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THE biosynthesis of estrogens appears to occur throughout the entire vertebrate phylum including mammals, birds, reptiles, amphibians, teleost and elasmobranch fish, and Agnatha (hagfish and lampreys) (1–3). It has also been described in the protochordate Amphioxus (4). To our knowledge, estrogen biosynthesis has not been reported in nonvertebrate animal phyla. In most vertebrate species that have been examined, aromatase expression occurs in the gonads and in the brain. This is true of the fish and avian species that have been examined as well as most mammals such as rodents. In many species estrogen biosynthesis in the brain has been implicated in sex-related behavior such as mating responses, and frequently a marked sexually dimorphic difference has been demonstrated. This is true, for example, in avian species in which the song of the male is important in courtship behavior (5). In the case of humans and a number of higher primates, there is a more extensive tissue distribution of estrogen biosynthesi...

The formation of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). In previous studies we have demonstrated that in adult human tissues and placenta, expression of the CYP19 gene is regulated in part by means of tissue-specific promoters through the use of alternative splicing mechanisms. In addition to placenta, a number of fetal tissues express aromatase, including liver, intestine, skin, and brain. To characterize the CYP19 transcripts present in these and other fetal tissues, we have used reverse transcription and polymerase chain reaction to amplify sequences corresponding to the various untranslated exons from RNA extracted from these tissues. In addition, we have prepared cDNA libraries using RNA from these tissues by the method of rapid amplification of cDNA ends. Sequencing of clones derived from these libraries has been employed to confirm the presence of sequence corresponding to untranslated exons at the 5'-ends of P450arom transcripts. Based on these findings, we conclude that in fetal tissues other than placenta, transcripts containing sequence found in the exon we have previously named I.4 appear to be the most common. Such sequences have been found in cells in which P450arom expression is stimulated by glucocorticoids. Thus, the presence of such transcripts in fetal liver RNA is consistent with our previous observations that aromatase activity in fetal hepatocytes is stimulated by glucocorticoids. Secondly, transcripts are present in the fetal adrenal, although no aromatase activity has ever been detected in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs—Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)—on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity. Principal Findings Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G0/G1 stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent. Conclusions CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

In humans, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory indicates that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus, the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start site. This promoter is not utilized in placenta; instead, the promoter used to drive aromatase expression in placenta is > or = 40 kb upstream from the translational start site. In addition, a minor promoter used in the expression of a small proportion of placental transcripts is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues, including placenta-related cells such as JEG-3 choriocarcinoma cells and hydatidiform moles and other fetal tissues such as fetal liver. In adipose tissue, on the other hand, expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

The conversion of androgens to estrogens is catalyzed by an enzyme complex known as aromatase, whose activity results in aromatization of the A ring of androgens, to form the phenolic A ring characteristic of estrogens with concomitant loss of the C19 angular methyl group. This enzyme complex is localized to the endoplasmic reticulum of cells in which it is expressed and consists of two components. The first is a form of cytochrome P450 known as aromatase cytochrome P450 (P450arom) (Mendelson et al. 1985; Chen et al. 1986; Kellis and Vickery 1987), the product of the CYP19 gene (Nebert et al. 1991). This heme protein is responsible for binding the C19 steroid substrate and catalyzing the concerted series of reactions leading to formation of the phenolic A ring. The second component is a flavoprotein, NADPH-cytochrome P450 reductase, which is a ubiquitous protein in the endoplasmic reticulum of most cell types and is responsible for transferring reducing equivalents from NADPH to any microsomal cytochrome P450 species with which it comes into contact. The aromatase reaction utilizes 3mol oxygen and 3mol NADPH for every mole of C19 steroid metabolized (Thompson and Siiteri 1974) (Fig. 1). There is general agreement that the first two oxygen molecules are utilized in the oxidation of the C19 angular methyl group. A growing consensus of opinion considers that the third oxygen attack is also on the C19 methyl group resulting in its loss as formic acid.

Ovarian cancer is the leading cause of death in women with gynecological malignancies and is often called the ‘silent killer’. Thus, new treatment targets are constantly being investigated. PPARγ (Peroxisome Proliferator Activated Receptor gamma) is a nuclear transcription factor that has been implicated in several diseases. However, little is known about PPARγ in ovarian cancer. Recently, PPARγ agonists such as the thiazolinediones (TZDs) have been studied for their potential use as a therapeutic treatment for cancer. In the present study, we investigated the effect of the four TZDs: Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio), on cell proliferation and PPARγ expression in an ovarian cancer cell line: Ovcar3. Treatment with 10 μM CGZ or TGZ for 24 hours decreased proliferation, whereas Rosi and Pio had no effect. We observed that this decrease in Ovcar3 cell proliferation is due to a higher fraction of cells in the G0/G1 stage of the cell cycle. In order to assess whether this was mediated by PPARγ, we investigated the effect of TGZ and CGZ on PPARγ mRNA, protein expression and activity. As Rosi and Pio had no effect on proliferation, only CGZ and TGZ were further investigated. CGZ and TGZ increased PPARγ mRNA expression in Ovcar3 cells (6.9 and 18.1 fold respectively). However, levels of PPARγ protein expression did not correlate with mRNA levels. Moreover, luciferase reporter assays showed that neither of the TZDs increased PPARγ activity. Ovcar3 cells were transfected with a PPARγ overexpression vector (WT) or a mutant dominant negative form of PPARγ (DN). Both CGZ and TGZ increased PPARγ activity when the receptor was over-expressed. Interestingly, the over-expression of WT in unstimulated cells caused an increase in reporter activity, indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. Cells exhibited no changes in proliferation when transfected with the WT or DN forms of PPARγ. Also, the effect on proliferation was not impacted when CGZ or TGZ were added to the WT or DN transfected cells. These findings demonstrate that PPARγ agonists, CGZ and TGZ, stimulate PPARγ expression. However, the decrease in cell proliferation and increased cell cycle arrest seems to be due to both PPARγ dependent and independent mechanisms. These data suggest that the non-PPAR targets of TZDs need to be further investigated. (Supported by NIH RO1 CA 95609-01, and NCRR-P20-RR15592). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1723.

Plasma-generated particulates are receiving increased attention as a source of contamination in plasma etching and deposition systems. These particles are suspended electrically in the plasma, and they are subject to electrical, thermophoretic, gravitational, and frictional drag forces from both plasma ions and neutrals. Consequently, they form complex spatial arrangements in the glow. We have investigated these particle arrangements by spatially mapping the particle distribution with a simple HeNe scattering system. We have also employed direct sampling as a way to determine particle size. The systems we have studied include copper and aluminum particles formed in an argon sputtering system, and carbon containing particles grown in a methane discharge. In addition we have modeled a particulate containing discharge in an effort to understand how the particulates act under the influence of electrical forces, ion drag, gravitational forces, and thermophoretic forces. The model is used as an aid in understanding the light scattering results.

Abstract #3055 Peroxisome proliferator-activated receptor gamma 1 (PPARγ1) is a member of the nuclear hormone receptor superfamily and is over expressed in breast cancer compared to human mammary epithelial cells (HMEC). Although synthetic ligands of PPARγ have been reported to mediate differentiation, inhibit proliferation, and induce apoptosis of cancer cells in culture it remains unclear whether these are mediated through PPARγ or by off target effects. Furthermore, the controversial nature of these studies and the disappointing results from clinical trials raise questions about the efficacy of these drugs in treating breast cancer. Recent work from our lab and others suggests that targeting the expression or endogenous transactivation of PPARγ represents a novel strategy in the treatment of breast cancer. We have previously demonstrated that the increase in PPARγ1 expression in seen in breast cancer cells is driven by a tumor-specific promoter. Furthermore, we have identified the myc-associated zinc finger protein (MAZ) as a critical mediator of PPARγ1 expression in these cells. In this study, we further demonstrated the significance of MAZ in the transcriptional regulation of PPARγ1 and confirmed that high expression of PPARγ1 in MCF-7 breast cancer cells is MAZ dependent. Additionally, using RNA interference (RNAi) techniques to knockdown PPARγ1 or MAZ or driving the expression of a dominant negative form of PPARγ1 in MCF-7 breast cancer cells, we demonstrated that blocking the expression of either protein resulted in reduced cellular proliferation and enhanced apoptosis. These findings suggest that in the absence of any exogenous ligand an increase in PPARγ1 signaling, driven at least in part by MAZ, contributes to an imbalance between proliferation and apoptosis, and may be an important mediator or of breast cancer progression. These results also highlight the need to understand the molecular mechanisms that drives tumor specific expression of PPARγ1 and development pure antagonist as a therapeutic treatment of this devastating disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3055.

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210–1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP 2 ) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.

Chemical composition of newborn larvae, muscle larvae and adult Trichinella spiralis. International Journal for Parasitology 16: 455–460. The chemical composition of newborn larvae (NBL), muscle larvae (ML) and 4-day-old and 7-day-old adult Trichinella spiralis were compared. Total protein constituted 44.1% of the dry wt of ML, 36.9% of NBL and 30.3% of 7-day-old adult worms. Glycogen content was 16.1% of worm dry wt in ML and 7.8% in NBL and 7.2% of worm dry wt in adults. Trehalose content was 5.2% in NBL, 8.3% in ML and 8.7% in 7-day-old adult worms. Significantly lower levels of trehalose and glycogen were found in 4-day-old worms than were present in 7-day-old worms. Free glucose amounted to 0.1% of dry wt in ML, 0.37% in NBL and 0.87% in 7-day-old adults. RNA accounted for 3.5% of the dry wt of ML, 3.2% of the dry wt of 7-day-old adults and 1.1% of the dry wt of NBL. The DNA content of NBL was 0.23% of worm dry wt, in ML 0.48% of worm dry wt and 0.51% of worm dry wt in 7-day-old adults. The three parasite stages examined agreed closely with regard to types of amino acids in the free pool. Exceptions were as follows: NBL lacked taurine which both adults and ML contained; adults lacked methionine which both ML and NBL contained; and, trace amounts of cysteine were present in ML but absent from the other two stages.

A relatively new noninvasive technique for the diagnosis of abnormalities affecting the central nervous system is presented. It is especially useful in patients with acoustic neuroma, brain stem disorder such as tumors of the posterior fossa with neuraxis impingement, as well as demyelinating disorders such as multiple sclerosis. It is certainly useful in the verification of neurologic normalcy in patients complaining of dizziness and vertigo.

Cloned cell lines have proven to be useful models in understanding the regulation of endocrine cells and steroid synthesis. In this study we report the isolation and characterization of a subclone of the MA-10 Leydig tumor cell line. Whereas there was no difference in basal steroid production between the clone (MA-10 LP) and the parent stock (MA-10), MA-10 LP produces very low levels of progesterone after stimulation by hCG or (Bu)2cAMP. In both cell populations, hCG stimulation resulted in the accumulation of comparable amounts of cAMP in the presence of a phosphodiesterase inhibitor, and similar levels of cAMP were measured at 30 min without inhibitor. Measurement of cholesterol side-chain cleavage activity using two separate methods demonstrated that the low steroid production in MA-10 LP could not be accounted for by a decrease in the activity of this enzyme complex. Additionally, no difference in 3 beta-hydroxysteroid dehydrogenase activity could be demonstrated between the two cell populations. Since the lesion that attenuates the ability of MA-10 to synthesize progesterone is somewhere after the production of cAMP and before cholesterol side-chain cleavage activity, this system may provide a useful model for understanding the regulatory mechanisms controlling steroid biosynthesis in Leydig cells.

Our findings in a relatively small series of cases seem to confirm a lack of statistically significant EEG changes when Carbidopa is combined with Levodopa in the therapy of patients with Parkinson's disease. There appears to be a slight increase in basic background frequency which was one of the earlier findings when Levodopa was first used clinically. From the literature surveyed there appears to be a definite lack of consistency in the effects of Levodopa therapy on the electroencephalogram and on the clinical status of the patients followed. We think this well may be explainable by the fact that no large study has been accomplished in which a neuroanatomical (pathological) correlation has been done with both the clinical and the EEG data. Neurological examination and the electroencephalogram are both clinical tools and have yet to be closely reviewed with the added parameter of neuro-pathologic investigation in this new day of therapy for Parkinson's disease.

The side-chain cleavage of cholesterol is the rate-limiting enzymic step in steroidogenesis and occurs in the mitochondria of steroid-producing tissues. In these studies, the effect of acute stimulation of MA-10 Leydig-tumour cells on the synthesis of mitochondrial proteins was investigated. Cells were incubated in the presence of stimulating levels of human choriogonadotropin (hCG) and [35S]methionine for 2 h periods. Mitochondria were isolated and their proteins analysed by two-dimensional polyacrylamide-gel electrophoresis and fluorography. At least three mitochondrial-specific proteins were found in cells exposed to gonadotropin, and these proteins were also found in cells treated with dibutyryl cyclic AMP (db cyclic AMP). The appearance of these proteins was prevented by the addition of cycloheximide to the cells, as was the production of progesterone, the major steroid produced in MA-10 cells. In addition, mitochondria isolated from cells stimulated with db cyclic AMP produced progesterone at a rate 3-fold greater than mitochondria isolated from control cells during 3 h of incubation. Lastly, mixing experiments demonstrated that sonicated mitochondria isolated from db cyclic AMP-treated cells stimulated progesterone production in control mitochondria 6-fold. These studies show that hCG and db cyclic AMP stimulation of MA-10 cells results in the rapid induction of cycloheximide-sensitive proteins located in the mitochondria which may be instrumental in the acute regulation of steroidogenesis.

The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.

SUMMARYAbsorption of glucose in vitro by enteric stages of Trichinella spiralis was examined in worms isolated at different times following infection. Glucose absorption by 6-h-old worms was similar to that seen during moulting (24 h post-infection (p.i.)). As embryogenesis began (48 h p.i.) glucose absorption in vitro by the parasite rose dramatically to a peak in 72-h-old worms. Absorption of glucose in vitro remained high in 96-h-old worms but as larviposition began (120 h p.i.) glucose uptake by T. spiralis dropped significantly. Glucose absorption by worms isolated from the host during the primary immune response was unaffected. Glucose absorption in vitro remained at similar levels in worms isolated between 120 and 312 h p.i. The percentage of absorbed label incorporated into parasite tissues was greatest in 48- and 312-h-old worms. Glucose uptake in vitro by pre-adult and adult worms changed with time and these changes accompanied specific events in the biology of the parasite.

Oxygen consumption, rectal temperature, and the level of activity in mice infected with Trichinella spiralis were significantly reduced below that seen in uninfected controls for periods of time during the first 30 days following infection. The differences in oxygen consumption between controls and infected animals were evident throughout the 24-hr period comprising day 7 postinfection. Both oxygen consumption and rectal temperature increased with decreasing level of infection. These changes in oxygen consumption, rectal temperature, and activity are discussed in terms of pathophysiologic and immunopathologic changes known to occur during the course of infection.

Background Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARγ has been identified as a potential target for breast cancer therapy based on the fact that its activation by synthetic ligands affects the differentiation, proliferation, and apoptosis of cancer cells. However, the controversial nature of current studies and disappointing results from clinical trials raise questions about the contribution of PPARγ signaling in breast cancer development in the absence of stimulation by exogenous ligands. Recent reports from both in vitro and in vivo studies are inconsistent and suggest that endogenous activation of PPARγ plays a much more complex role in initiation and progression of cancer than previously thought. Results We have previously demonstrated that an increase in expression of PPARγ1 in MCF-7 breast cancer cells is driven by a tumor-specific promoter. Myc-associated zinc finger protein (MAZ) was identified as a transcriptional mediator of PPARγ1 expression in these cells. In this study, using RNA interference (RNAi) to inhibit PPARγ1 expression directly or via down-regulation of MAZ, we report for the first time that a decrease in PPARγ1 expression results in reduced cellular proliferation in MCF-7 breast cancer cells. Furthermore, we demonstrate that these changes in proliferation are associated with a significant decrease in cell transition from G1 to the S phase. Using a dominant-negative mutant of PPARγ1, Δ462, we confirmed that PPARγ1 acts as a pro-survival factor and showed that this phenomenon is not limited to MCF-7 cells. Finally, we demonstrate that down-regulation of PPARγ1 expression leads to an induction of apoptosis in MCF-7 cells, confirmed by analyzing Bcl-2 expression and PARP-1 cleavage. Conclusion Thus, these findings suggest that an increase in PPARγ1 signaling observed in breast cancer contributes to an imbalance between proliferation and apoptosis, and may be an important hallmark of breast tumorigenesis. The results presented here also warrant further investigation regarding the use of PPARγ ligands in patients who are predisposed or already diagnosed with breast cancer.