Adaptive feedforward control of closed orbit distortion caused by fast helicity-switching undulators.

A new correction algorithm for closed orbit distortion based on an adaptive feedforward control (AFC) has been developed. At SPring-8, two helicity-switching twin-helical undulators (THUs) had been implemented with conventional feedforward corrections. However, the validity of these corrections turned out to be expiring due to unforeseen variation in the error magnetic fields with time. The developed AFC system has been applied to the THUs dynamically updating the feedforward table without stopping the helicity switching amid user experiments. The error sources in the two THUs are successfully resolved and corrected even while the two THUs are switching simultaneously with the same repetition period. The actual operation of the new AFC system enables us to keep the orbit variations suppressed with an accuracy at the sub-micrometre level in a transparent way for light source users.

Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT.

Glypican-5 (GPC5) is a heparan sulfate proteoglycan (HSPG) localized to the plasma membrane. We previously reported that in the human mesenchymal stem cell line UE6E7T-3, GPC5 is overexpressed in association with transformation and promotes cell proliferation by acting as a co-receptor for Sonic hedgehog signaling. In this study, we found using immunofluorescence microscopy that in transformed cells (U3DT), GPC5 localized not only at primary cilia on the cell surface, but also at the leading edge of migrating cells, at the intercellular bridge and blebs during cytokinesis, and in extracellular vesicles. In each subcellular region, GPC5 colocalized with fibroblast growth factor receptor (FGFR) and the small GTPases Rab11 and ARF6, indicating that GPC5 is delivered to these regions by Rab11-associated recycling endosomes. These colocalizations suggest that GPC5 plays an important role in FGF2 stimulation of cell migration, which was abrogated by knockdown of GPC5. Our findings indicate that GPC5 plays a role in regulation of U3DT cell migration and provides several insights into the functions of GPC5 that could be elucidated by future studies.

Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).

New soft X-ray beamline BL07LSU at SPring-8.

A new soft X-ray beamline, BL07LSU, has been constructed at SPring-8 to perform advanced soft X-ray spectroscopy for materials science. The beamline is designed to achieve high energy resolution (E/ΔE> 10000) and high photon flux [>10(12) photons s(-1) (0.01% bandwidth)(-1)] in the photon energy range 250-2000 eV with controllable polarization. To realise this state-of-the-art performance, a novel segmented cross undulator was developed and adopted as a light source. The details of the undulator light source and beamline monochromator design are described. The achieved performance of the beamline, such as the photon flux, energy resolution and the state of polarization, is reported.

Determination of HEL (Hexanoyl-lysine adduct): a novel biomarker for omega-6 PUFA oxidation.

Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies.

Performance upgrade in the JAEA actinide science beamline BL23SU at SPring-8 with a new twin-helical undulator.

The soft X-ray beamline BL23SU at SPring-8 has undergone an upgrade with a twin-helical undulator of in-vacuum type to enhance the experimental capabilities of the endstations. The new light source with a fast helicity-switching operation allows not only the data throughput but also the sensitivity in X-ray magnetic circular dichroism (XMCD) to be improved. The operational performance and potential are described by presenting XMCD results of paramagnetic ß-US(2) measured with a 10 T superconducting magnet.

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model.

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.

Analysis of antioxidant activities in vegetable oils and fat soluble vitamins and biofactors by the PAO-SO method.

Accumulated evidences indicate that reactive oxygen species (ROS) are involved in the pathophysiology of aging process. Antioxidants are believed to play an important role in the defense system to counteract ROS in the body. While excess hydrophilic antioxidants can be excreted easily in urine, lipophilic antioxidants can penetrate into blood lipoproteins and cell membranes, and may maintain long and high bioavailability. These lipophilic antioxidants are thus expected to contribute greatly to the prevention of age-related diseases. Oils extracted from plant seeds are known to contain various lipophilic antioxidants such as vitamin E (alpha-tocopherol) and carotenoids. They are known to not only decrease serum low-density-lipoprotein (LDL) level, but also prevent oxidation of LDL. In addition to vitamin E (alpha-tocopherol) and carotenoids, other lipophilic antioxidants such as gamma-oryzanol and sesaminol (from sesamolin) are in rice bran and sesame, respectively. They are sometimes called "vitamin-like food factors" or "biofactors." Although there are several methods for measuring the total antioxidant activities for various plant extracts, most of these methods are designed for hydrophilic antioxidants, and not for lipophilic antioxidants present in various plant seed oils. In this report, we present an assay method for the total potency of antioxidants that are soluble in oil (PAO-SO) utilizing bathocuproine (BC) as a chromogen. BC-based antioxidant activity assay shows good linearity (r(2) = 0.9986), good reproducibility (CV < 10%), and good recovery (86-91%) when dl-alpha-tocopherol, for example, is added to sesame oil. Total antioxidant activity of rape-seed oil, olive oil, and sesame oil could also be successfully measured.

Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture.

Aneuploidy (an abnormal number of chromosomes) is commonly observed in most human cancer cells, highlighting the need to examine chromosomal instability in tumorigenesis. Previously, the immortalized human mesenchymal stem cell line UE6E7T-3 was shown to undergo a preferential loss of one copy of chromosome 13 after prolonged culture. Here, the loss of chromosome 13 was found to be caused by chromosome missegregation during mitosis, which involved unequal segregation, exclusion of the misaligned chromosome 13 on the metaphase plate, and trapping of chromosome 13 in the midbody region, as observed by fluorescence in situ hybridization. Near-diploid aneuploidy, not tetraploidy, was the direct result. The loss of chromosome 13 was non-random, and was detected by analysis of microsatellites and single nucleotide polymorphism-based loss of heterozygosity (LOH). Of the five microsatellite loci on chromosome 13, four loci showed microsatellite instability at an early stage in culture, and LOH was apparent at a late stage in culture. These results suggest that the microsatellite mutations cause changes in centromere integrity provoking loss of this chromosome in the UE6E7T-3 cell line. Thus, these results support the use of this cell line as a useful model for understanding the mechanism of aneuploid formation in cell cultures.

Degree of circular polarization of soft X-rays emitted from a multi-polarization-mode undulator characterized by means of magnetic circular dichroism measurements.

The degree of circular polarization of soft X-rays emitted from the multi-polarization-mode undulator of BL17SU at SPring-8 has been deduced by means of magnetic circular dichroism in core-level X-ray absorption spectroscopy for a ferrimagnetic Gd-Fe-Co amorphous thin film. The results of reference measurements performed using well characterized undulator radiation of BL25SU at SPring-8 have also been utilized. The degrees of circular polarization were presumed for all the available operational modes and were compared with simple theoretical calculations. It was found that the calculated degrees of circular polarization were validated by the measurements reasonably well and will be useful in further experiments at BL17SU.

Green-tea polyphenol (-)-epigallocatechin-3-gallate provides resistance to apoptosis in isolated islets.

BACKGROUND/PURPOSE: Apoptosis resulting from disruption of the normal cell-matrix relationship (anoikis) during islet isolation, and the reactive oxygen and nitrogen species generated following hypoxia/reoxygenation (H/R) can lead to a loss of islet tissue in culture and the reduced survival of transplanted pancreatic islets. The aim of this study was to investigate the effect of (-)-epigallocatechin-3-gallate (EGCG), a well-known antiapoptotic agent, on inhibiting anoikis and H/R injury in an in vitro islet culture system. METHODS: Islets were isolated from F344 rats and cultured under normal or H/R condition with/without EGCG. RESULTS: EGCG inhibited apoptosis and lactate-dehydrogenase leakage from anoikis and H/R in a dose-dependent manner. Further, EGCG prevent increases in 8-hydroxy-2'-deoxyguanosine content and inhibited the decline of insulin secretory function induced by H/R. CONCLUSIONS: These results suggest that the addition of EGCG to an islet culture system may improve the survival rate of isolated islets and reduce the loss of functional islet mass that compromises the stable reversal of diabetes after islet transplantation.

Chromosomal instability in human mesenchymal stem cells immortalized with human papilloma virus E6, E7, and hTERT genes.

Human mesenchymal stem cells (hMSCs) are expected to be an enormous potential source for future cell therapy, because of their self-renewing divisions and also because of their multiple-lineage differentiation. The finite lifespan of these cells, however, is a hurdle for clinical application. Recently, several hMSC lines have been established by immortalized human telomerase reverse transcriptase gene (hTERT) alone or with hTERT in combination with human papillomavirus type 16 E6/E7 genes (E6/E7) and human proto-oncogene, Bmi-1, but have not so much been characterized their karyotypic stability in detail during extended lifespan under in vitro conditions. In this report, the cells immortalized with the hTERT gene alone exhibited little change in karyotype, whereas the cells immortalized with E6/E7 plus hTERT genes or Bmi-1, E6 plus hTERT genes were unstable regarding chromosome numbers, which altered markedly during prolonged culture. Interestingly, one unique chromosomal alteration was the preferential loss of chromosome 13 in three cell lines, observed by fluorescence in situ hybridization (FISH) and comparative-genomic hybridization (CGH) analysis. The four cell lines all maintained the ability to differentiate into both osteogenic and adipogenic lineages, and two cell lines underwent neuroblastic differentiation. Thus, our results were able to provide a step forward toward fulfilling the need for a sufficient number of cells for new therapeutic applications, and substantiate that these cell lines are a useful model for understanding the mechanisms of chromosomal instability and differentiation of hMSCs.

Species identification of animal cells by nested PCR targeted to mitochondrial DNA.

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons.

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.

Is phenol condensation one of the major pathways in the formation of polychlorinated dibenzofurans in municipal waste incinerators?: Model prediction vs. field observation.

The role of phenol condensation pathways in the formation of polychlorinated dibenzofurans (PCDF) in a municipal waste incinerator was assessed by comparing predicted PCDF homologue and isomer patterns with those obtained from the incinerator. A two-phenol condensation model, dependent only on the distribution of phenols, was used to predict the distributions of PCDF congeners in the incinerator. Complete distributions of phenols and PCDF congeners were obtained from the incinerator. To quantify the degree of agreement between obtained isomer distributions and those predicted by the model, R-squared values from linear correlations were calculated for the dichlorinated- through hexachlorinated-isomers. They ranged from 0.001 to 0.1. Agreement between obtained and predicted PCDF isomers was very poor for all homologues, suggesting that phenol condensation pathways are unlikely to be the primary route in the formation of PCDF in the incinerator. However, dibenzofuran (DF) is likely to be produced from a condensation of two phenols. This paper shows the use of PCDF homologue and isomer patterns calculated by the two-phenol condensation model for testing PCDF formation mechanism attribution in a municipal waste incinerator.

Homologue and isomer patterns of polychlorinated dibenzo-p-dioxins and dibenzofurans from phenol precursors: comparison with municipal waste incinerator data.

The role of phenol precursors in polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) formation in municipal waste incinerators is assessed on the basis of homologue and isomer patterns. Homologue and isomer patterns of PCDD and PCDF congeners formed from phenols both in the gas phase and via particle-mediated reactions were studied in an isothermal flow reactor. A mixture of unsubsitituted phenol and 19 chlorinated phenols in relative concentrations found in a municipal waste incinerator (MWI) stack gas was used for this study. PCDD and PCDF homologue and isomer patterns obtained from the phenol experiments were compared with those observed in MWI data. From the phenol experiments, gas-phase formation at 600-700 degrees C favors PCDF formation whereas particle-mediated formation at 400 degrees C favors PCDD formation. Unsubstituted phenol, which was present in high concentration, played a significant role in the formation of PCDD/F congeners under both sets of experimental conditions. PCDD/F distributions in MWI flue gas and fly ash samples were differentfrom those observed in the phenol experiments, suggesting that direct phenol condensation was not the primary route of PCDD/F formation at the incinerators. Gas-phase phenol condensation is a source of dibenzofuran, with subsequent particle-mediated chlorination resulting in PCDF formation. In the case of PCDD formation, phenol condensation may be responsible for the formation of certain highly chlorinated congeners. In this paper we demonstrate the use of homologue and isomer patterns for PCDD/F formation mechanism attribution in municipal waste incinerators.

CuCl2-catalyzed PCDD/F formation and congener patterns from phenols.

Homologue and isomer patterns of polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) in CuCl2-catalyzed formation were studied in an isothermal flow reactor using a distribution of 20 phenols as measured in municipal waste incinerator (MWI) exhaust gases. A mixture of 20 phenols was synthesized and used as reactants for this study because phenols are known to be key precursors in the formation of PCDD/F. Experiments were conducted at 400 degrees C. The 92% of nitrogen (N2) and 8% of oxygen (O2) were used as a carrier gas. PCDD/F homologue and isomer patterns with dibenzo-p-dioxin (DD) and dibenzofuran (DF) were obtained from a mixture of 20 phenols. DF+PCDF formation was favored over DD+PCDD formation. The major homologue groups formed were non-chlorinated DD and DF, and PCDD/F homologue fraction decreased with the degree of chlorination. PCDD/F homologue and isomer distributions were almost constant. Phenol and lower chlorinated phenols present in high amount played an important role in PCDD/F congener distributions. The results presented here can be used as characteristics or fingerprints for homologue and isomer patterns of PCDD/F formation attribution in CuCl2-catalyzed reaction from phenols.

Coexistence of salusin and vasopressin in the rat hypothalamo-hypophyseal system.

Salusins are two newly discovered TOR-related peptides consisting of 28 and 20 amino acids and designated salusin-alpha and salusin-beta, respectively. Using immunohistochemistry techniques, salusin-like immunoreactivity was detected in the rat hypothalamo-neurohypophyseal tract and immunopositive cells were distributed in the suprachiasmatic, supraoptic and paraventricular nucleus. In the paraventricular nucleus, salusin-like immunoreactivity was observed both in parvocellular and magnocellular neurons. Many salusin-positive nerve fibers and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Less intense salusin-positive staining of fibers and terminals was found in the suprachiasmatic nucleus and external layer of the median eminence. Dual immunostaining was performed to determine if salusin coexisted with vasopressin or oxytocin in the hypothalamus. Most of the salusin-like immunoreactivity was detected in vasopressin- but not in oxytocin-containing neurons in these nuclei. The functional significance of the coexistence of salusin with vasopressin is discussed, including the possibility that salusin participates in the regulation of blood pressure.

Role of chlorine in combustion field in formation of polychlorinated dibenzo-p-dioxins and dibenzofurans during waste incineration.

Combustion experiments performed in the presence of hydrogen chloride (HCl) in a laboratory-scale fluidized-bed reactor were carried out to elucidate the role of chlorine in the formation of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs; together: PCDD/Fs) in various sections of a municipal waste incinerator. We first demonstrated that the homologue profile and the pattern of the congener proportions of PCDD/Fs for a model waste containing poly(vinyl chloride) (PVC) combusted in the absence of HCl were similar to those for a PVC-free waste combusted in the presence of HCl. This showed no difference between PVC in the waste and injected HCl in the role as a chlorine source in PCDD/F formation during incineration. Next, to investigate PCDD/F formation in each section of the incinerator, we carried out combustion experiments with the PVC-free waste, injecting HCl at different locations of the incinerator. The amounts of PCDDs and PCDFs formed were significantly reduced when HCI was not supplied to the main combustion section. The presence of HCI in the main combustion section was essential for the formation of PCDD/Fs, even in the downstream sections. This finding indicates that compounds that were able to form PCDD/Fs in the downstream sections were mainly formed in the main combustion section in the presence of HCl.