Calibration of the microcalorimeter spectrometer on-board the Hitomi (Astro-H) observatory (invited).

The Hitomi Soft X-ray Spectrometer (SXS) was a pioneering non-dispersive imaging x-ray spectrometer with 5 eV FWHM energy resolution, consisting of an array of 36 silicon-thermistor microcalorimeters at the focus of a high-throughput soft x-ray telescope. The instrument enabled astrophysical plasma diagnostics in the 0.3-12 keV band. We introduce the SXS calibration strategy and corresponding ground calibration measurements that took place from 2012-2015, including both the characterization of the microcalorimeter array and measurements of the x-ray transmission of optical blocking filters.

Active galaxies. A fast and long-lived outflow from the supermassive black hole in NGC 5548.

Supermassive black holes in the nuclei of active galaxies expel large amounts of matter through powerful winds of ionized gas. The archetypal active galaxy NGC 5548 has been studied for decades, and high-resolution x-ray and ultraviolet (UV) observations have previously shown a persistent ionized outflow. An observing campaign in 2013 with six space observatories shows the nucleus to be obscured by a long-lasting, clumpy stream of ionized gas not seen before. It blocks 90% of the soft x-ray emission and causes simultaneous deep, broad UV absorption troughs. The outflow velocities of this gas are up to five times faster than those in the persistent outflow, and, at a distance of only a few light days from the nucleus, it may likely originate from the accretion disk.

Growth hormone binds to a single high affinity receptor site on mouse osteoblasts: modulation by retinoic acid and cell differentiation.

Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent Ka = 5.74 x 10(9) M-1) with 2200 sites per cell. Affinity crosslinking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4.2 and 1.2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1.45 +/- 1.0% vs 2.39 +/- 0.9%, P = 0.03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P = 0.02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Dexamethasone increases and serum decreases growth hormone receptor binding to UMR-106.01 rat osteosarcoma cells.

Dexamethasone (DEX) is known to exert major effects on functions of osteoblast-like cells. We investigated its action on the regulation of GH receptors in the osteoblast-like osteosarcoma cells UMR-106.01. DEX stimulated [125I]human GH (hGH) binding to UMR-106.01 cells. This effect was dose dependent and significant in a concentration range of 10(-8)-10(-6) M. The maximum effect was an increase of 42 +/- 1.4% (n = 3; mean +/- SE) above control, P < 0.01, at 10(-7) M DEX. Time dependence of this stimulation was observed, with a peak between the 12th and the 16th h of incubation, an effect being still detectable at 48 h. Cycloheximide decreased [125I]hGH binding and completely abolished the stimulating effect of DEX, suggesting that modulation of [125I]hGH binding by DEX is fully dependent on protein synthesis. Addition of fetal calf serum (FCS) resulted in a dose-dependent decrease of [125I]hGH binding to 24 +/- 2% of control (n = 3; mean +/- SE), P < 0.001, without interfering with the stimulatory effect of DEX, the ratio of DEX vs. control being higher with increasing FCS doses. Taken together, these results suggest the existence of different pathways for the regulation of GH receptor binding to UMR-106.01 cells, including a stimulatory one at the pretranslational level for DEX and an inhibitory one for (growth) factors present in FCS.

Influence of inhibition of insulin degradation on Scatchard analysis in H35 rat hepatoma cells.

Inhibition of insulin degradation by the use of chloroquine and bacitracin leads to a straight line in the Scatchard plot after correction for internalized insulin (r = 0.978 +/- 0.011). Analysis according to a model with two groups of heterogeneous binding sites showed a significant reduction in the total number of binding sites (p less than 0.01) in the chloroquine-bacitracin treated cells, with a disappearance of the low affinity binding sites and an increase in the assessment of the number of high affinity binding sites (p less than 0.002). The assessment of the high affinity association constant showed a decrease in the chloroquine-bacitracin treated cells (p less than 0.05). Analysis according to a model with a homogeneous group of binding sites with negative cooperativity also showed a decrease in the assessment of the number of binding sites (p less than 0.05) with a significant reduction in the Ke/Kf ratio from 4.5 +/- 1.5 to 1.16 +/- 0.22 (p less than 0.002). These results indicate that inhibition of insulin degradation leads to disappearance of negative cooperativity; the data are inconsistent with the two site model.

Demonstration of insulin induced enhancement of insulin dissociation from its receptor in H35 rat hepatoma cells in spite of a linear Scatchard plot.

The dissociation of insulin from its receptor has been reported to be enhanced in the presence of unlabeled insulin. This phenomenon has been used as a proof for the existence of negative cooperativity. However, when insulin degradation was inhibited by the use of a combination of bacitracin and chloroquine or prevented by a short incubation time in insulin binding studies in H35 rat hepatoma cells, linear Scatchard plots were obtained. This indicates that in the absence of insulin degradation negative cooperativity does not occur in these cells. Still the dissociation of insulin from its receptor was enhanced in the presence of 167 nM unlabeled insulin. This seriously questions the use of demonstration of insulin induced enhancement of insulin dissociation from its receptor as proof for the existence of negative cooperativity.

Effect of the insulin degradation inhibitors bacitracin and chloroquine on insulin binding data to H35 rat hepatoma cells.

The influence of correction for internalization and of inhibition degradation by the use of bacitracin or chloroquine on the insulin binding data of H35 rat hepatoma cells has been assessed. Internalized insulin represented about 50% of total cell bound insulin at tracer concentration and is not affected by bacitracin or chloroquine. The use of the insulin degradation inhibitors bacitracin and chloroquine resulted in Scatchard plots that were strikingly less curvilinear than control cells (as indicated by a decreased K1/K2 ratio (p less than 0.001) and Ke/Kf ratio (p less than 0.01)). In the two site model there was a significant increase in the number of high affinity binding sites concomitant with a significant decrease in the high affinity association constant; there was also a significant decrease in the number of the low affinity binding sites. The total number of binding sites did not change. For the negative cooperativity model the use of each drug resulted in a significant increase in the affinity constant of the filled site. These results indicate that insulin degradation induces negative cooperativity and that the results are inconsistent with the two site model.

A short incubation time in insulin binding experiments leads to disappearance of negative cooperativity in H35 hepatoma cells.

The effects of different periods of incubation (8 min vs 20 min) on insulin binding kinetics were examined in a H35 hepatoma cell line. Scatchard plots from cells incubated for 8 min were linear (r = 0.987 +/- 0.006), in contrast to curvilinear Scatchard plots from cells incubated for 20 min. Hill plots showed a slope of 1.006 +/- 0.024 for the 8 min incubation, whereas the slope was 0.827 +/- 0.0026 (p less than 0.0005) for the 20 min incubation. TCA precipitation of the medium showed minimal insulin degradation products at 8 min with a significant increase at 20 min (1.38 +/- 0.11% vs. 3.06 +/- 0.37%, p less than 0.0005). Internalized insulin was also significantly increased at 20 min as compared to 8 min incubation (48.9 +/- 5.6% vs. 32.4 +/- 3.0%, p less than 0.0005) These data indicate that after 8 min of incubation no appreciable cooperativity of insulin binding was present, while negative cooperativity was present after 20 min of incubation. As significantly more insulin degradation has taken place after prolonged incubation these data support the hypothesis that insulin degradation leads to negative cooperativity of insulin receptors.

Characterization of insulin-binding to the hepatoma cell line H35.

We have studied the characteristics of insulin-binding to its receptor in H35 hepatoma cells. Insulin-binding was time, temperature and pH dependent. Optimum pH was 8.0-8.2. Binding at 21 degrees C reached a steady state after 90 min of incubation at a level of 30.0 +/- 2.6% per mg protein. Pre-incubation of the cells with unlabelled exogenous insulin resulted in a decrease of insulin-binding which was time and concentration dependent. Pre-incubation with 10 micrograms/ml for 24 h resulted in a decrease to 35-40% of initial binding. Scatchard plots of the binding data were curvilinear in control as well as in down regulated cells. Analysis of the Scatchard plots revealed that decrease of insulin-binding to down regulated cells was due to a decrease of insulin-binding sites, while affinity constants did not change.

Sulphonylureas and biguanides do not affect insulin binding in H35 hepatoma cells.

Six sulphonylureas (tolbutamide, tolazamide, chlorpropamide, glibornuride, glipizide and gliquidone) and 2 biguanides (metformin and buformin) were tested for possible effects on insulin binding to H 35 rat hepatoma cells in culture. Insulin binding was measured after 24 and 72 hr of culturing cells in medium containing the drugs. Buformin and gliquidone were tested in concentrations from 10(-8)-5 X 10(-5) M, the other drugs in concentrations from 10(-7)-5 X 10(-4) M. All 24-hr experiments were repeated in cells down-regulated with 10 micrograms/ml insulin. None of the oral hypoglycemic agents tested had any significant influence on insulin binding to H 35 hepatoma cells, either in the presence or absence of insulin. We suggest that the insulin receptor status, at least in this type of liver cell, is not influenced by sulphonylureas or biguanides.

The effect of cyproterone acetate alone and in combination with ethinylestradiol on the hypothalamic pituitary adrenal axis, prolactin and growth hormone release in male-to-female transsexuals.

This study investigated the effects of long-term cyproterone-acetate (CA) treatment on the hypothalamus-pituitary-adrenal axis and further on the release of prolactin and growth hormone. Thirty-one male-to-female transsexuals, treated at least 3 months either with 100 mg CA alone or in combination with 50 micrograms ethinyloestradiol (EO)/day, were studied. In all of them the cortisol response to 250 micrograms Synacthen administration was comparable to that measured in controls. Six subjects treated with CA alone (group I) and six subjects treated with CA + EO (group II) underwent an insulin-hypoglycemia test. Seven eugonadal men served as controls. Compared with control values: in two subjects of group I and one subject of group II a low cortisol response was found. The mean growth hormone response was found to be lower in both groups, whereas the mean prolactin response was lower in group I.

Characteristics of insulin binding to H35 hepatoma cells.

Well differentiated hepatoma cells in culture exhibit insulin binding and insulin effects. We have studied insulin binding in control and in H35 hepatoma cells down-regulated with insulin. H35 cells were grown in monolayers in alpha MEM. Insulin binding was measured with A14 mono 125I labelled insulin 72 h after seeding. Binding was time, temperature and pH-dependent. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabelled insulin. Monolayers preincubated with 10 micrograms/ml unlabelled insulin for 24 h showed a decrease of 65% in the number of insulin binding sites. There was no change in affinity.

Effects of short- and long-term administration of tamoxifen on hCG-induced testicular steroidogenesis in man: no evidence for an oestradiol-induced steroidogenic lesion.

hCG-induced testicular desensitization is characterized by inhibition at the level of the C-17,20-lyase enzyme. This defect has been attributed to an early rise in oestradiol (E2) following hCG administration. To test this hypothesis the E2-receptor antagonist, tamoxifen, was employed. From in vitro studies the evidence suggests that tamoxifen depletes the E2-receptor within 24 h. In this in vivo study, short-term (36 h) administration of tamoxifen (to 6 eugonadal men) did not affect basal plasma levels of LH, FSH, 17 alpha-hydroxyprogesterone (17-OHP), testosterone (T) and E2, whereas long-term (3 months) tamoxifen with treatment of 6 normogonadotrophic oligozoospermic men increased LH and T levels, indicating a biological effect of tamoxifen. The response of 17-OHP, T, E2 and the 17-OHP/T ratio to hCG was similar in short-term and long-term tamoxifen-treated men as well as in 6 untreated eugonadal male controls. These results do not suggest a role for endogenous E2 in the hCG-induced testicular steroidogenic block.

Basal and LHRH-stimulated gonadotropin levels and the circadian rhythm of testosterone and the effect of exogenous testosterone thereon.

In seven eugonadal men, aged 20-26 years, a fall in plasma and saliva testosterone (T) levels between 8.00 and 16.00 h of the day was observed, but plasma oestradiol-17 beta levels did not show a significant variation. These findings substantiate the existence of a circadian rhythm in T levels. Concurrent with the decrease of T levels over the day, a small but significant rise in basal LH, but not in LHRH-stimulated LH levels were observed. Then the fall of plasma and saliva T levels over the day was prevented by the administration of 80 mg testosterone undecanoate (Andriol, Organon) by mouth at 8.00 h. A rise in plasma T and even more in saliva T levels was measured, which persisted till at least 16.00 h. At this hour basal LH, but not LHRH-stimulated LH levels appeared to be slightly, though significantly depressed. From our data we conclude that fluctuations of T levels of the magnitude of 25% around the baseline values, affect slightly basal LH levels, but not LHRH-stimulated LH levels.

Haemodialysis and testicular function.

Gonadal dysfunction in men with chronic renal failure is well established. However, the effect of haemodialysis on the function of the testis has not been studied in detail, so we have investigated the effect of haemodialysis by measuring the plasma levels of testosterone and dihydrotestosterone, SHBG binding capacity and salivary levels of testosterone. Plasma levels of testosterone, dihydrotestosterone and salivary testosterone levels were significantly lower in patients on haemodialysis than in controls. There was also a reduction in 5 alpha-reductase activity as evidenced by a reduced 5 alpha DHT/T ratio. SHBG levels were comparable to those found in eugonadal men. No differences were found in the parameters studied before and after 6 h haemodialysis. It is concluded that haemodialysis is not of obvious benefit to gonadal function.