scholarly journals Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 954-960 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Binding Affinity ◽  
Half Life ◽  
Chinese Hamster ◽  
Β Subunit ◽  
Wild Type ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

158 PRODUCTION OF RECOMBINANT LH FOR USE IN TIGER (TIGRIS ALTAICA) ASSISTED REPRODUCTION:A PRELIMINARY REPORT

2008 ◽  
Vol 20 (1) ◽  
pp. 159
Author(s):  
J. T. Aaltonen ◽  
E. Bedows ◽  
K. A. Estes ◽  
V. Y. Butnev ◽  
G. Bousfield ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Chinese Hamster ◽  
Granulosa Cell Tumor ◽  
Methionine Sulfoximine ◽  
Β Subunit ◽  
Wild Type ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Cell Assay ◽  
Cell Assays

Genetically, porcine LH is the closest commercially available gonadotropin analog to tiger LH (93% homologous); however, its use may lead to possible autoimmune reactions, lessening ovarian responses in stimulated tigresses over time (Crichton et al. 2005 Biol. Reprod. 68, 105–113). To overcome this problem for use in assisted reproduction, we produced recombinant tiger LH (tLH), and tested the bioactivity of several tLH constructs using heterologous (rat) and homologous (cat) Leydig cell assays. To clone tLH, mRNA was isolated from an Amur tiger pituitary by TRIzol extraction (Invitrogen, Carlsbad, CA). DNA was synthesized from the mRNA using reverse transcriptase (Stratagene, La Jolla, CA) and PCR was performed using tiger-specific primers for glycoprotein hormone α subunit or LH β subunit. The α subunit was cloned into the double-expression vector pIRES (Invitrogen). The tLH β subunit was cloned into the second site of pIRES and also into the plasmid pGS. Chinese hamster ovary (CHO) K1 and human granulosa cell tumor (COV 434) cells were transfected with plasmid DNA by calcium-phosphate precipitation: (1) pIRES containing α and pGS containing LH β, or (2) pIRES containing α and LH β. Cells were grown in selection media (250 µg mL–1 geneticine for pIRES, or 25 µm methionine sulfoximine for pGS). Media was collected and clarified at 1500g for 30 min. An immature rat Leydig cell assay protocol (Bousfield et al. 2001 Biol. Reprod. 64, 136–147) detected biological activity (testosterone production) by RIA. Of 14 tLH constructs created, 1 wild-type construct (LH WTCHO8) had LH activity 3 times greater than any other. A domestic kitten Leydig cell assay was performed in order to assess comparative sensitivities and specificities. Domestic kitten testicles, obtained from a local spay clinic, were disassociated with collagenase (225 U mg–1, Worthington Biochemical, Lakewood, NJ); however, the cells were more difficult to disperse than rat testicles, leading to low Leydig cell yields as determined histologically. Modification of the Leydig cell collagenase protocol for the cat was achieved by increasing the temperature and surface area, and agitating the minced tissue in medium on a stir plate. Samples of the 14 tLH constructs were run in parallel using rat and cat Leydig cell assays. Although rat Leydig cell testosterone concentrations (3.4 ng mL–1) were nearly 10-fold greater, the same trend for the different constructs was found in the cat Leydig cells with the same wild-type construct (LH WTCHO8, 0.37 ng mL–1) having greater LH activity than any other. The lower testosterone concentrations in the cat bioassay may be explained by insufficient Leydig cell numbers, age (sensitivity), damaging effect(s) of collagenase, or felid specificity. Still, these results lend validity to the use of the heterologous rat Leydig cell bioassay for recombinant tiger LH.

scholarly journals A Naturally Occurring Genetic Variant in the Human Chorionic Gonadotropin-β Gene 5 Is Assembly Inefficient*

Endocrinology ◽  
1999 ◽  
Vol 140 (8) ◽  
pp. 3496-3506 ◽  
Author(s):  
Amanda K. Miller-Lindholm ◽  
Elliott Bedows ◽  
Cynthia F. Bartels ◽  
Jacques Ramey ◽  
Victoria Maclin ◽  
...  
Keyword(s):  
Genetic Variant ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Polymorphism Analysis ◽  
Β Subunit ◽  
Wild Type ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Naturally Occurring ◽  
Single Stranded Conformational Polymorphism

Abstract The hCGβ gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGβ gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGβ gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGβ gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in β gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether βV79M hCG had biological properties that differed from those of wild-type hCG, a β-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone α-subunit in Chinese hamster ovary cells and 293T cells. When we examined βV79M hCG biosynthesis, we detected atypical βV79M hCG folding intermediates, including a βV79M conformational variant that resulted in aβ -subunit with impaired ability to assemble with the α-subunit. The inefficient assembly of βV79M hCG appeared to be independent ofβ -subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the βV79M subunit to fold correctly. The majority of the V79M β-subunit synthesized was secreted as unassembled free β. Although the amount of αβ hCG heterodimer formed and secreted by βV79M-producing cells was less than that by wild-type β-producing cells, the hCG that was secreted as αβ V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.

scholarly journals Human Thyrotropin (TSH) Variants Designed by Site-Directed Mutagenesis Block TSH Activity in Vitro and in Vivo

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2845-2850 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Zaki Kraiem ◽  
Fuad Fares
Keyword(s):  
Chorionic Gonadotropin ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Human Thyroid ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

Abstract TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific β-subunit and a common α-subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-β subunit as a linker [hTSHβ-carboxyl-terminal peptide (CTP)-α]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the α-subunit (hTSHβ-CTP-αdeg) and the other lacking also the oligosaccharides on the β-subunit (hTSHβdeg-CTP-αdeg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.

scholarly journals A Novel Follitropin Analog Inhibits Follitropin Activity In Vitro

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 325
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Fsh Receptor ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Reproductive Process ◽  
Α Subunit ◽  
Human Choriogonadotropin ◽  
Clinical Conditions

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique β specific subunit. Both α and FSHβ subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and β subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

scholarly journals Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the α-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1437-1446 ◽  
Author(s):  
Albina Jablonka-Shariff ◽  
T. Rajendra Kumar ◽  
Joshua Eklund ◽  
Anna Comstock ◽  
Irving Boime
Keyword(s):  
Posttranslational Modifications ◽  
Secretory Pathway ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Receptor Recognition ◽  
Transient Intermediates ◽  
Intersubunit Interactions

Abstract The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common α-subunit and a hormone-specific β-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the α-subunit sequences required for association with either β-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both β-subunits assembled with a single α-subunit. To address whether the α-subunit can interact with more than one β-subunit simultaneously, we genetically linked the FSHβ- and CGβ-subunit genes to the common α-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHβ-CGβ-α), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the α-subunit shuttles between the two β-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHβ- and CGβ-subunits and an α-subunit with cystine bond mutations (cys10–60 or cys32–84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the α- and both CGβ- and FSHβ-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the α-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the α/β-contacts are not a prerequisite for receptor recognition.

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 3167-3176 ◽  
Author(s):  
Magali Kitzmann ◽  
Marie Vandromme ◽  
Valerie Schaeffer ◽  
Gilles Carnac ◽  
Jean-Claude Labbé ◽  
...  
Keyword(s):  
Half Life ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Wild Type ◽  
E Box ◽  
Integral Role ◽  
Phosphopeptide Mapping

ABSTRACT We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

scholarly journals Source of the Fitness Defect in Rifamycin-ResistantMycobacterium tuberculosisRNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit

2018 ◽  
Vol 62 (6) ◽  
pp. e00164-18 ◽  
Author(s):  
Maxwell A. Stefan ◽  
Fatima S. Ugur ◽  
George A. Garcia
Keyword(s):  
Amino Acid Substitutions ◽  
Β Subunit ◽  
Wild Type ◽  
Rna Transcription ◽  
Content Type ◽  
Rna Hydrolysis ◽  
Rifampin Resistance ◽  
Compensatory Mutations

ABSTRACTMycobacterium tuberculosisis a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPrmutants with amino acid substitutions in the β subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β′ subunit of RNAP. To identify factors contributing to the fitness defects observedin vivo, severalin vitroRNA transcription assays were utilized to probe initiation, elongation, termination, and 3′-RNA hydrolysis with the wild-type and RMPrM. tuberculosisRNAPs. We found that the less prevalent RMPrmutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMPrmutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β′ subunit.

scholarly journals A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention

2013 ◽  
Vol 305 (12) ◽  
pp. F1697-F1704 ◽  
Author(s):  
Jacob Richards ◽  
Kit-Yan Cheng ◽  
Sean All ◽  
George Skopis ◽  
Lauren Jeffers ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Circadian Clock ◽  
Target Genes ◽  
Collecting Duct ◽  
Specific Rate ◽  
Wild Type ◽  
Α Subunit ◽  
Aldosterone Production

The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na+ transport genes in the collecting duct, including the α-subunit of the renal epithelial Na+ channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3β-dehydrogenase isomerase (3β-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na+ retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal Na+-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na+ compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na+ retention.

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