Accelerator Measurements of Magnetically Induced Radio Emission from Particle Cascades with Applications to Cosmic-Ray Air Showers.

For 50 years, cosmic-ray air showers have been detected by their radio emission. We present the first laboratory measurements that validate electrodynamics simulations used in air shower modeling. An experiment at SLAC provides a beam test of radio-frequency (rf) radiation from charged particle cascades in the presence of a magnetic field, a model system of a cosmic-ray air shower. This experiment provides a suite of controlled laboratory measurements to compare to particle-level simulations of rf emission, which are relied upon in ultrahigh-energy cosmic-ray air shower detection. We compare simulations to data for intensity, linearity with magnetic field, angular distribution, polarization, and spectral content. In particular, we confirm modern predictions that the magnetically induced emission in a dielectric forms a cone that peaks at the Cherenkov angle and show that the simulations reproduce the data within systematic uncertainties.

Taurine supplement in early life altered islet morphology, decreased insulitis and delayed the onset of diabetes in non-obese diabetic mice.

AIMS/HYPOTHESIS: We hypothesised that nutritional taurine, which is important for the development of the endocrine pancreas and reduces cytokine-induced apoptosis in pancreatic beta cells, would prevent or delay the onset of autoimmune diabetes, if given early in life to the non-obese diabetic (NOD) mouse. METHODS: Pregnant NOD mice received a diet supplemented with taurine throughout gestation or until weaning, and the pancreas of the offspring was examined using immunohistochemistry. This was done at postnatal day 14 and after 8 weeks (assessment of insulitis). The animals were also monitored until they became diabetic. RESULTS: At 14 days, pancreatic islet mass was significantly greater in animals treated with taurine than in controls. This finding was associated with a greater incidence of islet cell proliferation and a lower incidence of apoptosis. At age 8 weeks the number of islets manifesting insulitis was reduced by more than half, and the area of insulitis was reduced by 90%. Taurine treatment delayed the mean onset time of diabetes from 18 to 30 weeks in females, and from 30 to 38 weeks in males, while 20% of treated females remained free of diabetes after one year. CONCLUSIONS/INTERPRETATION: Taurine supplementation in early life altered islet development, reduced insulitis and delayed the onset of diabetes in NOD mice.

Expression of steroid-converting enzymes in osteoblasts derived from rat vertebrae.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

Insulin attenuates the stimulatory effects of tumor necrosis factor alpha on 11beta-hydroxysteroid dehydrogenase 1 in human adipose stromal cells.

Obesity is frequently associated with insulin-resistance and abnormal glucose homeostasis. Recent evidence indicates that TNFalpha may play a role in mediating the insulin-resistance of obesity through its overexpression in adipose tissue. Previously, we have shown that human adipose stromal cells contain 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) mRNA and activity. The present study was designed to examine the effects of insulin on 11beta-HSD1 expression in human adipose stromal cells under basal and TNFalpha-stimulated conditions. The cells were obtained from breast adipose tissue by collagenase digestion, and grown to confluence under replicating conditions in 10% fetal bovine serum. The cells were transferred to serum-free medium for 24 h prior to treatment with either TNFalpha, insulin or both for a further 24 h. The level of 11beta-HSD1 reductase activity was determined by measuring the conversion of [(3)H]-cortisone to [(3)H]-cortisol at a substrate concentration of 10 nM. Treatment with TNFalpha at concentrations of 0.1-10 ng/ml resulted in a dose dependent increase in 11beta-HSD1 reductase activity from 1.5 to 10-fold. Insulin (0.1-100 nM) had no effect under basal conditions, but inhibited the stimulatory effects of TNFalpha (5 ng/ml) on 11beta-HSD1 reductase activity in a dose dependent fashion (8-66%) inhibition). Northern blot analysis revealed corresponding changes in the level of 11beta-HSD1 mRNA, suggesting that the effects of TNFalpha and insulin on 11beta-HSD1 activity are mediated at the level of gene transcription. The interaction between insulin and TNFalpha suggests that local and systemic factors may act in a concerted fashion to modulate glucocorticoid activity in adipose and other peripheral tissues.

Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets.

In rats, a proportion of pancreatic beta-cells are deleted by apoptosis in the second week of postnatal life and replaced by endocrine cell neogenesis from pancreatic ductal epithelium. This coincides with a reduction in pancreatic insulin-like growth factor II (IGF-II) expression, and IGF-II has been shown to act as a beta-cell survival factor in vitro. To examine whether IGF-II regulates beta-cell apoptosis in vivo, an IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter, so that circulating IGF-II is retained postnatally. Mice were killed between postnatal days 7 and 26, and the pancreas was examined histologically. Apoptotic cells were visualized by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling method, and proliferating cells were examined by immunohistochemistry for proliferating cell nuclear antigen. In nontransgenic mice, serum IGF-II was absent by 26 days, but mean (+/-SEM) values were 45+/-9 ng/ml (n = 5) in transgenic animals. A 2- to 3-fold rise in islet cell apoptosis was seen in normal animals between days 11 and 16, but this was substantially decreased in IGF-II transgenic mice (day 11; control, 12+/-1%; transgenic, 6+/-1%; P < 0.01; n = 5). Consequently, islets from IGF-II transgenic mice had a significantly greater mean area from days 11-16, but the proportions of beta- and alpha-cells and circulating insulin levels were not changed. Islet cell DNA synthesis was increased in transgenic mice on days 13 and 16. The total islet number per section did not alter. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.

11 beta-Hydroxysteroid dehydrogenase 1 activity and gene expression in human adipose stromal cells: effect on aromatase activity.

The biological activity of glucocorticoids in target tissues can be influenced by locally produced 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme responsible for the interconversion of cortisol and its inactive metabolite cortisone. In human adipose stromal cells, glucocorticoids are potent stimulators of the conversion of androgens to estrogens (aromatase activity). The present study was designed to determine whether 11 beta-HSD activity was present in human adipose stromal cells, and if changes in the activity of this enzyme could influence aromatase activity. 11 beta-HSD activity was determined by a radiometric conversion assay in breast adipose tissue from six patients. It was found that both dehydrogenase (cortisol to cortisone) and reductase (cortisone to cortisol) activities were present in all six subjects, and the reductase activity was always predominant. Carbenoxolone (CBX), a potent inhibitor of 11 beta-HSD, added to the culture medium at 50 and 200 microM, resulted in 39 +/- 4% and 85 +/- 1% inhibition, respectively, of both reductase and dehydrogenase activity of 11 beta-HSD. To determine whether alterations in 11 beta-HSD could influence aromatase activity, the effect of CBX (200 microM) on cortisol- and cortisone-induced changes in the conversion of androstenedione to estrone was examined. CBX prevented the stimulatory effect of cortisone and minimally potentiated the stimulatory effect of cortisol on aromatase activity, reflecting an inhibition of the local activation of cortisone and the local metabolism of cortisol, respectively. In order to determine whether the product of the 11 beta-HSD 1 gene was responsible for the observed 11 beta-HSD activity, total RNA extracts from these cells were subjected to Northern blot analysis using human 11 beta-HSD 1 cDNA as the probe. A single 1.8 11 beta-HSD 1 transcript was detected, and its abundance was reduced by CBX. No 11 beta-HSD 2 mRNA was detected. The present results demonstrate that the 11 beta-HSD 1 gene is expressed and functional in human breast adipose stromal cells and that changes in 11 beta-HSD 1 activity result in alterations in aromatase activity.

Growth and differentiation of human adipose stromal cells in culture.

Human adipose stromal cells provide an excellent model for studying a variety of metabolic processes in an in vitro system These are normal cells derived from subcutaneous or omental adipose tissue. Under specific culture condition, they will differentiate without replication into cells resembling mature adipocytes or will replicate, become confluent, and grow in subculture. The initial observation that the stromal vascular fraction of human omental adipose tissue contained a fibroblast-like cell that was a possible adipocyte precursor was made by Poznanski et al (1). Subsequent studies demonstrated enzymological and morphological properties that developed during differentiation (2-4). The primary interest has focused on the conditions required to stimulate these cells to differentiate into adipocytes, which can accumulate lipid and possess the enzymes involved in lipogenesis, including glycerol-3-phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL).

Estrone formation from dehydroepiandrosterone in cultured human breast adipose stromal cells.

The metabolism of dehydroepiandrosterone (DHA) and androstenedione (A-dione) was studied in cultured human adipose stromal cells obtained from breast tissue of six premenopausal patients undergoing reduction mammoplasty. Cells were maintained in culture in the presence of 10% fetal bovine serum. Studies were carried out during the proliferative and confluent phases of culture with radiolabelled substrates (2 microCi, 10 nM). During the early phases of replication 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA) was formed from DHA. As the cells reached confluence, the major metabolite of DHA in cells from all patients was A-dione indicating the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD). The conversion of DHA to A-dione was variable among patients when cells were confluent with 30-80% of substrate being metabolized to this product. Adipose stromal cells synthesized estrone (E1) from DHA once A-dione formation was established. Under basal conditions E1 was obtained in cells from three of the six patients examined with up to 36% substrate converted to this product. Dexamethasone (Dex 10(-7) M) stimulated E1 formation in cells from all subjects with up to 50% of substrate being converted. Parallel studies comparing the conversion of DHA with A-dione to E1 revealed that as the cells became confluent, E1 formation from both substrates was similar. The pattern of steroid metabolism was also examined in primary culture and in subculture. Passage 1 cells continued to form A-dione as a major metabolite of DHA, and did not revert to the pattern of metabolism found in primary cells during the early stages of replication, when 7 alpha-hydroxylation predominated. Human adipose stromal cells actively metabolize DHA, producing 7 alpha-OHDHA, A-dione and E1 as principal metabolites. Changes in the circulating levels of DHA may directly influence the formation of E1 in peripheral tissues. This source of E1 will be modulated by factors controlling 3 beta-HSD and aromatase activities.

Effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-hydroxydehydroepiandrosterone by human adipose stromal cells.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with < 30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Metabolism of dehydroepiandrosterone by cultured human adipose stromal cells: identification of 7 alpha-hydroxydehydroepiandrosterone as a major metabolite using high performance liquid chromatography and mass spectrometry.

Studies of the metabolism of dehydroepiandrosterone (DHA) by cultured human adipose stromal cells revealed that the most abundant metabolite detected by HPLC was a polar compound accounting for up to 45% of total radioactivity. This metabolite was isolated by chromatography on Lipidex 5000 from the culture medium of breast adipose stromal cells cultured with unlabelled DHA (5 microM) and identified by combined capillary gas chromatography and mass spectrometry as 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA). In breast adipose stromal cells, the conversion of DHA to 7 alpha-OHDHA was linear from a substrate concentration of 10 nM to 1 microM. At 1 microM substrate concentration, the formation of 7 alpha-OHDHA in four patients ranged from 6.1 to 22.5 ng/10(5) cells/24 h. Incubations carried out in primary culture and up to the fifth subculture revealed continued formation of 7 alpha-OHDHA. Adipose stromal cells from abdomen, flank and perinephric fat also produced 7 alpha-OHDHA from DHA. These studies have shown that 7 alpha-OHDHA is a major metabolite of DHA in human adipose stromal cells. The variability from patient to patient and the magnitude of this conversion suggests that this pathway may play an important role in the peripheral metabolism of DHA.

Genetic activity of X chromosomes in pluripotent female teratocarcinoma cells and their differentiated progeny.

We have induced teratocarcinomas from female embryos heterozygous for electrophoretic variants of the X-linked gene coding for phosphoglycerate kinase (PGK). An embryonal carcinoma cell line, P10, has been isolated from such a teratocarcinoma. It has a normal female karyotype and cultures contain both PGK isoenzymic forms. Clonal populations derived from P10 also contain both PGK electrophoretic variants. In addition, both X chromosomes in these cells replicate in synchrony with the autosomes during early S phase of the cell cycle. These data indicate that the undifferentiated P10 embryonal carcinoma cells contain two active X chromosomes. When cultured under the appropriate conditions, the P10 cells differentiate to form a variety of tissue types. At least some of these differentiated cells contain an inactive X chromosome as determined by cytogenetic analysis. Apparently X chromosome inactivation accompanies the differentiation of these female embryonal carcinoma cells.