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.

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.

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)

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.

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.

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.

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.