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Using collocated NASA Cloud Physics Lidar (CPL) and Research Scanning Polarimeter (RSP) data from the Studies of Emissions and Atmospheric Composition, Clouds and Climate Coupling by Regional Surveys (SEAC4RS) campaign, a new observational-based method was developed which uses aK-means clustering technique to classify ice crystal habit types into seven categories: column, plates, rosettes, spheroids, and three different type of irregulars. Intercompared with the collocated SPEC, Inc., Cloud Particle Imager (CPI) data, the frequency of the detected ice crystal habits from the proposed method presented in the study agrees within 5% with the CPI-reported values for columns, irregulars, rosettes, and spheroids, with more disagreement for plates. This study suggests that a detailed ice crystal habit retrieval could be applied to combined space-based lidar and polarimeter observations such asCALIPSOand POLDER in addition to future missions such as the Aerosols, Clouds, Convection, and Precipitation (A-CCP).

The translocator protein (TSPO; 18k Da) is an evolutionarily conserved outer mitochondrial membrane (OMM) protein highly expressed in steroid-synthesizing cells and found to possess a number of physiological and drug-binding partners. Extensive pharmacological, biochemical and cell biological research over the years has led to a model of TSPO involvement in mitochondrial cholesterol transport and promotion of steroid synthesis, a model guiding the design of drugs useful in stimulating neurosteroid synthesis and alleviating psychopathological symptoms. The involvement of TSPO in these processes has been called into question; however, with the publication of TSPO-deletion mouse models which saw no changes in steroid production. Here, we review work characterizing TSPO in steroidogenesis and offer perspective to research into TSPO pharmacology and its involvement in steroid biosynthesis.

This paper will report on the data collected by a Sigma Space Micropulse Lidar (MPL-111), and how these measurements, when integrated with other data, helps to inform our analysis of two cases of the Great Plains nocturnal Low-Level Jet (LLJ) in the vicinity of FP3.

Inhibition of GSK-3β has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3β resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3β inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3β inhibitors, in particular analogs 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube® behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, i.p.) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.

Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the “transduceosome,” in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.

A homology model of the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain of STARD1 was built, and the cholesterol binding site was identified. Structure-based design studies were performed to identify small molecule inhibitors of the START domain. The lead compounds were selected based on cAMP-induced, but not 22R-hydroxycholesterol-supported, inhibition of steroid synthesis by 50% at 10 μM. The results obtained by molecular docking & dynamics show a good correlation between bioactivity, docking scores and calculated binding energies of ligand–protein complexes. The best active compounds will be optimized further and used to develop potential drugs to control excessive steroid formation.

Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic cell-targeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo(fl/fl);Amhr2-Cre(/+)). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two- and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspo-floxed mice. The resulting Tspo(fl/fl);Nr5a1-Cre(/+) mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.

Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation.

Steroid hormones, bioactive oxysterols and bile acids are all derived from the biological metabolism of lipid cholesterol. The enzymatic pathways generating these compounds have been an area of intense research for almost a century, as cholesterol and its metabolites have substantial impacts on human health. Owing to its high degree of hydrophobicity and the chemical properties that it confers to biological membranes, the distribution of cholesterol in cells is tightly controlled, with subcellular organelles exhibiting highly divergent levels of cholesterol. The manners in which cells maintain such sterol distributions are of great interest in the study of steroid and bile acid synthesis, as limiting cholesterol substrate to the enzymatic pathways is the principal mechanism by which production of steroids and bile acids is regulated. The mechanisms by which cholesterol moves within cells, however, remain poorly understood. In this review, we examine the subcellular machinery involved in cholesterol metabolism to steroid hormones and bile acid, relating it to both lipid- and protein-based mechanisms facilitating intracellular and intraorganellar cholesterol movement and delivery to these pathways. In particular, we examine evidence for the involvement of specific protein domains involved in cholesterol binding, which impact cholesterol movement and metabolism in steroidogenesis and bile acid synthesis. A better understanding of the physical mechanisms by which these protein- and lipid-based systems function is of fundamental importance to understanding physiological homeostasis and its perturbation.

Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4′-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

Steroids metabolically derive from lipid cholesterol, and vertebrate steroids additionally derive from the steroid pregnenolone. Pregnenolone is derived from cholesterol by hydrolytic cleavage of the aliphatic tail by mitochondrial cytochrome P450 enzyme CYP11A1, located in the inner mitochondrial membrane. Delivery of cholesterol to CYP11A1 comprises the principal control step of steroidogenesis, and requires a series of proteins spanning the mitochondrial double membranes. A critical member of this cholesterol translocation machinery is the integral outer mitochondrial membrane translocator protein (18kDa, TSPO), a high-affinity drug- and cholesterol-binding protein. The cholesterol-binding site of TSPO consists of a phylogenetically conserved cholesterol recognition/interaction amino acid consensus (CRAC). Previous studies from our group identified 5-androsten-3β,17,19-triol (19-Atriol) as drug ligand for the TSPO CRAC motif inhibiting cholesterol binding to CRAC domain and steroidogenesis. To further understand 19-Atriol's mechanism of action as well as the molecular recognition by the TSPO CRAC motif, we undertook structure-activity relationship (SAR) analysis of the 19-Atriol molecule with a variety of substituted steroids oxygenated at positions around the steroid backbone. We found that in addition to steroids hydroxylated at carbon C19, hydroxylations at C4, C7, and C11 contributed to inhibition of cAMP-mediated steroidogenesis in a minimal steroidogenic cell model. However, only substituted steroids with C19 hydroxylations exhibited specificity to TSPO, its CRAC motif, and mitochondrial cholesterol transport, as the C4, C7, and C11 hydroxylated steroids inhibited the metabolic transformation of cholesterol by CYP11A1. We thus provide new insights into structure-activity relationships of steroids inhibiting mitochondrial cholesterol transport and steroidogenic cholesterol metabolic enzymes.

Previous studies in MA-10 tumor Leydig cells demonstrated that disruption of the mitochondrial electron-transport chain (ETC), membrane potential (ΔΨ(m)), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ΔΨ(m), and ATP synthesis cooperatively affected steroidogenesis. These results suggest significant differences between the two systems and call into question the extent to which results from tumor Leydig cells relate to primary cells. Thus, to further understand the similarities and differences between the two systems as well as the impact of ATP disruption on steroidogenesis, we performed comparative studies of MA-10 and primary Leydig cells under similar conditions of mitochondrial disruption. We show that mitochondrial ATP synthesis is critical for steroidogenesis in both primary and tumor Leydig cells. However, in striking contrast to primary cells, perturbation of ΔΨ(m) in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ΔΨ(m) powers the mitochondrial ATP synthase. Further studies revealed that a significant proportion of cellular ATP in MA-10 cells derives from glycolysis. In contrast, primary cells appear to be almost completely dependent on mitochondrial respiration for their energy provision. Inhibitor studies also suggested that the MA-10 ETC is impaired. This work underscores the importance of mitochondrial ATP for hormone-stimulated steroid production in both MA-10 and primary Leydig cells while indicating that caution must be exercised in extrapolating data from tumor cells to primary tissue.

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.

The principal site of regulation of steroid hormone biosynthesis is the transfer of cholesterol from the outer to inner mitochondrial membrane. Hormonal stimulation of steroidogenic cells promotes this mitochondrial lipid import through a multi-protein complex, termed the transduceosome, spanning the two membranes. The transduceosome complex is assembled from multiple proteins, such as the steroidogenic acute regulatory (STAR) protein and translocator protein (TSPO), and requires their targeting to the mitochondria for transduceosome function. The vast majority of mitochondrial proteins, including those participating in cholesterol import, are encoded in the nucleus. Their subsequent mitochondrial incorporation is performed through a series of protein import machineries located in the outer and inner mitochondrial membranes. Here we review our current knowledge of the mitochondrial cholesterol import machinery of the transduceosome. This is complemented with descriptions of mitochondrial protein import machineries and mechanisms by which these machineries assemble the transduceosome in steroidogenic mitochondria.

MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated (or =50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p0.05). Decreased MED1 transcript levels were observed in matched normal mucosa when compared to controls (median fold difference 8.0). Additional decreased expression was seen between mucosa and matched tumor (median fold decrease 4.4). Thus, MED1 promoter methylation and gene silencing occur in sporadic CRC patients and represent an early event in CRC tumorigenesis. Detection of MED1 methylation and gene suppression in normal colon mucosa may contribute to identifying patients at higher risk of developing CRC during screening procedures.