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Supplementary Figure 5 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Supplementary Figure Legends 1-5 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Supplementary Figure 1 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Supplementary Figure 4 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Iridium complex is a promising organic light–emitting diode material for next generation video displays that emits phosphorescence quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light–emitting Ir(btp)2(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor-1α images. BTP was further applied to imaging five nude mouse transplanted with tumors. All tumors presented a bright BTP-emitting image even 5 minutes after injection. The minimal image recognition size was ∼2 mm in diameter. By morphologic examination and phosphorescence lifetime measurement, BTP appeared to localize to the tumor cells. Because BTP is easily modifiable, we synthesized BTP analogues with a longer excitation/emission wavelength. One of them, BTPHSA, depicted clear imaging from tumors transplanted 6 to 7 mm deep from the skin surface. We suggest that iridium complex materials have a vast potential for imaging hypoxic lesions such as tumor tissues. Cancer Res; 70(11); 4490–8. ©2010 AACR.

Supplementary Figure 3 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Supplementary Table 1 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Supplementary Figure 2 from Phosphorescent Light–Emitting Iridium Complexes Serve as a Hypoxia-Sensing Probe for Tumor Imaging in Living Animals

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping ( SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system

Secretogranin II (SgII) and III (SgIII) function within peptide hormone-producing cells and are involved in secretory granule formation. However, their function in active amine-producing cells is not fully understood. In this study, we analyzed the expression profiles of SgII and SgIII in canine adrenal medulla and pheochromocytomas by immunohistochemical staining. In normal adrenal tissues, the intensity of coexpression of these two secretogranins (Sgs) differed from each chromaffin cell, although a complete match was not observed. The coexpression of vesicular monoamine transporter 2 (VMAT2) with SgIII was similar to that with chromogranin A, but there was a subpopulation of VMAT2-expressing cells that were negative or hardly detectable for SgII. These results are the first to indicate that there are distinct expression patterns for SgII and SgIII in adrenal chromaffin cells. Furthermore, the expression of these two Sgs varied in intensity among pheochromocytomas and did not necessarily correlate with clinical plasma catecholamine levels in patients. However, compared with SgIII, the expression of SgII was shown to be strong at the single-cell level in some tumor tissues. These findings provide a fundamental understanding of the expression differences between SgII and SgIII in normal adrenal chromaffin cells and pheochromocytomas.

To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2. To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2. Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse β-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system

The future time of emergence when precipitation changes due to anthropogenic influences begins to continuously exceed the previous maximum value is defined as the 'tipping year' Historical experiments and future experiments simulated by state-of-the-art climate models were utilized. A total of 510,000 time series from year 1856 to 2095 were generated by sampling the natural internal variability in precipitation. The time evolutions of internal variability in the whole time period were estimated from the combination of past and future experiments with preindustrial control experiments. A large ensemble size enabled an estimation of the probability density function of the tipping year at each grid point, providing precise information on the uncertainty of the projection. The tipping year of average precipitation emerges earlier in high latitudes than in lower latitudes. In some regions in lower latitudes and mid-latitudes, the tipping year of intense precipitation emerges faster than that of average precipitation. The tipping years of average and intense precipitation are earlier for higher anthropogenic forcing scenarios than for lower scenarios. The global average of the tipping year for intense precipitation might be attributed to the enhancement of the thermodynamic effect (moisture) rather than the dynamic effect (vertical motion).

The future time of emergence when precipitation changes due to anthropogenic influences begins to continuously exceed the previous maximum value is defined as the ‘tipping year’ Historical experiments and future experiments simulated by state-of-the-art climate models were utilized. A total of 510,000 time series from year 1856 to 2095 were generated by sampling the natural internal variability in precipitation. The time evolutions of internal variability in the whole time period were estimated from the combination of past and future experiments with preindustrial control experiments. A large ensemble size enabled an estimation of the probability density function of the tipping year at each grid point, providing precise information on the uncertainty of the projection. The tipping year of average precipitation emerges earlier in high latitudes than in lower latitudes. In some regions in lower latitudes and mid-latitudes, the tipping year of intense precipitation emerges faster than that of average precipitation. The tipping years of average and intense precipitation are earlier for higher anthropogenic forcing scenarios than for lower scenarios. The global average of the tipping year for intense precipitation might be attributed to the enhancement of the thermodynamic effect (moisture) rather than the dynamic effect (vertical motion).

During spring 2017, we conducted research expeditions to the SIGMA-A site, which is located on the northwestern Greenland Ice Sheet. We maintained an automated weather station (AWS) to enable continuous meteorological observations. We extended 1.5-m long poles of the AWS and replaced two aerovane sensors, two thermo-hydrometers and an ultrasonic snow gauge. We also drilled an ice core and recovered a core with a total length of 60.06 m, conducted stratigraphic observations, and measured the density of the ice core. In addition, we conducted snow-pit observations and snow sampling, measured the specific surface area of snow using near-infrared reflectance, performed sunphotometry observations, and measured the spectral albedo. To schedule research activities in the field camp and helicopter pick-up flights, we received weather forecasts from the Meteorological Research Institute of Japan through the Internet using a satellite phone every day. We took a male dog to the field camp to alert us to approaching animals.

Oxygen imaging of biological cells and tissues is becoming increasingly important in cell biology and in the pathophysiology of various hypoxia-related diseases. The optical oxygen-sensing method using luminescent probes provides very useful, high spatial resolution information regarding oxygen distribution in living cells and tissues. This review focuses on recent advances in biological oxygen measurements based on the phosphorescence quenching of probe molecules by oxygen, and on hypoxia-sensitive fluorescent probes. Special attention is devoted to metal complex probes, Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes. Current knowledge regarding the mechanism of phosphorescence quenching of metal complexes by oxygen is described in relation to the oxygen sensitivity of the probes, and recent advances in optical oxygen probes and detection techniques for intracellular and tissue oxygen measurements are reviewed, emphasizing the usefulness of chemical modifications for improving probe properties. Tissue oxygen imaging and hypoxic tumor imaging using these metal complex probes demonstrate the vast potential of optical oxygen-sensing methods using luminescent probes.

Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells.

The Ninth Symposium on Polar Science/Ordinary sessions: [OM] Polar meteorology and glaciology, Wed. 5 Dec. / Entrance Hall (1st floor), National Institute of Polar Research

The Ninth Symposium on Polar Science/Ordinary sessions: [OM] Polar meteorology and glaciology, Wed. 5 Dec. / Entrance Hall (1st floor), National Institute of Polar Research

To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although

Purpose: The main purpose in this study was to present the results of beam modeling and how the authors systematically investigated the influence of double and triple Gaussian proton kernel models on the accuracy of dose calculations for spot scanning technique. Methods: The accuracy of calculations was important for treatment planning software (TPS) because the energy, spot position, and absolute dose had to be determined by TPS for the spot scanning technique. The dose distribution was calculated by convolving in-air fluence with the dose kernel. The dose kernel was the in-water 3D dose distribution of an infinitesimal pencil beam and consisted of an integral depth dose (IDD) and a lateral distribution. Accurate modeling of the low-dose region was important for spot scanning technique because the dose distribution was formed by cumulating hundreds or thousands of delivered beams. The authors employed a double Gaussian function as the in-air fluence model of an individual beam. Double and triple Gaussian kernel models were also prepared for comparison. The parameters of the kernel lateral model were derived by fitting a simulated in-water lateral dose profile induced by an infinitesimal proton beam, whose emittance was zero, at various depths using Monte Carlo (MC) simulation. The fitted parameters were interpolated as a function of depth in water and stored as a separate look-up table. These stored parameters for each energy and depth in water were acquired from the look-up table when incorporating them into the TPS. The modeling process for the in-air fluence and IDD was based on the method proposed in the literature. These were derived using MC simulation and measured data. The authors compared the measured and calculated absolute doses at the center of the spread-out Bragg peak (SOBP) under various volumetric irradiation conditions to systematically investigate the influence of the two types of kernel models on the dose calculations. Results: The authors investigated the difference between double and triple Gaussian kernel models. The authors found that the difference between the two studied kernel models appeared at mid-depths and the accuracy of predicting the double Gaussian model deteriorated at the low-dose bump that appeared at mid-depths. When the authors employed the double Gaussian kernel model, the accuracy of calculations for the absolute dose at the center of the SOBP varied with irradiation conditions and the maximum difference was 3.4%. In contrast, the results obtained from calculations with the triple Gaussian kernel model indicated good agreement with the measurements within ±1.1%, regardless of the irradiation conditions. Conclusions: The difference between the results obtained with the two types of studied kernel models was distinct in the high energy region. The accuracy of calculations with the double Gaussian kernel model varied with the field size and SOBP width because the accuracy of prediction with the double Gaussian model was insufficient at the low-dose bump. The evaluation was only qualitative under limited volumetric irradiation conditions. Further accumulation of measured data would be needed to quantitatively comprehend what influence the double and triple Gaussian kernel models had on the accuracy of dose calculations.

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

Future changes in precipitation intensity over the Arctic were calculated based on three-member ensemble simulations using a global atmospheric model with a high horizontal resolution (60-km grid) for the period 1872–2099 (228 years). During 1872–2005, the model was forced with observed historical sea surface temperature (SST) data, while during 2006–2099, boundary SST data were estimated using the multi-model ensemble (MME) of the Coupled Model Intercomparison Project, Phase 3 (CMIP3) model, assuming the A1B emission scenario. The annual mean precipitation (PAVE), the simple daily precipitation intensity index (SDII), and the maximum 5-day precipitation total (R5d) averaged over the Arctic increased monotonically towards the end of the 21st century. Over the Arctic, the conversion rate from water vapor to precipitation per one degree temperature increase is larger for PAVE than for R5d, which is opposite to the tropics and mid-latitudes. The increases in PAVE, SDII, and R5d can be partly attributed to an increase in water vapor associated with increasing temperatures, and to an increase in the horizontal transport of water vapor from low to high latitudes associated with transient eddies.

As part of the terrestrial branch of the Japan-funded Arctic Climate Change Research Project (GRENE-TEA), which aims to clarify the role and function of the Arctic terrestrial system in the climate system, and assess the influence of its changes on a global scale, this model intercomparison project (GTMIP) is planned and being conducted to (1) enhance communication and understanding between the "minds and hands" (i.e., between the modelling and field scientists) and (2) assess the uncertainty and variations stemming from variability in model implementation/design and in model outputs due to climatic and historical conditions in the Arctic terrestrial regions. This paper provides an overview and the experiment protocol of Stage 1 of the project, site simulations driven by statistically fitted data created using the GRENE-TEA site observations for the last three decades. The target metrics for the model evaluation cover key processes in both physics and biogeochemistry, including energy budgets, snow, permafrost, phenology, and carbon budgets. The preliminary results on four metrics (annual mean latent heat flux, annual maximum snow depth, gross primary production, and net ecosystem production) already demonstrate the range of variations in reproducibility among existing models and sites. Full analysis on annual as well as seasonal time scales, to be conducted upon completion of model outputs submission, will delineate inter-dependence among the key processes, and provide the clue for improving the model performance.

A cationic iridium(III) complex (btp)2Ir(phen-NH2) (btp = benzothienylpyridine; phen-NH2 = 5-amino-1,10-phenanthroline) and its analog with extended π-electronic structures (btq)2Ir(phen-NH2) (btq = benzothienylquinoline), (ttph)2Ir(phen-NH2) (ttph = thienothiophenylphenanthridine), (btph)2Ir(phen-NH2) (btph = benzothienylphenanthridine) have been designed and synthesized to develop new oxygen probes for living cells. The photophysical and cellular properties of these complexes were systematically investigated by using (btp)2Ir(acac) and its derivatives as reference compounds. The extension of the π-electronic systems of ligands resulted in remarkable red shifts in the absorption and phosphorescence spectra; the phosphorescence bands of (ttph)2Ir(phen-NH2) and (btph)2Ir(phen-NH2) appeared in the near-infrared region, maintaining the relatively high phosphorescence quantum yields ( Φ p 0 = 0.14–0.24). Dimethylation and diethylation of the amino group of the phen-NH2 ligand in (btp)2Ir(phen-NH2) and its analog significantly enhanced the cellular uptake efficiency as compared with their neutral analog with an acetylacetonate (acac) ligand. The cationic iridium(III) complex (btp)2Ir(phen-NH2) and its analog internalized into living HeLa cells showed selective distribution into mitochondria. Owing to the high cellular uptake, relatively high cytotoxicity was found for the cationic iridium complexes. The phosphorescence intensity of newly developed iridium complexes exhibited oxygen response in HeLa cells, demonstrating that these complexes have potential as mitochondria-selective oxygen sensors for biological cells and tissues.

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin–IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment–induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

author, In contrast to the widely accepted images of the Golgi apparatus as a cup-like shape, the Golgi in pituitary gonadotropes is organized as a spherical shape in which the outer and inner faces are cis- and trans-Golgi elements, respectively. At the center of the spherical Golgi, a pair of centrioles is situated as a microtubule-organizing center from which radiating microtubules isotropically extend toward the cell periphery. This review focuses on the significance of the characteristic organization of the Golgi and microtubule network in gonadotropes, considering the roles of microtubule-dependent membrane transport in the formation and maintenance of the Golgi structure. Because the highly symmetrical organization of the Golgi is possibly perturbed in response to experimental treatments of gonadotropes, monitoring of the Golgi structure in gonadotropes under various experimental conditions will be a novel in vivo approach to elucidate the biogenesis of the Golgi apparatus.

Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells.

author, Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules (SGs) at the trans-Golgi network (TGN) in endocrine cells. Secretogranin III (SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A (CgA) and peptide hormones to the cholesterol-rich SG membrane (SGM). Here, we evaluated the role of SgIII in SG sorting in AtT-20 cells transfected with small interfering RNA targeting SgIII. In the SgIII-knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA-containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin (POMC) was considerably impaired in SgIII-knockdown cells, residual adrenocorticotropic hormone (ACTH)/POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II (SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol-dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides. © 2012 John Wiley & Sons A/S.

© 2016, Springer-Verlag Berlin Heidelberg. The second West African Monsoon Modeling and Evaluation Project Experiment (WAMME II) is designed to improve understanding of the possible roles and feedbacks of sea surface temperature (SST), land use land cover change (LULCC), and aerosols forcings in the Sahel climate system at seasonal to decadal scales. The project’s strategy is to apply prescribed observationally based anomaly forcing, i.e., “idealized but realistic” forcing, in simulations by climate models. The goal is to assess these forcings’ effects in producing/amplifying seasonal and decadal climate variability in the Sahel between the 1950s and the 1980s, which is selected to characterize the great drought period of the last century. This is the first multi-model experiment specifically designed to simultaneously evaluate such relative contributions. The WAMME II models have consistently demonstrated that SST forcing is a major contributor to the twentieth century Sahel drought. Under the influence of the maximum possible SST forcing, the ensemble mean of WAMME II models can produce up to 60 % of the precipitation difference during the period. The present paper also addresses the role of SSTs in triggering and maintaining the Sahel drought. In this regard, the consensus of WAMME II models is that both Indian and Pacific Ocean SSTs greatly contributed to the drought, with the former producing an anomalous displacement of the Intertropical Convergence Zone before the WAM onset, and the latter mainly contributes to the summer WAM drought. The WAMME II models also show that the impact of LULCC forcing on the Sahel climate system is weaker than that of SST forcing, but still of first order magnitude. According to the results, under LULCC forcing the ensemble mean of WAMME II models can produces about 40 % of the precipitation difference between the 1980s and the 1950s. The role of land surface processes in responding to and amplifying the drought is also identified. The results suggest that catastrophic consequences are likely to occur in the regional Sahel climate when SST anomalies in individual ocean basins and in land conditions combine synergistically to favor drought.

Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules (SGs) at the trans-Golgi network (TGN) in endocrine cells. Secretogranin III (SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A (CgA) and peptide hormones to the cholesterol-rich SG membrane (SGM). Here, we evaluated the role of SgIII in SG sorting in AtT-20 cells transfected with small interfering RNA targeting SgIII. In the SgIII-knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA-containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin (POMC) was considerably impaired in SgIII-knockdown cells, residual adrenocorticotropic hormone (ACTH)/POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II (SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol-dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides.

New versions of the high-resolution 20- and 60-km-mesh Meteorological Research Institute (MRI) atmospheric general circulation models (MRI-AGCM version 3.2) have been developed and used to investigate potential future changes in tropical cyclone (TC) activity. Compared with the previous version (version 3.1), version 3.2 yields a more realistic simulation of the present-day (1979–2003) global distribution of TCs. Moreover, the 20-km-mesh model version 3.2 is able to simulate extremely intense TCs (categories 4 and 5), which is the first time a global climate model has been able to simulate such extremely intense TCs through a multidecadal simulation. Future (2075–99) projections under the Intergovernmental Panel on Climate Change (IPCC) A1B scenario are conducted using versions 3.1 and 3.2, showing consistent decreases in the number of TCs globally and in both hemispheres as climate warms. Although projected future changes in basin-scale TC numbers show some differences between the two versions, the projected frequency of TC occurrence shows a consistent decrease in the western part of the western North Pacific (WNP) and in the South Pacific Ocean (SPO), while it shows a marked increase in the central Pacific. Both versions project a future increase in the frequency of intense TCs globally; however, the degree of increase is smaller in version 3.2 than in version 3.1. This difference arises partly because version 3.2 projects a pronounced decrease in mean TC intensity in the SPO. The 20-km-mesh model version 3.2 projects a northward shift in the most intense TCs (category 5) in the WNP, indicating an increasing potential for future catastrophic damage due to TCs in this region.

Singlet oxygen sensitization of the water-soluble silylated tetraphenylporphyrin derivative has been studied. Quantum yield of singlet oxygen sensitization of the silylated compound was improved compared with that of the non-silylated one. To clarify the mechanism of the improvement, photophysical processes have been studied. The silylation increased the fraction of the triplet state quenched responsible for the formation of singlet oxygen, and resultantly, this effect improved the quantum yield of singlet oxygen sensitization. To demonstrate the silylated compound suitable for a photosensitizer in photodynamic therapy for cancer, a cell culture study was carried out with a human cancer cell line. We found that the silylated compound displayed much higher photodynamic activity than the non-silylated one. We conclude that this high activity was caused by the improvements of both quantum yield of singlet oxygen sensitization and cellular uptake efficiency. We emphasize that improved lipophilicity by silylation contributes much to the high cellar uptake efficiency of porphyrin derivatives.

We have synthesized novel perylene derivatives bearing a silyl function with modifiable functional groups as a feasible material for visualizing biological substances. The fluorescent perylene derivative was coupled to C-5 position of pyrimidine nucleotide and the nucleotide was subsequently incorporated to the middle position of oligoDNA using an automated DNA synthesizer. Also, the derivative was coupled with a cholesterol analog with different length of linking alkyl function. The resulting modified fluorescent oligoDNA exhibited a small change in its fluorescent signal upon hybridization with the complementary oligoDNA. While the cholesterol analogs bearing the silylated perylene exhibited marked fluorescent signal in living cells. The signal is stronger in the analog having longer linker function compared to the analog having shorter linker function. In each case, however, the signal was much prominent compared to that of dehydroergosterol (DHE), a fluorescent cholesterol analog widely used in biological study.

Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by β-hydroxy-β-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 β-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40–50 mol% is crucial for insulin secretion and SG formation functions.

Secretory granules in endocrine cells selectively store bioactive peptide hormones and amines, which are secreted in a regulated manner upon appropriate stimulation. In addition to bioactive substances, various proteins and lipids characteristic of secretory granules are likely recruited to a restricted space at the trans-Golgi Network (TGN), and the space then matures to the secretory granule. Although experimental findings so far have strongly suggested that aggregation- and receptor-mediated processes are essential for the formation of secretory granules, the putative link between these two processes remains to be clarified. Recently, secretogranin III (SgIII) has been identified as a specific binding protein for chromogranin A (CgA), a representative constituent of the core aggregate within secretory granules, and it was later revealed that SgIII can also bind to the cholesterol-rich membrane domain at the TGN. Based on its multifaceted binding properties, SgIII may act as a central player in the formation of cholesterol-rich membrane platforms. Upon these platforms, essential processes for secretory granule biogenesis coordinately occur; that is, selective recruitment of prohormones, processing and modifying of prohormones, and condensation of mature hormones as an aggregate. This review summarizes the findings and theoretical concepts on the issue to date and then focuses on the putative role of SgIII in secretory granule biogenesis in endocrine cells.

Functional variations in the secretogranin III (SCG3) gene are associated with susceptibility to obesity. SCG3 forms secretory granules with orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), and POMC in the hypothalamus. In this study, we screened proteins for SCG3-binding activity and identified secretogranin II (SCG2) using a yeast two-hybrid system. Immunoprecipitation revealed that SCG2 interacts with SCG3. In situ hybridization and immunohistochemistry indicated that SCG2 was highly expressed in the lateral hypothalamic area, paraventricular nucleus, and arcuate nucleus of the hypothalamus. Double-labeling immunohistochemical analysis demonstrated that SCG2 was expressed in orexin-, MCH-, NPY-, and POMC-expressing neurons. SCG2 was also coexpressed with SCG3. Upon introduction into neuroblastoma cells, SCG2 was expressed in the cytosol and formed granule-like structures with SCG3, orexin, NPY, or POMC. SCG3 bound to POMC; however, it did not bind to orexin, MCH, or NPY. By contrast, SCG2 formed aggregates with orexin, MCH, NPY, and POMC. SCG2 may act as a hormone carrier for orexin, MCH, NPY, and POMC by binding with SCG3, which targets proteins to the secretory granules. SCG2 mRNA levels increased along with those of SCG3, orexin, MCH, and NPY after a 24-h fast, suggesting that the SCG2/SCG3 system may respond in an adaptive manner to acute body weight changes. However, this SCG2/SCG3 system appears to be unresponsive to chronic body weight changes, such as diet-induced obesity or obesity in ob/ob mice. We suggest that SCG2, as well as SCG3, may be a potential regulator of food intake based on its capacity to accumulate appetite-related hormones into secretory granules.

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.

In the present review, we discuss the sorting mechanism of peptide hormones and the biogenesis mechanism of secretory granules in view of the significance of the high cholesterol composition of secretory granule membranes. Peptide hormones and granin-family proteins are sorted to immature budding granules at the trans-Golgi network in neuroendocrine cells. Two models have been proposed for granule protein sorting: "aggregation-mediated sorting" and "receptor-mediated sorting". In the aggregation-mediated sorting model, granin-family proteins such as chromogranin A and B form aggregates with peptide hormones in weakly acidic, high calcium milieu of the budding granules. Chromogranins have a disulfide loop at their N-terminal at which they bind to the budding granular membrane, and bring hormones to the granules. In the receptor-mediated sorting model, carboxypeptidase E and/or secretogranin III function as a sorting receptor for peptide hormones. They bind peptide hormones, such as proopiomelanocortin, and have a high-cholesterol-binding domain. Since secretory granule membranes contain high levels of cholesterol, peptide hormones are brought to the secretory granules by these receptors. Although the two models have been conflicting, we suggest that both are cooperative and compensating each other for the sorting of peptide hormones and the biogenesis of secretory granules.

Pancreatic beta-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse beta-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased beta-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in beta-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to beta-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 beta-cell death. We suggest that beta-cell death is regulated by interactions between JNK and its specific MKPs.

This study modifies a Global River-flow model (GRiveT) to more realistically represent groundwater and river-flow processes and examines the effects of these modifications on the reproducibility of the hydrological processes. These modifications include assigning calibrated spatially distributed current speeds and implementing a groundwater scheme. A current speed calibration method is proposed to eliminate river discharge phase differences (RPDs) between the observation and the simulation. We performed nine-year integrations of the modified version of GRiveT. The experimental results were then compared with the observed data for 70 of the world’s major rivers. The proposed calibration method provides reasonable calibrated speeds that eliminate RPD for most of the 70 rivers considered. The calibration significantly improves the river discharge correlation coefficient and phase difference and improves the terrestrial water storage (TWS) correlation coefficient and phase difference to a lesser extent. However, there was little improvement to river discharge and TWS amplitudes. The implementation of the groundwater scheme improves river discharge and TWS correlation coefficients and phase differences for experiments without calibration. The calibration can compensate for the disadvantages of not implementing the groundwater scheme.