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Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Circadian proteins also regulate genes involved in either cell division or death; and a perturbation of the balance among these processes leads to cancer development and progression. A key aspect of cancer research is identifying new regulatory pathways involved in proliferation and differentiation of cell. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression, nor which, if any, of its components is either the ultimate effector that influences the fate of the cell. Initial experiments were devoted to identifying new interacting partners for Per2 using a two -hybrid system. Interestingly, among the positive clones analyzed was the oncogenic protein Mdm2. This result was validated by immunoprecipitation of recombinant and endogenous Per2/Mdm2 complexes from unstressed cells. Pull-down assays using tagged-expressed proteins fragments and labeled proteins were later used to map the interacting regions between Per2 and Mdm2. Our results show Mdm2 binds to the central flexible region of Per2 known to interact with various protein partners. Thus, we hypothesized that binding of Mdm2 to Per2 might act by mediating its ubiquitination and therefore altering Per2 stability. We next examined the formation of the Mdm2/p53/Per2 complex by immunoprecipitation. Our data show anti-p53 antibody is able to co-immunoprecipitate Per2 and Mdm2. Moreover, in vitro and in vivo ubiquitination assays show that binding of Per2 to p53 prevented ubiquitination of p53 by Mdm2 without altering their binding. Immunofluorescence studies using H1299 cells (p53-) confirmed Per2 role in p53 stabilization and for localization. Overall our results suggest that Mdm2 modulates the stability of Per2 and p53 in unstressed cells, and might be responsible for the oscillatory levels of these proteins observed in a 24 h cycle. [ABSTRACT FROM AUTHOR]

ABSTRACT Mutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N , E , and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance.Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.

Nitric oxide (NO) synthase (NOS) expression was analyzed in rat adrenal zona fasciculata. Both neuronal NOS and endothelial NOS mRNAs were detected by RT-PCR, immunohistochemistry, and immunoblot analysis. The biochemical characterization of adrenal zona fasciculata NOS enzymatic activity confirmed the presence of a constitutive isoform. In a cell line derived from mouse adrenal cortex, only endothelial NOS expression was detected by both RT-PCR and immunoblot analysis. Nitrate plus nitrite levels in Y1 cell incubation medium were increased in the presence of L-arginine and the calcium ionophore A23187, but not D-arginine, indicating enzymatic activity. Moreover, a low, but significant, conversion of Larginine to L-citrulline, abolished by the NOS inhibitor, N(G)-nitro-L-arginine, was detected in Y1 cells. The effect of L-arginine on pregnenolone production was examined. L-Arginine decreased both basal and ACTH-stimulated pregnenolone production in Y1 cells. The inhibitory effect of L-arginine could be attributed to endogenously generated NO, because it was blocked by N(G)-nitro-L-arginine, and it was mimicked by the addition of a NO donor, diethylenetriamine-NO. An inhibitory effect of NO on pregnenolone production from 22Rhydroxycholesterol and on steroidogenic acute regulatory protein expression was also determined. Taken together, these results suggest that at least part of the adrenal NO could derive from steroidogenic cells and modulate their function.

Apoptosis is controlled by a complex interplay between regulatory proteins. Previous work has shown that Xenopus embryos remove damaged cells by apoptosis when irradiated before, but not after, the midblastula transition (MBT). Here we demonstrate that Akt/protein kinase B is activated and mediates an antiapoptotic signal only in embryos irradiated after the MBT. In addition, an increase in xBcl-2/xBax oligomerization and a decrease in xBax homodimerization promote a protective effect against apoptosis only after the MBT. The post-MBT survival mechanism arrests cells in G(1) phase by increasing expression of the cyclin-dependent kinase inhibitor p27(Xic1). p27(Xic1) associates with cyclin D/Cdk4 and cyclin A/Cdk2 complexes to cause G(1)/S arrest, perhaps allowing more time for DNA repair. Taken together, the results define the DNA damage response as an element of the MBT and indicate that multiple mechanisms prevent apoptosis after the MBT.

Xenopus oocytes and embryos undergo two major maternally controlled cell-cycle transitions: oocyte maturation and the mid-blastula transition (MBT). During maturation, the essential order of events in the cell cycle is perturbed in that the M phases of Meiosis I and II occur consecutively without an intervening S phase. Use of U0126, a new potent inhibitor of MAPK kinase (MEK), shows that MAPK activation is essential to inhibit the anaphase-promoting complex and cyclin B degradation at the MI/MII transition. If MAPK is inactivated, cyclin B is degraded, S phase commences and meiotic spindles do not form. These events are restored in U0126-treated oocytes by a constitutively active form of the protein kinase p90Rsk. Thus all actions of MAPK during maturation are mediated solely by activation of p90Rsk. At the MBT, commencing with the 13th cleavage division, there are profound changes in the cell cycle. MBT events such as maternal cyclin E degradation and sensitivity to apoptosis are regulated by a developmental timer insensitive to inhibition of DNA, RNA or protein synthesis. Other events, such as zygotic transcription and the DNA replication checkpoint, are controlled by the nuclear:cytoplasmic ratio. Lengthening of the cell cycle at the MBT is caused by increased Tyr15 phosphorylation of Cdc2 resulting from degradation of the maternal phosphatase Cdc25A and continued expression of maternal Wee1. Ionizing radiation causes activation of a checkpoint mediating apoptosis when administered before but not after the MBT. Resistance to apoptosis is associated with increased p27Xic1, the relative fraction of Bcl-2 or Bax in pro- versus anti-apoptotic complexes, and the activity of the protein kinase Akt.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur J. Biochem. 224, 709-716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues.

We have reported the purification of a phosphoprotein (p43) intermediary in arachidonic acid release and steroid synthesis. Here we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein is homologous to a family of novel acyl-CoA thioesterases and identical to a peroxisome proliferator-inducible mitochondrial acyl-CoA thioesterase that shows highest substrate specificity for very-long-chain fatty acids such as arachidoyl- and palmitoyl-CoA. The deduced amino acid sequence of the protein has consensus sites for phosphorylation by different protein kinases, and a putative lipase serine motif. This motif is conserved in several species such as mouse, rat and human. Antibodies raised against a synthetic peptide that includes the lipase serine motif block the action of the protein. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect of ACTH was rapid (5 min), reached a maximum (62%) at 15 min and returned to basal levels at 30 min. The effect was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases, with specificity for very-long-chain acids, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues. Given the obligatory role of the protein in the activation of steroidogenesis through arachidonic acid release, we propose the name Arachidonic acid- Related Thioesterase Involved in Steroidogenesis (ARTISt) for p43.

Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.

We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.

It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confirming the role of mitochondrial proteins in intramitochondrial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 +/- 0.5 vs. 13.0 +/- 1.0 ng P4/tube for CHx-ACTH-treated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 +/- 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.

We have investigated the effect of the proteinase inhibitors 1,10-phenantroline (OP) and phenylmethylsulfonyl fluoride (PMSF) on steroidogenesis in rat adrenal cortex. Both PMSF and OP inhibited adrenocorticotropin (ACTH)- and 8-Br cAMP-induced stimulation of corticosterone synthesis. On the contrary, arachidonic acid-induced stimulation of corticosterone synthesis was only slightly inhibited by PMSF and unchanged by OP. Intra- and extracellular cAMP levels were determined by radioimmunoassay. While PMSF did not affect neither the intra- nor the extracellular cAMP levels, OP decreased the intra- and extracellular levels of unstimulated as well as ACTH-stimulated cells. The site of action of the proteinase inhibitors was also studied by recombination of mitochondria with the different subcellular fractions in vitro. Addition of PMSF abolished the stimulation achieved by in vitro activation of cytosol by cAMP and PKA. On the other hand, OP completely inhibited the activation of mitochondria. Our results provide evidence for the involvement of proteinases in ACTH-induced stimulation of steroidogenesis in adrenal cortex both prior to the release of arachidonic acid and at the level of cholesterol transport from the outer to the inner mitochondrial membrane.

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A2 inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDS/PAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearance of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.