Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

Effects of anticonvulsants on human p450c17 (17alpha-hydroxylase/17,20 lyase) and 3beta-hydroxysteroid dehydrogenase type 2.

PURPOSE Women with epilepsy apparently have a higher incidence of polycystic ovary syndrome (PCOS) than do women without epilepsy. Whether the underlying disease or the antiepileptic drug (AED) treatment is responsible for this increased risk is unknown, although clinical reports implicate valproic acid (VPA) as a potential cause. The steroidogenic enzymes 3beta HSDII (3beta-hydroxysteroid dehydrogenase) and P450c17 (17alpha-hydroxylase/17,20 lyase) are essential for C19 steroid biosynthesis, which is enhanced during adrenarche and in PCOS. METHODS To determine whether the AEDs VPA, carbamazepine (CBZ), topiramate (TPM), or lamotrigine (LYG) directly affect the activities of human 3beta HSDII and P450c17, we added them to yeast expressing human P450c17 or 3beta HSDII and assayed enzymatic activities in the microsomal fraction. RESULTS Concentrations of VPA < or = 10 mM had no effect on activities of P450c17; however, VPA inhibited 3beta HSDII activity starting at 0.3 mM (reference serum unbound concentration, 0.035-0.1 mM) with an IC50 of 10.1 mM. CBZ, TPM, and LTG did not influence 3beta HSDII or P450c17 activities at typical reference serum unbound concentrations, but did inhibit 3beta HSDII and P450c17 at concentrations >10-fold higher. CONCLUSIONS None of the tested AEDs influenced 3beta HSDII or P450c17 activities at concentrations normally used in AED therapy. However, VPA started to inhibit 3beta HSDII activity at concentrations 3 times above the typical reference serum unbound concentration. Because inhibition of 3beta HSDII activity will shift steroidogenesis toward C19 steroid production when P450c17 activities are unchanged, very high doses of VPA may promote C19 steroid biosynthesis, thus resembling PCOS. CBZ, TPM, and LTG influenced 3beta HSDII and P450c17 only at toxic concentrations.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Clinical, genetic, and functional characterization of adrenocorticotropin receptor mutations using a novel receptor assay.

The ACTH receptor (MC2R) is expressed predominantly in the adrenal cortex, but is one of five G protein-coupled, seven-transmembrane melanocortin receptors (MCRs), all of which bind ACTH to some degree. Testing of MC2R activity is difficult because most cells express endogenous MCRs; hence, ACTH will elicit background activation of assayable reporter systems. Inactivating mutations of MC2R lead to hereditary unresponsiveness to ACTH, also known as familial glucocorticoid deficiency (FGD). These patients are usually seen in early childhood with very low cortisol concentrations, normal mineralocorticoids, hyperpigmentation, and increased bodily growth. Several MC2R mutations have been reported in FGD, but assays of the activities of these mutants are cumbersome. We saw two patients with typical clinical findings of FGD. Genetic analysis showed that patient 1 was homozygous for the mutation R137W, and patient 2 was a compound heterozygote for S74I and Y254C. We tested the activity of these mutations in OS-3 cells, which are unresponsive to ACTH but have intact downstream cAMP signal transduction. OS-3 cells transfected with a cAMP-responsive luciferase reporter plasmid (pCREluc) were unresponsive to ACTH, but cotransfection with a vector expressing human MC2R increased luciferase activity more than 40-fold. Addition of ACTH to cells cotransfected with the pCREluc reporter and wild-type MC2R activated luciferase expression with a 50% effective concentration of 5.5 x 10(-9) M ACTH, which is similar to previously reported values. By contrast, the MC2R mutant R137W had low activity, and the S74I or Y254C mutants elicited no measurable response. This assay provides excellent sensitivity in an easily assayed transient transfection system, providing a more rapid and efficient measurement of ACTH receptor activity.

Hispanic physicians' tobacco intervention practices: a cross-sectional survey study

Background U.S. Hispanic physicians constitute a considerable professional collective, and they may be most suited to attend to the health education needs of the growing U.S. Hispanic population. These educational needs include tobacco use prevention and smoking cessation. However, there is a lack of information on Hispanic physicians' tobacco intervention practices, their level of awareness and use of cessation protocols, and the type of programs that would best address their tobacco training needs. The purpose of this study was to assess the tobacco intervention practices and training needs of Hispanic physicians. Methods Data was collected through a validated survey instrument among a cross-sectional sample of self-reported Hispanic physicians. Data analyses included frequencies, descriptive statistics, and factorial analyses of variance. Results The response rate was 55.5%. The majority of respondents (73.3%) were middle-age males. Less than half of respondents routinely performed the most basic intervention: asking patients about smoking status (44.4%) and advising smoking patients to quit (42.2%). Twenty-five percent assisted smoking patients by talking to them about the health risks of smoking, providing education materials or referring them to cessation programs. Only 4.4% routinely arranged follow-up visits or phone calls for smoking patients. The majority of respondents (64.4%) indicated that they prescribe cessation treatments to less than 20% of smoking patients. A few (4.4%) routinely used behavioral change techniques or programs. A minority (15.6%) indicated that they routinely ask their patients about exposure to tobacco smoke, and 6.7% assisted patients exposed to secondhand smoke in understanding the health risks associated with environmental tobacco smoke (ETS). The most frequently encountered barriers preventing respondents from intervening with patients who smoke included: time, lack of training, lack of receptivity by patients, and lack of reimbursement by third party payers. There was no significant main effect of type of physician, nor was there an interaction effect (gender by type of physician), on tobacco-related practices. Conclusion The results indicate that Hispanic physicians, similarly to U.S. physicians in general, do not meet the level of intervention recommended by health care agencies. The results presented will assist in the development of tobacco training initiatives for Hispanic physicians.

Recruitment of Ascophyllum Nodosum: Wave Action as a Source of Mortality

The brown alga Ascophyllum nodosum is a dominant rocky intertidal organism throughout much of the North Atlantic Ocean, yet its inability to colonize exposed or denuded shores is well recognized. Our experimental data show that wave action is a major source of mortality to recently settled zygotes. Artificially recruited zygotes consistently exhibited a Type IV survivorship curve in the presence of moving water. As few as 10, but often only 1 relatively low energy wave removed 85 to 99% of recently settled zygotes. Increasing the setting time for attachment of zygotes (prior to disturbance from water movement) had a positive effect on survival. However, survival was significantly lower at high densities, and decreased at long (24 h) setting times, probably as a result of bacteria on the surface of zygotes. Spatial refuges provided significant protection from gentle water movement but relatively little protection from waves.

Petrology and diagenesis of sandstones at DSDP Hole 58-445

The sandstone succession in the lower 240 meters of DSDP Site 445, on the Daito Ridge, provided an opportunity to evaluate the effect of burial diagenesis of sandstones in a deep hole in a tectonic environment (remnant arc) characterized by a history of high heat flow. This report provides preliminary data concerning the petrology and diagenesis of these sandstones and records diagenetic changes which have occurred with increasing depth of burial. Methods used for this study included grain-size analysis (measured from thin sections using the method of Friedman, 1958), polarizing microscopy, X-ray diffraction, and scanning electron microscopy. A JEOL scanning electron microscope fitted with an energydispersive- X-ray detector was used for obtaining qualitative chemical data on certain minerals to aid in identification.

(Table 1) Compressional wave velocities for rocks from DSDP Hole 31-293 in the Philippine Sea

Compressional wave velocities measured in gabbroic rocks and metabasites recovered from Site 293 of Leg 31 in the Philippine Sea (on the Central Basin Fault) are correlative with seismic velocities determined for Layer 3. The lower crustal origin for these rocks suggested by this data is further supported by the similarity between these samples, dredge haul samples from fracture zones in the main ocean basins and rocks found in ophiolite complexes. These plutonic rocks were possibly introduced to the sea floor by movements along the Central Basin Fault, a major tectonic feature in the Philippine Sea, or formed as part of new ocean crust within a leaky transform fault.

Summary of shipboard physical properties of mudstones, carbonates, and clastic at DSDP Holes 72-515B and 72-516F

Compressional-wave velocity, wet-bulk density, and porosity were measured on sediments and rocks recovered from Deep Sea Drilling Project Holes 515B and 516F. Wet-bulk densities were measured by both gravimetric and GRAPE methods. Velocities were measured on trimmed samples with the Hamilton frame velocimeter. The shipboard measurement techniques are discussed in the explanatory notes chapter (Coulbourn, this volume) and are described in detail by Boyce (1976a). Only the shipboard measurements are reported here.

Anisotropy and acoustic properties at DSDP Holes 62-463 and 62-465A

Variations of acoustic properties within the sediment column may significantly affect the propagation of acoustic energy in the upper portion of the oceanic crust. Moreover, the acoustic properties of sediments reflect their mineral compositions, fabrics, and degrees of compaction and cementation. Hence, the physical properties of indurated deep-sea sediments are of considerable geophysical and geological interest. Chalks and limestones are particularly important because substantial accumulations of biogenic carbonates are generally present at the base of the deep-sea sediment column, and high-standing features such as Hess Rise are capped by calcareous deposits. This paper constitutes a preliminary report of the compressional-wave velocities and densities of 31 indurated calcareous sediment samples recovered at DSDP Sites 463 and 465, in the Mid-Pacific Mountains and on Hess Rise, respectively. The sample set includes nine pairs of samples in which velocities were measured parallel and perpendicular to bedding to determine the velocity anisotropy of the sediment. This research is part of an ongoing study of the seismic properties of indurated deep-sea carbonates.

Compressional and shear wave velocities and elastic constants at DSDP Leg 59 Holes

Prior to the Deep Sea Drilling Project the composition of the oceanic crust could only be inferred from seismic-refraction and gravity data and the recovery of a wide variety of dredged rocks. Through the success of the Deep Sea Drilling Project, it is now clear that the top of oceanic Layer 2 usually consists of basalt. Several laboratory studies (e.g., Fox et al., 1972; Christensen and Shaw, 1970; Hyndman and Drury, 1976) have demonstrated that the seismic velocities of oceanic basalt are similar to velocities reported from refraction studies of Layer 2 and that the variability in Layer 2 velocities has many causes, the most important being fracturing and sea-floor alteration produced by the interaction of basalt and sea water (Christensen and Salisbury, 1973). To date, most reported measurements of velocities in oceanic basalts are from samples obtained from the main ocean basins. With the exception of an earlier study of velocities and related elastic properties of a suite of rocks from DSDP Sites 292, 293, 294, and 296 located in the Philippine Sea (Christensen et al., 1975; Fountain et al., 1975), elastic properties have not been determined for oceanic rocks from marginal basins. In this chapter compressional- and shear-wave velocities and elastic constants are reported at elevated confining pressures for basalt and volcanic breccias from Holes 447A, 448, and 448A.

(Table 1) Bulk and grain density, porosity, and p- and s-wave velocity characteristics of ODP Hole 163-990A sediments

Seismic velocities in rocks are influenced by the properties of the solid, the pore fluid, and the pore space. Cracks dramatically affect seismic velocities in rocks; their influence on the effective elastic moduli of rocks depends on their shape and concentration. Thin cracks (or fractures) substantially lower the moduli of a rock relative to the effect of spherical voids (or vesicles), and lower moduli are reflected by lower P- and S-wave velocities. The objective of this research is to determine the types and concentrations of cracks and their influence on the seismic properties of subaerially erupted basalts drilled from Hole 990A on the Southeast Greenland margin during Ocean Drilling Program Leg 163. Ellipsoidal cracks are used to model the voids in the rocks. The elastic moduli of the solid (grains) are also free parameters in the inverse modeling procedure. The apparent grain moduli reflect a weighted average of the moduli of the constituent minerals (e.g., plagioclase, augite, and clay minerals). The results indicate that (1) there is a strong relationship between P-wave velocity and porosity, suggesting a similarity of pore shape distributions, (2) the distribution of crack types within the massive, central region of aa flows from Hole 990A is independent of total porosity, (3) thin cracks are the first to be effectively sealed by alteration products, and (4) grain densities (an alteration index) and apparent grain moduli of the basalt samples are directly related.