scholarly journals Testosterone inhibits human wild-type and chimeric aldosterone synthase activity in vitro

2020 ◽  
Author(s):  
Andrea Vecchiola ◽  
Cristóbal A. Fuentes ◽  
Cristian A. Carvajal ◽  
Carmen Campino ◽  
Fidel Allende ◽  
...  
Keyword(s):  
Active Sites ◽  
Direct Action ◽  
Inhibitory Effect ◽  
Type I ◽  
Wild Type ◽  
Aldosterone Synthase ◽  
Hek 293 ◽  
Aldosterone Production ◽  
Modelling Studies

Abstract BackgroundFamilial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol did not affect.AimTo explore the direct action of testosterone on ASWT and ASCE enzymes.MethodsHEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities were evaluated incubating HEK-cells transfected with enzymes vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed.ResultsIn this system, testosterone inhibited ASWT (90% inhibition at five µM, IC50=1.690 µM) with higher efficacy and potency than ASCE (80% inhibition at five µM, IC50=3.176 µM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures.ConclusionsThe inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.

Evidence for persistent dysfunction of wild-type aldosterone synthase gene in glucocorticoid-treated familial hyperaldosteronism type I

1997 ◽  
Vol 15 (12) ◽  
pp. 1729-1733 ◽  
Author(s):  
Michael Stowasser ◽  
Wendy L. Taylor ◽  
Michael G. Gartside ◽  
Terry J. Tunny ◽  
Richard D. Gordon
Keyword(s):  
Type I ◽  
Wild Type ◽  
Aldosterone Synthase ◽  
Familial Hyperaldosteronism ◽  
Familial Hyperaldosteronism Type I

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Abstract 128: Leptin: A Regulator of Aldosterone Synthase Expression & Aldosterone Secretion in Visceral Adipocytes, in Mice

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Eric J Belin de Chantemele ◽  
Anne-Cecile Huby ◽  
P. T Menk ◽  
Weiqin Chen ◽  
Brian Lane ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Cross Sections ◽  
Leptin Receptor ◽  
Zona Glomerulosa ◽  
Aldosterone Synthase ◽  
Leptin Receptors ◽  
Fat Depots ◽  
Physiological Relevance ◽  
Aldosterone Production

Obesity is associated with inappropriately high aldosterone levels, which contribute to the development of metabolic and cardiovascular disorders. The origin of these high aldosterone levels is incompletely understood. We recently demonstrated that the adipocyte-derived hormone leptin regulates aldosterone synthase (CYP11B2) expression and stimulates aldosterone release from adrenal zona glomerulosa cells. Recent studies demonstrate that adipocytes express CYP11B2 and secrete aldosterone. However, the mechanisms regulating aldosterone release from adipocytes remain unclear. Likewise, whether visceral (Visc) and subcutaneous (SubQ) adipose tissue contribute to a similar extent to aldosterone production is unknown. We tested the hypothesis that leptin increases adipocyte CYP11B2 expression and aldosterone production and investigated whether Visc and SubQ adipose tissues respond similarly to leptin. Immunostaining of mouse adipose tissue cross-sections and isolated mature adipocytes revealed that Visc and SubQ adipose tissue express leptin receptors. Treatment of mouse freshly isolated mature adipocytes, non-differenciated (stromal fraction) and differentiated adipocytes revealed that leptin dose-dependently increased CYP11B2 expression and aldosterone production in Visc adipose tissue only. Although leptin receptor and CYP11B2 levels were similar in SubQ and Visc adipocytes, SubQ adipocytes were unresponsive to leptin. The physiological relevance of these in vitro data was tested by measuring plasma aldosterone levels in mice deprived of adipose tissue (lipodystrophic mice) treated with leptin. Absence of adipose tissue in lipodystrophic mice blunted leptin-induced increases in aldosterone levels (WT-vehicle: 471±82 vs. WT-Leptin: 1699±396, p<0.05; KO-vehicle: 539±71 vs. KO+leptin: 787±156, NS). The human relevance of these data was determined by reporting that CYP11B2 expression gradually increased with body mass index in human mediastinal and omental fat depots. In summary these data strongly suggest that leptin regulates CYP11B2 levels and aldosterone release in visceral adipose tissue and that leptin-induced, adipocyte-derived aldosterone may contribute to obesity-associated hyperaldosteronism.

Effect of CO2/pH on the aldosterone response to hypoxia in bovine adrenal cells in vitro

1993 ◽  
Vol 265 (4) ◽  
pp. R820-R825
Author(s):  
H. Raff ◽  
B. Jankowski
Keyword(s):  
Respiratory Acidosis ◽  
Inhibitory Effect ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Adrenal Cells ◽  
Aldosterone Production ◽  
Per Se ◽  
Air Control

Acidosis increases and hypoxia decreases aldosterone production from the adrenal zona glomulerosa in vivo, in situ, and in vitro. These effects appear to be located at different steps in the steroidogenic process. Because respiratory acidosis and hypoxemia are common sequelae of chronic lung disease, the present experiments evaluated the interaction of hypoxia and CO2 (with uncompensated or compensated extracellular pH) on aldosteronogenesis in vitro. Bovine adrenal zona glomerulosa cells were stimulated with angiotensin II (ANG II) or adenosine 3',5'-cyclic monophosphate under room air control (21% O2-0% CO2), CO2 per se (21% O2-10% CO2), hypoxia per se (10% O2-0% CO2), and the combination of CO2 and hypoxia (10% O2-10% CO2). Furthermore, under CO2, pH was either allowed to decrease from 7.2 to 6.8 (uncompensated) or its decrease was minimized (> 7.05) with NaOH (compensated). CO2 without pH compensation led to a significant increase in ANG II-stimulated aldosterone release; when the decrease in pH was minimized, CO2 inhibited ANG II-stimulated aldosterone release. Hypoxia inhibited aldosterone release; the inhibitory effect of hypoxia predominated when combined with CO2. In the presence of cyanoketone, pregnenolone production from endogenous precursors (early pathway) was unaffected. However, the conversion of corticosterone to aldosterone (late pathway) was inhibited by low O2 but unaffected by CO2. It is concluded that the inhibitory effect of low O2 on the late pathway predominates over the effects of uncompensated or compensated simulated respiratory acidosis on aldosteronogenesis.

scholarly journals Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2912 ◽  
Author(s):  
Sherif T. S. Hassan ◽  
Miroslava Šudomová ◽  
Kateřina Berchová-Bímová ◽  
Karel Šmejkal ◽  
Javier Echeverría
Keyword(s):  
Dna Polymerase ◽  
Active Sites ◽  
Inhibitory Action ◽  
Inhibitory Concentration ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Standard Drug ◽  
Key Enzyme ◽  
Hsv 1

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 μM; SI: 114.8) compared with that of ACV (EC50: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 μM; inhibition constant (Ki): 0.3 μM) compared with reference drugs aphidicolin (IC50: 0.8 μM; Ki: 0.4 μM) and ACV triphosphate (ACV-TP) (IC50: 0.9 μM; Ki: 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

2001 ◽  
Vol 354 (3) ◽  
pp. 613-625 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Baruch VELAN ◽  
Avigdor SHAFFERMAN
Keyword(s):  
Laser Desorption Ionization ◽  
Wild Type ◽  
Ionization Time ◽  
Hek 293 ◽  
Enzyme Preparations ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Differential Behaviour ◽  
Oligomeric Forms

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

Inhibition of Angiogenesis by Type I Interferons in Models of Kaposi'S Sarcoma

1999 ◽  
Vol 14 (4) ◽  
pp. 257-262 ◽  
Author(s):  
C. Marchisone ◽  
R. Benelli ◽  
A. Albini ◽  
L. Santi ◽  
D. M. Noonan
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Inhibitory Effect ◽  
Type I Interferons ◽  
Endothelial Cell Migration ◽  
Type I ◽  
Type I Ifns ◽  
Hiv 1

Kaposi's Sarcoma (KS) is a pathology which occurs with increased frequency and in a particularly aggressive form in AIDS patients. The HIV-1 Tat protein appears to be an important co-factor in the induction of the extensive neo-vascularization associated with AIDS-KS. Tat acts as a chemoattractant for endothelial cells in vitro, inducing both chemotactic and invasive responses. Several clinical trials have been performed testing the effectiveness of diverse biological agents in therapy of KS, among these the type I interferons. Type I IFNs have diverse biological functions besides their anti-viral activity, including anti-angiogenic properties. We have shown that IFNα and IFNβ are potent inhibitors of both primary and immortalized endothelial cell migration and morphogenesis in vitro as well as neo-angiogenesis induced by HIV-1 Tat in vivo. The inhibitory effect of IFN class I on HIV-Tat associated angiogenesis further supports its use as a therapy for epidemic Kaposi's sarcoma. The use of recombinant IFNs at the levels required to obtain a therapeutic effect are associated with side effects and toxicity, therefore we are now developing a gene therapy approach for constant and local delivery type I IFNs.

Inhibition of aldosterone release by hypoxia in vitro: interaction with carbon monoxide

1994 ◽  
Vol 76 (2) ◽  
pp. 689-693 ◽  
Author(s):  
H. Raff ◽  
B. Jankowski
Keyword(s):  
Carbon Monoxide ◽  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Michaelis Constant ◽  
Aldosterone Synthase ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
In Vitro Interaction ◽  
Zona Glomerulosa Cells

We have demonstrated that the aldosteronogenic pathway of the zona glomerulosa is unusually sensitive to modest changes in PO2 (Michaelis constant for O2 approximately 95 Torr). The current study evaluated the interaction of CO (the classic ligand for P-450 enzymes) and the decreases in O2 on aldosteronogenesis in vitro. Bovine adrenocortical zona glomerulosa cells were incubated for 2 h and stimulated with either adenosine 3′,5′-cyclic monophosphate (cAMP) or angiotensin II. Ten and 20% CO led to significant decreases in cAMP- and angiotensin II-stimulated aldosteronogenesis. The combination of 20% CO and moderate decreases in PO2 (from approximately 140 to approximately 100 Torr) led to an interactive decrease in aldosterone production. The conversion of corticosterone to aldosterone catalyzed by aldosterone synthase, which is the site of O2 sensitivity, was not significantly inhibited by CO. We conclude that the aldosterone pathway is not exceptionally sensitive to CO compared with other steroidogenic pathways. This observation suggests that the unique O2-sensitive properties of the aldosterone pathway located primarily within aldosterone synthase may not reside in its CO binding site (i.e., heme).

scholarly journals Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Il Young Kim ◽  
Min Young Lee ◽  
Mi Wha Park ◽  
Eun Young Seong ◽  
Dong Won Lee ◽  
...  
Keyword(s):  
Murine Model ◽  
Ureteral Obstruction ◽  
Renal Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Type I ◽  
Wild Type ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Hk2 Cells

We investigated the role of Akt1, one of the three isoforms of Akt, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). We subjected wild type and Akt1−/− mice to UUO. The Akt1 gene was silenced in vitro using short hairpin RNA delivered via a lentiviral vector in human proximal tubular cells (HK2 cells) and kidney fibroblasts (NRK-49F cells). The obstructive kidneys of Akt1−/− mice showed more severe tubulointerstitial fibrosis than those of wild type mice. The expression of fibronectin and type I collagen was significantly increased in obstructed kidneys of Akt1−/− mice compared to those of wild type mice. The important finding was that the expression of transforming growth factor β1 (TGFβ1) was significantly increased in the Akt1−/− mice compared to the wild type mice. The knockdown of Akt1 enhanced the expression of TGFβ1 in HK2 cells. Interestingly, the upregulation of TGFβ1 due to genetic knockdown of Akt1 was associated with activation of signal transducer and activator of transcript 3 (STAT3) independently of the Smad pathway in NRK-49F and HK2 cells. Immunohistochemical staining also showed that expression of phosphorylated STAT3 was more increased in Akt1−/− mice than in wild type mice after UUO. Additionally, the deletion of Akt1 led to apoptosis of the renal tubular cells in both in vivo and in vitro studies. Conclusively, these results suggest that the deletion of Akt1 may contribute to renal fibrosis via induction of the TGFβ1/STAT3 pathway in a murine model of UUO.

scholarly journals Vitamin D Potentiates the Inhibitory Effect of MicroRNA-130a in Hepatitis C Virus Replication Independent of Type I Interferon Signaling Pathway

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Xiaoqiong Duan ◽  
Yujuan Guan ◽  
Yujia Li ◽  
Shan Chen ◽  
Shilin Li ◽  
...  
Keyword(s):  
Vitamin D ◽  
Type I Interferon ◽  
Inhibitory Effect ◽  
Therapeutic Strategies ◽  
Innate Immune ◽  
Type I ◽  
Interferon Signaling ◽  
Host Innate Immune Response ◽  
Antiviral Mechanism

Calcitriol, the bioactive metabolite of vitamin D, was reported to inhibit HCV production in a synergistic fashion with interferon, a treatmentin vitro. Our previous study established that miR-130a inhibits HCV replication by restoring the host innate immune response. We aimed to determine whether there is additive inhibitory effect of calcitriol and miR-130a on HCV replication. Here we showed that calcitriol potentiates the anti-HCV effect of miR-130a in both Con1b replicon and J6/JFH1 culture systems. Intriguingly, this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/βproduction. All these findings may contribute to the development of novel anti-HCV therapeutic strategies although the antiviral mechanism needs to be further investigated.

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