scholarly journals In vivo effects of adrenocorticotrophin on the expression of the hamster steroidogenic acute regulatory protein

1998 ◽  
Vol 21 (2) ◽  
pp. 131-139 ◽  
Author(s):  
A Fleury ◽  
L Ducharme ◽  
JG LeHoux
Keyword(s):  
Untranslated Region ◽  
Regulatory Protein ◽  
Northern Blotting ◽  
Polyadenylation Signal ◽  
Amino Acid Residues ◽  
Star Protein ◽  
Blotting Analysis ◽  
Steroidogenic Acute Regulatory ◽  
In Vivo Effects

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

scholarly journals In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*

Endocrinology ◽  
1997 ◽  
Vol 138 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Patricia Grasso ◽  
Matthew C. Leinung ◽  
Stacy P. Ingher ◽  
Daniel W. Lee
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Amino Acid ◽  
Food Intake ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Residues ◽  
Inactive Form ◽  
In Vivo Effects

Abstract In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

scholarly journals The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression

2004 ◽  
Vol 380 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Teruo SUGAWARA ◽  
Seiichiro FUJIMOTO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Expression Vector ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroid Production ◽  
Star Protein ◽  
Protein Levels ◽  
Steroid Sulphatase ◽  
Steroidogenic Acute Regulatory

The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3β-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription–translation reaction mixture. Pulse–chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly (P<0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production.

scholarly journals The Steroidogenic Acute Regulatory Protein Is Expressed in Steroidogenic Cells of the Day-Old Brain

Endocrinology ◽  
2004 ◽  
Vol 145 (10) ◽  
pp. 4775-4780 ◽  
Author(s):  
Steven R. King ◽  
Stephen D. Ginsberg ◽  
Tomohiro Ishii ◽  
Roy G. Smith ◽  
Keith L. Parker ◽  
...  
Keyword(s):  
De Novo ◽  
Metastatic Lymph Node ◽  
Regulatory Protein ◽  
Cell Types ◽  
Adult Brain ◽  
Peripheral Tissues ◽  
Cytochrome P450scc ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory ◽  
The Brain

Abstract Although recent research has focused on the fundamental role(s) of steroids synthesized de novo in the brain on development, the mechanism by which production of these neurosteroids is regulated remains unclear. Steroid production in peripheral tissues is acutely regulated by the steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroid biosynthesis: the intramitochondrial delivery of cholesterol to cytochrome P450scc for conversion to steroid. We recently demonstrated that StAR is present in discrete cell types in the adult brain, suggesting that neurosteroid production is mediated by StAR. Nevertheless, little is known regarding the presence of StAR in the developing brain. In the present study, the presence of StAR and for the first time, its homolog, the putative cholesterol transport protein metastatic lymph node 64 (MLN64), were defined in the neonatal mouse brain using immunocytochemical techniques. Both StAR and MLN64 were found to be present in the brain with staining patterns characteristic to each protein, indicating the authenticity of StAR and MLN64 immunoreactivity. Furthermore, we found MLN64 to be expressed in the adult brain as well, apparently at higher levels than StAR. Importantly, StAR protein is present in cells that also express P450scc. These data suggest that, as with the adult, neurosteroid production during development occurs through a StAR-mediated pathway.

scholarly journals The Acute and Chronic Effects of Adrenocorticotropin on the Levels of Messenger Ribonucleic Acid and Protein of Steroidogenic Enzymes in Rat Adrenal in Vivo*

Endocrinology ◽  
1998 ◽  
Vol 139 (9) ◽  
pp. 3913-3922 ◽  
Author(s):  
Jean-Guy Lehoux ◽  
Alain Fleury ◽  
Lyne Ducharme
Keyword(s):  
Cytochrome P450 ◽  
Western Blotting ◽  
Steroidogenic Enzymes ◽  
Chronic Stimulation ◽  
Western Blotting Analysis ◽  
Star Protein ◽  
Protein Levels ◽  
Blotting Analysis ◽  
Acth Treatment

Abstract The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220–370% and 300–350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5–1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11β-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3β-hydroxysteroid dehydrogenase (3βHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210–270%) and a decrease in the levels of 3βHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3βHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570–870%, respectively). The levels of 3βHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3βHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3βHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3βHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. Under chronic stimulation by ACTH, adrenals were hypertrophied, and the expression of many steroidogenic enzymes was modified, particularly the level of StAR protein was increased in the ZG and ZFR, confirming the importance of this protein in the control of steroidogenesis in a situation similar to that of Cushing’s syndrome.

scholarly journals Cloning and regulation of the rat activin betaE subunit

2000 ◽  
Vol 24 (3) ◽  
pp. 409-418 ◽  
Author(s):  
MK O'Bryan ◽  
KL Sebire ◽  
O Gerdprasert ◽  
MP Hedger ◽  
MT Hearn ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Alternative Polyadenylation ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Mrna Transcripts ◽  
Polymerase Chain

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

scholarly journals Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

2008 ◽  
Vol 22 (4) ◽  
pp. 951-964 ◽  
Author(s):  
Goro Sasaki ◽  
Tomohiro Ishii ◽  
Pancharatnam Jeyasuria ◽  
Youngah Jo ◽  
Assaf Bahat ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Adrenal Cortex ◽  
Regulatory Protein ◽  
Artificial Chromosome ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Steroidogenic Cells ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

scholarly journals Genetic Transformation and siRNA-Mediated Gene Silencing for Aphid Resistance in Tomato

Agronomy ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 893
Author(s):  
Mohammad Faisal ◽  
Eslam M. Abdel-Salam ◽  
Abdulrahman A. Alatar ◽  
Quaiser Saquib ◽  
Hend A. Alwathnani ◽  
...  
Keyword(s):  
Target Gene ◽  
Northern Blotting ◽  
Small Interfering Rnas ◽  
Aphid Infestation ◽  
Tomato Plants ◽  
Feeding Experiments ◽  
Transgenic Tomato Plants ◽  
Tomato Cultivars ◽  
In Vivo Effects

We explored the ability of RNA interference (RNAi) to silence the Acetylcholinesterase 1 (Ace 1) gene in aphid Myzus persicae and developed transgenic tomato plants resistant to aphid infestation. Three plasmid constructs, T-449: a single Ace 1 fragment (forward orientation), T-452: two Ace 1 fragments (reverse and forward orientations), and T455: a single inverted Ace 1 fragment, were developed and transformed into two tomato cultivars, Jamila and Tomaland. PCR, northern blotting, and small interfering RNAs (siRNA) analysis were performed to validate the success of Agrobacterium-mediated transformation. The efficiency of transformation was highest for the T-452 construct. In vivo effects of the transformed constructs were confirmed in feeding experiments, and there was significant downregulation of the Ace 1 gene. In addition, an aphid challenge assay was conducted to investigate the siRNA-mediated silencing of the target gene (Ace 1) in the inhibition of fecundity in M. persicae. We found that the plants that were transformed with the T-452 vector had 37.5% and 26.4% lower fecundity at 27 °C in the Jamila and Tomaland, respectively. Our results strongly indicated that the plant-mediated silencing of aphid-RNA might be a robust and effective approach for developing pest and disease resistant in plants.

Effects of daidzein on testosterone secretion in cultured immature mouse testis

2014 ◽  
Vol 8 (2) ◽  
pp. 247-253
Author(s):  
Liuping Zhang ◽  
Genbao Shao ◽  
Yaoqian Pan
Keyword(s):  
Direct Action ◽  
Regulatory Protein ◽  
Mouse Testis ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Testosterone Secretion ◽  
Immature Male ◽  
Testosterone Production ◽  
Steroidogenic Acute Regulatory

AbstractBackground: Daidzein is a major isoflavone in soybeans. Several in vivo studies have showed that daidzein can affect immature male testosterone production. However, whether daidzein has direct action on immature male testis is unknown.Objective: We investigated the effects of daidzein on testosterone secretion in 3-day-old and 21-day-old mouse Leydig cells with organotypic culture model.Materials and Methods: The testes were exposed to different concentrations (10-7to 10-4M) of daidzein for 72 h with medium changed every 24 h. From 72 to 75 h of culture, 100 ng/ml human chorionic gonadotropin (hCG) was added. The testosterone production was determined, and the related mechanisms of daidzein action were also evaluated by measuring the mRNA levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD-1) involved in testosterone biosynthesis.Results: The results revealed that in the presence of 100 ng/ml hCG, 10-7to 10-5M daidzein had no significant effect on testosterone secretion in cultured 3-day-old mouse testis. But 10-4M daidzein significantly increased testosterone concentration (p < 0.05). Daidzein in range of studied doses had no obvious influence on testosterone production in cultured 21-day-old mouse testis. RT-PCR results showed that 10-4M daidzein had obvious influence on the mRNA levels of StAR, P450scc and 3β-HSD-1 in cultured 3-day-old mouse testis (p < 0.05).Conclusion: These results suggest that daidzein mainly influences neonatal mouse testis function, and the influence is partially related to the upregulation of StAR, P450scc, and 3β-HSD-1 mRNA levels.

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