rat LH receptor antisense RNA probe. The numbers of LH receptors per cell measured by the binding study were 2,623 +/- 1,110 in PLC, 9,024 +/- 1,992 in ILC, and 39,896 +/- 15,234 in ALC (mean +/- SEM of four replicate experiments; ALC significantly greater than either PLC or ILC at P less than 0.05). The Northern blotting revealed three major bands [6.7, 2.6, and 2.3 kilobases (kb)] that were present in Leydig cells at all three ages and were not detected in HepG2 cells. When the steady state levels of the predominant 6.7-kb species were normalized to actin mRNA, PLC were 6.3-fold lower than ILC and 1.7-fold lower than ALC (n = 3 replicate isolations of poly(A) RNA). The 2.6- and 2.3-kb species exhibited similar trends. Levels of AR were estimated by immunoblotting using a polyclonal antibody against a synthetic peptide of the receptor (residues 14-32) that detected a 110-kilodalton AR protein. Levels of AR mRNA were estimated by Northern blot analysis, using a rat AR antisense RNA probe that detected a single 10-kb AR mRNA. The relative levels of AR protein were 1.0, 1.5, and 0.5 in PLC, ILC, and ALC, respectively (n = 3). Similar trends were observed for AR mRNA (n = 3). The observation that both LH and AR levels were lower in PLC compared to ILC is consistent with the hypothesis that the former are progenitors of Leydig cells.

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.