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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.01.009 Byline: Hyun Ae Woo (1), Sun Hee Yim (1), Dong Hae Shin (1), Dongmin Kang (1), Dae-Yeul Yu (2), Sue Goo Rhee (1) Keywords: SIGNALING; PROTEINS Abstract: Despite its toxicity, H.sub.2O.sub.2 is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H.sub.2O.sub.2 concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H.sub.2O.sub.2 around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H.sub.2O.sub.2 elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress. Author Affiliation: (1) Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea (2) Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea Article History: Received 24 July 2009; Revised 6 November 2009; Accepted 4 January 2010 Article Note: (miscellaneous) Published: February 18, 2010

The nanos-class gene of the leech Helobdella robusta (Hro-nos) is present as a maternal transcript whose levels decay during cleavage; HRO-NOS protein is more abundant in the D quadrant cells relative to the A, B, and C quadrants; and HRO-NOS is more abundant in the ectodermal precursor cell (DNOPQ) than in its sister mesodermal precursor (DM) (Pilon and Weisblat, 1997). Here, using in situ hybridization, we show that Hro-nos mRNA is broadly distributed throughout the zygote, is concentrated in both animal and vegetal teloplasm during stage I and is at higher levels in DNOPQ than in DM at stage 4b. Hro-nos expression increases after stage 7, as judged by in situ hybridization, developmental RT-PCR, and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9 and 10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive 'spots' comprising of one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin, arising in a segmentally iterated manner from the M lineage, and correspond to cells previously proposed as primordial germ cells (PGCs; Burger, 1891; Weisblat and Shankland, 1985). These results support the proposal that nanos-class genes functioned in the specification of germline cells in the ancestral bilaterian and possibly in a separate process related to embryonic polarity in the ancestral protostome. Key Words: annelid; leech; Helobdella; germline; nanos.

In Ecdysozoan taxa, e.g. Drosophila, Caenorhabditis and others, nanos and its homologs function in various aspects of germline development such as migration and transcriptional repression of primordial germ. To gain information on how and when this function might have arisen during evolution, we have been working to further characterize the expression and function of a nanos homolog (Hro-nos) in a lophotrochozoan species, the leech Helobdella robusta (phylum Annelida), using in situ hybridization, developmental RT-PCR, western blots and immunostaining. Here, we present data suggesting that zygotic Hro-nos is involved in the development of primordial germ cells, in particular the precursors of the several bilateral pairs of testisacs (male sex organ) and a pair of ovisacs (female sex organ). Previous work used Northern blots, western blots and immunostaining to show that Hro-nos is present at high levels as a maternal transcript that declines dramatically during cleavage. We are now able to confirm these results by in situ hybridization. Moreover, we find that Hro-nos levels increase after stage 7, as judged by in situ hybridization, developmental RT-PCR and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9-10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive spots comprising one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin and correspond to cells previously proposed as germline. These results support the conclusion that an ancestral role of nanos-class genes was in the specification of germline cells. Support from NSF (to DAW) and Korean Research Foundation (to DK)

The hedgehog signaling pathway is essential for embryogenesis in organisms ranging from Drosophila to human. A hedgehog homolog (Hro-hh) has been isolated from glossiphoniid leech, Helobdella robusta and its expression was examined by reverse transcription polymerase chain reaction (RT-PCR) and whole mount in situ hybridization. The peak of Hro-hh expression occurs during neurulation and organo-genesis (stages 10-11). In stage 10 embryos, its transcripts appear in primordial gut and esophagus. The pattern of expression is clearer and more refined in early stage 11 embryos, as segmentation of gut begins. From sectioned embryos stained for Hro-hh, we confirm that the mRNA is expressed in foregut (the outer layer of the proboscis and esophagus), midgut (epithelium and/or visceral mesoderm), hindgut (a pattern of stripes at marking the midgut/hindgut boundary and the rectum), and in an iterated pattern of ganglionic neurons. We also found evidence that Hro-hh regulates gut formation, using the steroidal alkaloids cyclopamine, which is known to produce cyclopia and holoprosencephaly in vertebrate by blocking hedgehog signaling. When cyclopamine was bath-applied to gastrulation stage embryos (early/mid stage 8), malformation of foregut was observed at concentrations as low as 5 uM; deformation of both foregut and midgut were observed at slightly higher concentrations (10 uM). Solanidine gave similar results at 10 uM. Hindgut and segmental tissues appeared to develop normally in all concentrations tested for either drug. Support from NSF (to DAW) and Korean Research Foundation (to DK)

Summary: Sestrins (Sesns) protect cells from oxidative stress. The mechanism underlying the antioxidant effect of Sesns has remained unknown, however. The Nrf2-Keap1 pathway provides cellular defense against oxidative stress by controlling the expression of antioxidant enzymes. We now show that Sesn1 and Sesn2 interact with the Nrf2 suppressor Keap1, the autophagy substrate p62, and the ubiquitin ligase Rbx1 and that the antioxidant function of Sesns is mediated through activation of Nrf2 in a manner reliant on p62-dependent autophagic degradation of Keap1. Sesn2 was upregulated in the liver of mice subjected to fasting or subsequent refeeding with a high-carbohydrate, fat-free diet, whereas only refeeding promoted Keap1 degradation and Nrf2 activation, because only refeeding induced p62 expression. Ablation of Sesn2 blocked Keap1 degradation and Nrf2 activation induced by refeeding and thereby increased the susceptibility of the liver to oxidative damage resulting from the acute stimulation of lipogenesis associated with refeeding. [Copyright &y& Elsevier]

Abstract: Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion. Both the transmembrane and cytoplasmic domains of syndecan-4 are known to contribute to its functions, but the regulatory mechanisms underlying the functional interplay between the two domains were previously unclear. Here, we examined the functional relationship between these two domains. Fluorescence resonance energy transfer (FRET)-based assays showed that syndecan-4 expression enhanced RhoA activation. Furthermore, rat embryonic fibroblasts (REFs) plated on fibronectin fragments lacking the heparin-binding domain that interacts with syndecan-4 showed much lower RhoA activation than that in cells plated on full-length fibronectin, indicating that RhoA is involved in syndecan-4-mediated cell adhesion signaling. Syndecan-4 mutants defective in transmembrane domain-induced oligomerization and syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed decreases in RhoA activation and RhoA-related functions, such as adhesion, spreading and focal adhesion formation, and subsequent increase in cell migration, but the inhibitory effect was much higher in cells expressing the transmembrane domain mutants. The cytoplasmic domain mutants (but not the transmembrane domain mutants) retained the capacity to form SDS-resistant dimers, and the cytoplasmic mutants showed less inhibition of syndecan-4-mediated protein kinase C activation compared to the transmembrane domain mutants. Finally, cytoplasmic domain activation failed to overcome the inhibition conferred by mutation of the transmembrane domain. Taken together, these data suggest that the transmembrane domain plays a major role in regulating syndecan-4 functions, and further show that a domain hierarchy exists in the regulation of syndecan-4. [Copyright &y& Elsevier]

Hydrogen peroxide (HO) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of HO requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of HO in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3'5'-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular HO, methods based on dihydro compounds such as 2',7'-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to HO. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on HO-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of HO in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of HO can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with HO, they cannot be used to monitor transient changes in HO concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with HO and can be targeted to various compartments of the cell, but they are not selective for HO because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of HO-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR. [ABSTRACT FROM AUTHOR]

Summary There are abundant peroxiredoxin (Prx) enzymes, but an increase of cellular H 2 O 2 level always happens in apoptotic cells. Here, we show that cellular H 2 O 2 switches different apoptosis pathways depending on which type of Prx enzyme is absent. TNF-α-induced H 2 O 2 burst preferentially activates the DNA damage-dependent apoptosis pathway in the absence of PrxI. By contrast, the same H 2 O 2 burst stimulates the RIPK1-dependent apoptosis pathway in the absence of PrxII by inducing the destruction of cIAP1 in caveolar membrane. Specifically, H 2 O 2 induces the oxidation of Cys308 residue in the cIAP1-BIR3 domain, which induces the dimerization-dependent E3 ligase activation. Thus, the reduction in cIAP level by the absence of PrxII triggers cell-autonomous apoptosis in cancer cells and tumors. Such differential functions of PrxI and PrxII are mediated by interaction with H2AX and cIAP1, respectively. Collectively, this study reveals the distinct switch roles of 2-Cys Prx isoforms in apoptosis signaling. Graphical Abstract Highlights • Absence of Prx I and Prx II exacerbates TNF-α-induced apoptosis in cancer cells. • Prx I binds histone H2AX and inhibits DNA damage-dependent apoptosis. • Prx II binds and prevents the oxidative activation of cIAP1 E3 ligase. • Absence of Prx II induces cell-autonomous apoptosis and inhibits tumor growth. Lee et al. show that the 2-Cys peroxiredoxin (Prx) isoforms, Prx I and Prx II, discretely regulate different apoptosis pathways. Specifically, the absence of Prx I augments apoptosis through the DNA damage response, while the absence of Prx II switches RIPK1-dependent apoptosis by causing cIAP1 depletion. [ABSTRACT FROM AUTHOR]

Abstract: The dynamics of the melt-quenched amorphization in TiO x mixed Ge2Sb2Te5 films were studied using real-time reflectivity measurements with a nanosecond laser pulse and various amounts of TiO x . It was observed that the laser-power required for amorphization was significantly reduced by 27% when 9.1mol% of TiO x was incorporated with Ge2Sb2Te5. The lowering of the thermal conductivity of the films, due to the increase in the number of interface between the Ge2Sb2Te5 and TiO x phases, led to an increase in the thermal efficiency. Such enhanced thermal efficiency is of great technological importance in phase change storage applications. [Copyright &y& Elsevier]

Abstract: Chondroitin sulfate (CsA) is an acidic mucopolysaccharide, which is able to form ionic complexes with positively charged proteins. In this study, a protein–CsA complex was constructed to nano-sized particles. Zeta potential measurements revealed that a CsA-to-protein fraction of greater than 0.1 results in a neutralization of the positive charge on lysozyme (Lys). Based on this preliminary study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring Lys/CsA complexes via the multi-emulsion method. Protein stability in the PLGA microspheres was preserved during both microsphere preparation and protein release. The profiles of Lys release from the PLGA microspheres evidenced nearly zero-order kinetics, depending on the quantity of CsA. An in vivo fluorescent image of experimental mouse tissue showed that the PLGA microspheres with the Lys/CsA complex had released the entirety of their Lys without no residual amount after 23 days, but microspheres without the complex harbored a great deal of residual Lys, which is attributable to its degradation by acidic PLGA degradates. The tissue reaction evidenced by the PLGA microspheres stabilized with CsA showed minimal foreign body reaction and little configuration of immune cells including neutrophils and macrophages, but the reactions of the PLGA microspheres without CsA were characterized by a relatively elevated inflammation. These results show that CsA is a viable candidate for long-acting micro-particular protein delivery. [Copyright &y& Elsevier]

Peroxisomes are essential organelles for maintaining the homeostasis of lipids and reactive oxygen species (ROS). While oxidative stress-induced endoplasmic reticulum (ER) stress plays an important role in nonalcoholic fatty liver disease (NAFLD), the role of peroxisomes in ROS-mediated ER stress in the development of NAFLD remains elusive. We investigated whether an impaired peroxisomal redox state accelerates NAFLD by activating ER stress by inhibiting catalase, an antioxidant expressed exclusively in peroxisomes. Wild-type (WT) and catalase knockout (CKO) mice were fed either a normal diet or a high-fat diet (HFD) for 11 weeks. HFD-induced phenotype changes and liver injury accompanied by ER stress and peroxisomal dysfunction were accelerated in CKO mice compared to WT mice. Interestingly, these changes were also significantly increased in CKO mice fed a normal diet. Inhibition of catalase by 3-aminotriazole in hepatocytes resulted in the following effects: (i) increased peroxisomal H 2 O 2 levels as measured by a peroxisome-targeted H 2 O 2 probe (HyPer-P); (ii) elevated intracellular ROS; (iii) decreased peroxisomal biogenesis; (iv) activated ER stress; (v) induced lipogenic genes and neutral lipid accumulation; and (vi) suppressed insulin signaling cascade associated with JNK activation. N-acetylcysteine or 4-phenylbutyric acid effectively prevented those alterations. These results suggest that a redox imbalance in peroxisomes perturbs cellular metabolism through the activation of ER stress in the liver. Image 1 • Catalase deficiency accelerates nonalcoholic fatty liver disease (NAFLD) in mice. • Catalase deficiency impairs redox balance of peroxisomes in NAFLD. • Peroxisomal redox imbalance accelerates ER stress-mediated NAFLD. [ABSTRACT FROM AUTHOR]

Abstract Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H 2 O 2 , which serves as an intracellular messenger. We have shown that exogenous H 2 O 2 reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca2+ concentration and on H 2 O 2. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca2+-dependent members of the NADPH oxidase family as the major source of H 2 O 2 for Sac1 oxidation. Expression of a Golgi-targeted H 2 O 2 probe revealed transient EGF-induced H 2 O 2 production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca2+ mobilization to events at the Golgi including Duox activation, H 2 O 2 production, Sac1 oxidation, and PtdIns(4)P accumulation. Graphical abstract fx1 Highlights • EGF elicits transient H 2 O 2 production at the Golgi by Ca2+-dependent Duox. • This H 2 O 2 inactivates Sac1 at the Golgi by transiently oxidizing its catalytic Cys. • Transient inactivation of Sac1 results in a transient increase in Golgi PtdIns(4)P. • This new EGF signaling pathway links Ca2+ to Golgi PtdIns(4)P via Duox-H 2 O 2 -Sac1. [ABSTRACT FROM AUTHOR]

Abstract Phosphatidylinositol 4-phosphate [PtdIns(4)P] plays a key role in the biogenesis of transport vesicles at the Golgi complex by recruiting coat proteins and their accessory factors. The PtdIns(4)P content of the Golgi is determined by the concerted action of PtdIns 4-kinase (PI4K) and PtdIns(4)P phosphatase enzymes. Sac1 (suppressor of actin 1) is the major PtdIns(4)P phosphatase and is localized to the Golgi and endoplasmic reticulum. The targeting of both PI4Ks and Sac1 to the Golgi membrane is extensively regulated, as is the catalytic activity of PI4Ks at the Golgi. However, regulation of the catalytic activity of Sac1 has been largely unexplored. Here we show that Sac1undergoes reversible inactivation in mammalian cells when its catalytic Cys389 residue is oxidized by exogenous H 2 O 2 to form an intramolecular disulfide with Cys392. The oxidative inactivation of Sac1 results in the accumulation of PtdIns(4)P at the Golgi, with this effect also being supported by the H 2 O 2 -induced activation of p38 mitogen-activated protein kinase (MAPK), which was previously shown to promote the translocation of Sac1 from the Golgi to the endoplasmic reticulum. The increase in Golgi PtdIns(4)P due to Sac1 inactivation, however, is faster than that due to Sac1 translocation. Exposure of cells to H 2 O 2 also increased membrane protein trafficking from the Golgi to the plasma membrane as well as protein secretion. Graphical abstract fx1 Highlights • PtdIns(4)P levels at the Golgi rise rapidly in mammalian cells exposed to H 2 O 2. • This rise results from oxidative inactivation of the PtdIns(4)P phosphatase Sac1. • The catalytic Cys389 of oxidized Sac1 forms an intramolecular disulfide with Cys392. • H 2 O 2 increases protein trafficking from the Golgi to the plasma membrane. [ABSTRACT FROM AUTHOR]

Summary: All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. Here, we show that phospholipase C-β (PLCβ) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCβ through Gαq, which in turn mobilizes intracellular Ca2+ through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCβ mutant flies had a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCβ restored efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Gαq-PLCβ-Ca2+-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle. [Copyright &y& Elsevier]

Abstract: The blood–brain barrier (BBB) is essential for maintaining brain homeostasis and protecting the brain from toxic substances. Breakdown of this barrier results in severe brain pathologies, whereas impermeability of the BBB is a major obstacle for drug delivery to the brain. Despite its importance, our understanding of the maturation and modulation of the BBB is limited. Zebrafish (Danio rerio) has emerged as a useful model organism for studying vertebrate development and disease mechanisms, as well as for preclinical drug screening. However, the nature of the BBB has not yet been examined in teleost fish. In this paper, we report that with the exception of the circumventricular organs, the cerebral microvessels in zebrafish are impermeable to sulfo-NHS-biotin and horseradish peroxidase (HRP). Brain endothelial cells show immunoreactivity to Claudin-5 and Zonula Occludens-1 (ZO-1), implying the presence of tight junctions in these cells. The expression of Claudin-5 and ZO-1 was detected in cerebral microvessels from 3 days post-fertilization (dpf), concomitant with maturation of the BBB, as determined by restricted permeability to HRP and various fluorescent tracers. Real-time analysis of fluorescent tracer leakage in embryonic zebrafish suggests that they may be used as an in vivo model for BBB breakdown. Taken together, our results show that the endothelial tight junction-based BBB of zebrafish is similar to that of higher vertebrates and thus, zebrafish may be an excellent genetic and experimental model organism for studying development and maintenance of the BBB. [Copyright &y& Elsevier]

Abstract: In order to design an effective intratumoral radioisotope carrier, a self-assembled nanoparticle evidencing ionic strength (IS)-sensitivity from a polysaccharide derivative (pullulan acetate nanoparticle (PAN)) was prepared via dialysis. The PAN had a spherical shape in a range of size of 50–130nm and a low critical aggregation concentration (CAC) (<8μg/mL). With increases in the IS of the dialysis media (ISdia), the CAC of PAN was reduced gradually and the rigidity of the hydrophobic core in PAN was increased. This suggests that the property of PAN was altered more hydrophobically at high IS values. The stabilities of PANs prepared from various ISdia were also monitored with changes in the turbidity and particle size in different IS solutions. In the case of PAN prepared at an ISdia =0.0, the turbidity was dramatically reduced with increasing IS due to the facilitation of aggregation between the particles, whereas in the other cases, these changes were negligible. This finding indicates that PAN prepared in distilled water (IS=0.0) can be readily injected as the consequence of its nano-size, and accumulates quickly, then remains in the tumor site for a considerable period (IS=0.15). In order to closely estimate the potential of PAN as a radioisotope carrier, the radioisotope labeling efficiency of PAN with no chelating agents was evaluated. PAN evidenced a high degree of 99mTechnetium (99mTc) labeling efficiency (approximately 98%). The percentage retention rate (%RR) of the 99mTc-labeled PAN was significantly longer than that of the free 99mTc (p <0.05), due largely to PAN''s IS-sensitivity. In conclusion, PAN may constitute a new approach to the achievement of maximal radioisotope efficiency with regard to intratumoral administration. [Copyright &y& Elsevier]

Abstract: We have recently demonstrated that human follicular dendritic cells (FDCs) strongly express prostacyclin synthase. The purpose of this study is to investigate the production mechanism of prostacyclin using the established human FDC line, HK. The levels of PGIS protein expression did not vary during the different stages of the cell cycle. We stimulated HK cells with various inflammatory cytokines but, none of the tested stimuli modulated PGIS expression significantly. However, incubation of HK cells with tumor necrosis factor (TNF)-α gave rise to a significant increase in the protein level of cyclooxygenase (COX)-2. Furthermore, elevated levels of prostacyclin secretion stimulated by TNF-α were markedly down-regulated by indomethacin and a selective COX-2 inhibitor. These results suggest that the production of prostacyclin in FDC is controlled by the regulation of upstream COX-2 but not by terminal PGIS protein production. This study has important implications for the development of new anti-inflammatory drugs. [Copyright &y& Elsevier]