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Usher syndrome (USH) is a rare autosomal recessive disease characterized by the combination of hearing loss, visual impairment due to retinitis pigmentosa, and in some cases vestibular dysfunctions. Studies published in the 1980s reported that USH is associated with cellular radiosensitivity. However, the molecular basis of this particular phenotype has not yet been documented. The aim of this study was therefore to document the radiosensitivity of USH1-a subset of USH-by examining the radiation-induced nucleo-shuttling of ATM (RIANS), as well as the functionality of the repair and signaling pathways of the DNA double-strand breaks (DSBs) in three skin fibroblasts derived from USH1 patients. The clonogenic cell survival, the micronuclei, the nuclear foci formed by the phosphorylated forms of the X variant of the H2A histone (ɣH2AX), the phosphorylated forms of the ATM protein (pATM), and the meiotic recombination 11 nuclease (MRE11) were used as cellular and molecular endpoints. The interaction between the ATM and USH1 proteins was also examined by proximity ligation assay. The results showed that USH1 fibroblasts were associated with moderate but significant radiosensitivity, high yield of micronuclei, and impaired DSB recognition but normal DSB repair, likely caused by a delayed RIANS, suggesting a possible sequestration of ATM by some USH1 proteins overexpressed in the cytoplasm. To our knowledge, this report is the first radiobiological characterization of cells from USH1 patients at both molecular and cellular scales.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis ( Arabidopsis thaliana ) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Mitochondrial dysfunction plays an important role in gamma-radiation-induced mediating oxidative stress. Scavenging radiation-induced reactive oxygen species (ROS) can help mitochondria to maintain their physiological function. Rosmarinic acid is a polyphenol antioxidant that can scavenge radiation-induced ROS, but the structure prevents it from accumulating in mitochondria. In this study, we designed and synthesized a novel rosmarinic acid derivative (Mito-RA) that could use the mitochondrial membrane potential to enter the organelle and scavenge ROS. The DCFH-DA assay revealed that Mito-RA was more effective than rosmarinic acid at scavenging ROS. DNA double-strand breaks, chromosomal aberration, micronucleus and comet assays demonstrated the ability of Mito-RA to protect against radiation-induced oxidative stress in vitro. These findings demonstrate the potential of Mito-RA as an antioxidant, which can penetrate mitochondria, scavenge ROS and protect cells against radiation-induced oxidative damage.

Gold nanoparticles (GNPs) have been demonstrated as effective radiosensitizing agents in a range of preclinical models using broad field sources of various energies. This study aimed to distinguish between these mechanisms by applying subcellular targeting using a soft X-ray microbeam in combination with GNPs. DNA damage and repair kinetics were determined following nuclear and cytoplasmic irradiation using a soft X-ray (carbon K-shell, 278 eV) microbeam in MDA-MB-231 breast cancer and AG01522 fibroblast cells with and without GNPs. To investigate the mechanism of the GNP induced radiosensitization, GNP-induced mitochondrial depolarisation was quantified by TMRE staining, and levels of DNA damage were compared in cells with depolarised and functional mitochondria. Differential effects were observed following radiation exposure between the two cell lines. These findings were validated 24 hours after removal of GNPs by flow cytometry analysis of mitochondrial depolarisation. This study provides further evidence that GNP radiosensitisation is mediated by mitochondrial function and it is the first report applying a soft X-ray microbeam to study the radiobiological effects of GNPs to enable the separation of physical and biological effects.

Expression of rRNA affects cell growth and proliferation, but mechanisms that modulate rRNA levels are poorly understood. We conducted a genetic screen for factors that negatively regulate generation of endogenous short interfering RNA (endo-siRNA) in Caenorhabditis elegans and identified a suppressor of siRNA (susi-1) and antisense ribosomal siRNAs (risiRNAs). risiRNAs show sequence complementary to 18S and 26S rRNAs and require RNA-dependent RNA polymerases (RdRPs) for their production. They act through the nuclear RNA interference (RNAi) pathway to downregulate pre-rRNA. Stress stimuli, including low temperature and UV irradiation, induced the accumulation of risiRNAs. SUSI-1 is a homolog of the human DIS3L2 exonuclease involved in 3'-5' degradation of oligouridylated RNAs. In susi-1 mutant and in low temperature-treated animals, 3'-tail oligouridylated 26S rRNA accumulated. The injection of oligouridylated rRNA elicited nuclear accumulation of NRDE-3. Our findings identify a new subset of 22G-RNAs that regulate pre-rRNA expression and a mechanism to maintain rRNA homeostasis.

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again., (© 2015 John Wiley & Sons Ltd.)

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

Plants absorb sunlight to power the photochemical reactions of photosynthesis, which can potentially damage the photosynthetic machinery. However, the mechanism that protects chloroplasts from the damage remains unclear. In this work, we demonstrated that rice (Oryza sativa L.) SLAC7 is a generally expressed membrane protein. Loss-of-function of SLAC7 caused continuous damage to the chloroplasts of mutant leaves under normal light conditions. Ion leakage indicators related to leaf damage such as H2 O2 and abscisic acid levels were significantly higher in slac7-1 than in the wild type. Consistently, the photosynthesis efficiency and Fv/Fm ratio of slac7-1 were significantly decreased (similar to photoinhibition). In response to chloroplast damage, slac7-1 altered its leaf morphology (curled or fused leaf) by the synergy between plant hormones and transcriptional factors to decrease the absorption of light, suggesting that a photoprotection mechanism for chloroplast damage was activated in slac7-1. When grown in dark conditions, slac7-1 displayed a normal phenotype. SLAC7 under the control of the AtSLAC1 promoter could partially complement the phenotypes of Arabidopsis slac1 mutants, indicating a partial conservation of SLAC protein functions. These results suggest that SLAC7 is essential for maintaining the chloroplast stability in rice., (© 2015 Huazhong Agricultural University. Journal of Integrative Plant Biology Published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, The Chinese Academy of Sciences.)

Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens., (© 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Background: Nanoparticles made from aluminum phthalocyanine (AlPc) are non-fluorescent in the nanoparticle form. Once AlPc molecules become detached from the particle, fluorescence occurs. Preliminary work showed the benefit of using aluminum phthalocyanine nanoparticles (nAlPc) for the rating of the rejection risk of skin autografts in mice by measuring fluorescence intensities of detached AlPc. Skin autografts showing a high fluorescence intensity were finally rejected suggesting an inflammatory process. In contrast, autografts with normal autofluorescence were accepted. This work was focused on the mechanism of this finding. The aim is detecting inflammatory processes and the potential use of nAlPc for PDT as a new treatment modality., Methods: The effect of the lipopolysaccharide-stimulated monocyte/macrophage murine cell line J774A.1 on the monomerization of internalized nAlPc was tested. Further, we investigated the influence of J774A.1 cells and the normal skin cell lines L-929 or HaCaT on the dissolution of nAlPc by laser scanning microscopy and flow cytometry. Localization of AlPc molecules after uptake and dissolution of nanoparticles by the cells was surveyed., Results: In co-culture models composed of J774A.1 and HaCaT/L-929 cells, the AlPc fluorescence intensity in J774A.1 cells is 1.38/1.89 fold higher, respectively. According to localization measurements in J774A.1 cells it can be assumed that nAlPc is taken up via endocytosis and remains in endosomes and/or lysosomes dissolving there. Detached molecules of AlPc cause rapture of the endosomal and/or lysosomal membrane after irradiation to become quite uniformly distributed in the cytoplasm., Conclusions: Evidence for monocytes/macrophages being the origin of the measured AlPc fluorescence in rejected skin autografts was confirmed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.

Seedling deetiolation, a critical process in early plant development, is regulated by an intricate transcriptional network. Here, we identified VQ MOTIF-CONTAINING PROTEIN29 (VQ29) as a novel regulator of the photomorphogenic response in Arabidopsis (Arabidopsis thaliana). We showed that 29 of the 34 VQ proteins present in Arabidopsis exhibit transcriptional activity in plant cells and that mutations in the VQ motif affect the transcriptional activity of VQ29. We then functionally characterized VQ29 and showed that the hypocotyl growth of plants overexpressing VQ29 is hyposensitive to far-red and low-intensity white light, whereas a vq29 loss-of-function mutant exhibits decreased hypocotyl elongation under a low intensity of far-red or white light. Consistent with this, VQ29 expression is repressed by light in a phytochrome-dependent manner. Intriguingly, our yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that VQ29 physically interacts with PHYTOCHROME-INTERACTING FACTOR1 (PIF1). We then showed that VQ29 and PIF1 directly bind to the promoter of a cell elongation-related gene, XYLOGLUCAN ENDOTRANSGLYCOSYLASE7, and coactivate its expression. Furthermore, the vq29 pif1 double mutant has shorter hypocotyls than either of the corresponding single mutants. Therefore, our study reveals that VQ29 is a negative transcriptional regulator of light-mediated inhibition of hypocotyl elongation that likely promotes the transcriptional activity of PIF1 during early seedling development.

Radiosensitization efficacy of gold nanoparticles (AuNPs) with low energy radiations (88 keV) was evaluated in vitro and in vivo on rats bearing glioma. In vitro, a significant dose-enhancement factor was measured by clonogenic assays after irradiation with synchrotron radiation of F98 glioma cells in presence of AuNPs (1.9 and 15 nm in diameter). In vivo, 1.9 nm nanoparticles were found to be toxic following intracerebral delivery in rats bearing glioma, whether no toxicity was observed using 15 nm nanoparticles at the same concentration (50 mg/mL). The therapeutic efficacy of gold photoactivation was determined by irradiating the animals after intracerebral infusion of AuNPs. Survival of rats that had received the combination of treatments (AuNPs: 50 mg/mL, 15 Gy) was significantly increased in comparison with the survival of rats that had received irradiation alone. In conclusion, this experimental approach is promising and further studies are foreseen for improving its therapeutic efficacy., From the Clinical Editor: These investigators report that gold nanoparticles of the correct size can be used to enhance the effects of irradiation in the context of a glioma model. Since many of the glioma varieties are currently incurable, this or similar approaches may find their way to clinical trials in the near future., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cytosolic Ca(2+) in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca(2+) increases result from Ca(2+) influx through Ca(2+)-permeable channels in the plasma membrane and Ca(2+) release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca(2+)-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3',5'-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg(2+) as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba(2+), Ca(2+), and Na(+) ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca(2+)-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO2 concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca(2+)-permeable cation channels in the plasma membrane of Arabidopsis guard cells.

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7',8' position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

Photoconvertible fluorescent proteins such as Kaede are routinely used for tracking proteins, organelles, and whole cells. Kaede was the first identified photoconvertible fluorescent protein and has since become the most commonly used photoconvertible fluorescent protein in vertebrates. Kaede can be irreversibly converted from a green to a red fluorescent form upon UV/blue light irradiation and fluorescence of each form can be isolated separately by appropriate filter sets. Spectral properties of the Kaede forms allow Förster resonance energy transfer (FRET) from the green form as donor to the red form as acceptor. As a sample containing oligomerized Kaede-containing proteins is exposed to UV or blue light, FRET first increases as green Kaede is converted to red and then decreases as the green donor becomes depleted. Thus, FRET information is potentially obtained from a number of independent measurements taken as photoconversion proceeds. We demonstrate here the application of this approach to detect homo-aggregation and conformational dynamics of plant protein constructs. Structural alterations of 2-cys peroxiredoxin–Kaede were successfully detected depending on the redox state in living plant cells. Photoconversion was performed gradually and donor emission, acceptor emission, and FRET-derived sensitized acceptor emission were measured at each step of conversion. Since photoconvertible proteins have not been routinely used in plants, two plasmids have been designed to facilitate plant applications. The plasmids allow either transient expression of Kaede-containing protein constructs in plant cells or Gateway cloning and stable transformation of plants.

Compared with small heat shock proteins (sHSPs) in other organisms, those in plants are the most abundant and diverse. However, the molecular mechanisms by which sHSPs are involved in cell protection remain unknown. Here, we characterized the role of HSP21, a plastid nucleoid-localized sHSP, in chloroplast development under heat stress. We show that an Arabidopsis thaliana knockout mutant of HSP21 had an ivory phenotype under heat stress. Quantitative real-time RT-PCR, run-on transcription, RNA gel blot, and polysome association analyses demonstrated that HSP21 is involved in plastid-encoded RNA polymerase (PEP)-dependent transcription. We found that the plastid nucleoid protein pTAC5 was an HSP21 target. pTAC5 has a C4-type zinc finger similar to that of Escherichia coli DnaJ and zinc-dependent disulfide isomerase activity. Reduction of pTAC5 expression by RNA interference led to similar phenotypic effects as observed in hsp21. HSP21 and pTAC5 formed a complex that was associated mainly with the PEP complex. HSP21 and pTAC5 were associated with the PEP complex not only during transcription initiation, but also during elongation and termination. Our results suggest that HSP21 and pTAC5 are required for chloroplast development under heat stress by maintaining PEP function.

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards one apoptotic pathway, the intrinsic mitochondrial one where Bcl-2 family of proteins plays the major role. We are particularly interested in two pro-apoptotic players Bak and Bax from this family. Here we investigated their role in apoptosis triggered by photodynamic action. Targeted photodynamic therapy (PDT) is a promising approach to diagnose and treat different types of cancer. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action causes Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.

The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8(-/-) mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.

Heading date is a critical trait that determines cropping seasons and regional adaptability in rice (Oryza sativa). Research efforts during the last decade have identified some important photoperiod pathway genes that are conserved between Arabidopsis and rice. In this study, we identified a novel gene, Oryza sativa ELF3 (OsELF3), which is a putative homolog of the ELF3 gene in Arabidopsis thaliana. OsELF3 was required for the control of heading date under long-day conditions. Its Tos17-tagging mutants exhibited a delayed heading date phenotype only under long-day, but not short-day, conditions. OsELF3 was highly expressed in leaf blades, and the OsELF3 protein was localized in the nucleolus. An obvious diurnal rhythm of OsELF3 transcript level was observed, with a trough in the early day and a peak in the late night in wild-type plants. However, this expression pattern was disrupted in oself3 mutants. Further investigations showed that the expression of OsGI and Ghd7 was up-regulated in the oself3 mutant, indicating that OsELF3 acts as a negative regulator upstream of OsGI and Ghd7 in the flowering-time control under long-day conditions. The rhythmic expression of circadian clock-related genes, including some OsPRR members, was obviously affected in oself3 mutants. Our results indicated that OsELF3 acts as a floral activator in the long-day photoperiodic pathway via its crosstalk with the circadian clock in rice.

Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd⋅kg(-1) in the grain compared with a mean of 1.73 mg Cd⋅kg(-1) in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD(50) light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.

Light-induced damage is a widely used model to study retinal degeneration. We examined whether bacterial lipopolysaccharide (LPS) protects the retina against light-induced injury. One day before intense light exposure for 24 h, rats were intravitreally injected with LPS in one eye and vehicle in the contralateral eye. At several time points after light exposure, rats were subjected to electroretinography and histological analysis. Bax, Bcl-xL, p-Akt, and p-Stat3 levels were assessed by Western blotting, and retinal thiobarbituric acid reactive substances levels were measured as an index of lipid peroxidation. One group of animals received injections of dexamethasone, aminoguanidine (an inducible NOS inhibitor), 5-hydroxydecanoic acid (a mitochondrial K(+) /ATP channel blocker), or wortmannin [a phosphoinositide-3-kinase (PI3K) inhibitor] in order to analyze their effect on the protection induced by LPS. LPS afforded significant morphologic and functional protection in eyes exposed to intense light. Light damage induced an increase in mitochondrial Bax/cytoplasmic Bax ratio, and lipid peroxidation which were prevented by LPS. Dexamethasone and wortmannin (but not aminoguanidine or 5-hydroxydecanoic acid) prevented the effect of LPS. Moreover, wortmannin prevented the effect of LPS on p-Akt levels. These results indicate that LPS provides retinal protection against light-induced stress, probably through a PI3K/Akt-dependent mechanism., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Protein-protein interactions mediated by ubiquitin-like (Ubl) modifications occur as mono-Ubl or poly-Ubl chains. Proteins that regulate poly-SUMO (small ubiquitin-like modifier) chain conjugates play important roles in cellular response to DNA damage, such as those caused by cancer radiation therapy. Additionally, high atomic number metals, such as gold, preferentially absorb much more X-ray energy than soft tissues, and thus augment the effect of ionizing radiation when delivered to cells. In this study, we demonstrate that conjugation of a weak SUMO-2/3 ligand to gold nanoparticles facilitated selective multivalent interactions with poly-SUMO-2/3 chains leading to efficient inhibition of poly-SUMO-chain-mediated protein-protein interactions. The ligand-gold particle conjugate significantly sensitized cancer cells to radiation but was not toxic to normal cells. This study demonstrates a viable approach for selective targeting of poly-Ubl chains through multivalent interactions created by nanoparticles that can be chosen based on their properties, such as abilities to augment radiation effects.

The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.

In order to understand the consequences of radiation a thorough understanding of the radiobiological mechanisms of the molecular up to the clinical level is of importance. Radiobiology therefore combines the basic principles of physics as well as biology and medicine and is concerned with the action of radiation from the subcellular level up to the living organism. Topics of interest and relevance are covered in much more broadness as is possible in the short following article in the literature to which the interested reader is referred to. Classical books in this field were written by Steel et al. (1989) as well as by Hall (1994). Topics usually covered by radiobiological reviews are the classification of different types of radiation, cell cycle dependency of radiation effects, types of radiation damage and cell death, dose response curves, measurement of radiation damage, the oxygen effect, relative biological effectiveness, the influence of dose rate, and several other important research areas. This short overview will concentrate on a subset of radiobiological topics of high importance and relative novelty.

• In this study, we examined the biochemical and physiological functions of Nicotiana benthamiana S1 domain-containing Transcription-Stimulating Factor (STF) using virus-induced gene silencing (VIGS), cosuppression, and overexpression strategies. • STF : green fluorescent protein (GFP) fusion protein colocalized with sulfite reductase (SiR), a chloroplast nucleoid-associated protein also present in the stroma. Full-length STF and its S1 domain preferentially bound to RNA, probably in a sequence-nonspecific manner. • STF silencing by VIGS or cosuppression resulted in severe leaf yellowing caused by disrupted chloroplast development. STF deficiency significantly perturbed plastid-encoded multimeric RNA polymerase (PEP)-dependent transcript accumulation. Chloroplast transcription run-on assays revealed that the transcription rate of PEP-dependent plastid genes was reduced in the STF-silenced leaves. Conversely, the exogenously added recombinant STF protein increased the transcription rate, suggesting a direct role of STF in plastid transcription. Etiolated seedlings of STF cosuppression lines showed defects in the light-triggered transition from etioplasts to chloroplasts, accompanied by reduced light-induced expression of plastid-encoded genes. • These results suggest that STF plays a critical role as an auxiliary factor of the PEP transcription complex in the regulation of plastid transcription and chloroplast biogenesis in higher plants., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca(2+) homeostasis manifested by Ca(2+) rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

Class II histone deacetylases in humans and other model organisms undergo nucleocytoplasmic shuttling. This unique functional regulatory mechanism has been well elucidated in eukaryotic organisms except in plant systems. In this study, we have paved the baseline evidence for the cytoplasmic and nuclear localization of Class II HDAs as well as their mRNA expression patterns. RT-PCR analysis on the different vegetative parts and developmental stages reveal that Class II HDAs are ubiquitously expressed in all tissues with minimal developmental specificity. Moreover, stable and transient expression assays using HDA-YFP/GFP fusion constructs indicate cytoplasmic localization of HDA5, HDA8, and HDA14 further suggesting their potential for nuclear transport and deacetylating organellar and cytoplasmic proteins. Organelle markers and stains confirm HDA14 to abound in the mitochondria and chloroplasts while HDA5 localizes in the ER. HDA15, on the other hand, shuttles in and out of the nucleus upon light exposure. In the absence of light, it is exported out of the nucleus where further re-exposition to light treatments signals its nuclear import. Unlike HDA5 which binds with 14-3-3 proteins, HDA15 fails to interact with these chaperones. Instead, HDA15 relies on its own nuclear localization and export signals to navigate its subcellular compartmentalization classifying it as a Class IIb HDA. Our study indicates that nucleocytoplasmic shuttling is indeed a hallmark for all eukaryotic Class II histone deacetylases.

Background: The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth., Methods and Findings: Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism., Conclusions: Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.

Phytochrome B (phyB) is the major informational photoreceptor in light-grown plants. The phyB polypeptide is folded into two domains, the N-terminal domain and the C-terminal domain. The N-terminal domain covalently binds to the chromophore via a particular cysteine residue, which allows the holoprotein to absorb light and undergo a photoreversible conformational change. The N-terminal domain of phyB interacts with transcription factors, such as PIF3 (PHYTOCHROME-INTERACTING FACTOR 3), to transduce the light signal to downstream components. Since substitution of the chromophore attachment site, Cys357, with alanine (C357A) abolishes the biological activity of Arabidopsis phyB, the covalent attachment with the chromophore is widely assumed to be necessary for phyB signal transduction. In this study, we show that Arabidopsis phyB is capable of transducing signals with a non-covalently retained chromophore. Substituting the Tyr276 residue of phyB with histidine (Y276H) is known to confer constitutive phyB signaling. PhyB containing both Y276H and C357A substitutions exhibited light-independent biological activity in transgenic Arabidopsis plants in a chromophore-dependent manner. Spectrophotometric analysis showed that the N-terminal domain of phyB containing just the C357A substitution could retain the chromophore non-covalently. The N-terminal domain containing both the Y276H and C357A substitutions interacted with PIF3 in a light-independent but chromophore-dependent fashion in yeast two-hybrid assays. From these results, we conclude that the constitutive phyB signaling conferred by Y276H requires the chromophore, but that the chromophore does not need to be covalently bonded to phyB.

Background: Photodynamic therapy (PDT) is considered a promising new strategy for ovarian cancer treatment. As the key component in PDT, photosensitizer metabolism and localization in cancer cells is particularly important., Materials and Methods: The localization of the photosensitizers hematoporphyrin monomethyl ether (HMME) and hypocrellin B (HB) were determined in the ovarian cancer cell lines SKOV3 and NuTu-19 by fluorescence microscopy and laser scanning confocal microscopy(LSCM). A JD801 image analysis system was used to analyze the fluorescence intensity of the photosensitizers in the cells. The phototoxicity of both drugs to the cancer cells was determined by MTT assay., Results: Both photosensitizers were mainly distributed in the cytoplasm. Drug uptake reached a peak after 4 h incubation with HB and after 3 h incubation with HMME. Within a certain range, the higher the concentration, the stronger the fluorescence became and at 40 μg/ml, the intracellular photosensitizer had reached saturation. Based on these results PDT was applied to SKOV3 cells. All the cells were killed when the photosensizer dose reached 40 μg/ml., Conclusion: PDT is an effective therapy for ovarian cancer cells.

Track structure studies using PARTRAC have been performed with the aim to investigate the possibility of revealing information on initiating targets and mechanisms of bystander effects mediated by signals released into the culture medium. Dependences on radiation dose have been assessed for alternative signal emission scenarios, defined by required energy deposits in a number of subcellular targets, mimicking e.g. mitochondria as hypothetical targets for the release of signals. The simulation results agree with target theory, and elucidate the characteristic dose for signal emission as a function of target topology, size and activation energy. The observed dose dependence of bystander cell kill in medium transfer experiments is not as steep as predicted by the considered simple signal emission scenarios with a single or even multiple hits to the hypothetical targets. This has been resolved by accounting for variations in cellular characteristics among the irradiated cells.

Cell cycle checkpoints play an important role in regulation of DNA repair pathways. However, how the regulation occurs throughout the cell cycle remains largely unknown. Here we demonstrate that nucleotide excision repair (NER) is regulated by the ATR/p53 checkpoint via modulation of XPA nuclear import and that this regulation occurs in a cell cycle-dependent manner. We show that depletion of p53 abrogated the UV-induced nuclear translocation of XPA, while silencing of Chk1 or MAPKAP Kinase-2 (MK2) had no effect. Inhibition of p53 transcriptional activities and silencing of p53-Ser15 phosphorylation also reduced the damage-induced XPA nuclear import. Furthermore, in G1-phase cells the majority of XPA remained in the cytoplasm even after UV treatment. By contrast, while most of the XPA in S-phase cells was initially located in the cytoplasm before DNA damage, UV irradiation stimulated bulk import of XPA into the nucleus. Interestingly, the majority of XPA molecules always were located in the nucleus in G2-phase cells no matter whether the DNA was damaged or not. Consistently, the UV-induced Ser15 phosphorylation of p53 occurred mainly in S-phase cells, and removal of cyclobutane pyrimidine dimers (CPDs) was much more efficient in S-phase cells than in G1-phase cells. Our results suggest that upon DNA damage in S phase, NER could be regulated by the ATR/p53-dependent checkpoint via modulation of the XPA nuclear import process. In contrast, the nuclear import of XPA in G(1) or G(2) phase appears to be largely independent of DNA damage and p53.

Background: Rhodopseudomonas palustris (R. palustris) is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach., Methodology/principal Findings: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM) but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC) strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain., Conclusions/significance: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth through both common and divergent metabolic mechanisms.

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations.

Identification of biomarker proteins in the retina and retinal pigment epithelium (RPE) under oxidative stress may imply new insights into signaling mechanisms of retinal degeneration at the molecular level. Proteomic data from an in vivo mice model in constant light and an in vitro oxidative stress model are compared to controls under normal conditions. Our proteomic study shows that prohibitin is involved in oxidative stress signaling in the retina and RPE. The identity of prohibitin in the retina and RPE was studied using 2D electrophoresis, immunohistochemistry, western blot, and mass spectrometry analysis. Comparison of expression levels with apoptotic markers as well as translocation between mitochondria and the nucleus imply that the regulation of prohibitin is an early signaling event in the RPE and retina under oxidative stress. Immunohistochemical analysis of murine aged and diabetic eyes further suggests that the regulation of prohibitin in the RPE/retina is related to aging- and diabetes-induced oxidative stress. Our proteomic approach implies that prohibitin in the RPE and the retina could be a new biomarker protein of oxidative stress in aging and diabetes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanoparticles leads to endosome rupture and escape of the nanoparticles into the cytosol without affecting the cells' viability. The low light intensity of our experiments allows us to rule out photothermal effects as the underlying mechanism, and we present results that suggest photoinduced radicals as the photogenerated active species. This nonthermal mechanism may also be important in the context of cell death at higher laser intensities, which had been reported previously.

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.

The messenger RNA of the rice seed storage protein prolamine is targeted to the endoplasmic reticulum (ER) membranes surrounding prolamine protein bodies via a mechanism, which is dependent upon both RNA sorting signals and the actin cytoskeleton. In this study we have used an RNA bait corresponding to the previously characterized 5'CDS prolamine cis-localization sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the cis-localization RNA bait sequence led to the capture of a much larger number of proteins, 18 of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analyzed to date show good to excellent specificity for binding to cis-localization sequences over the control RNA bait. Temporal expression studies showed that steady state protein levels for one RNA binding protein, RBP-A, paralleled prolamine gene expression. Immunoprecipitation studies showed that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression. Using confocal immunofluorescence microscopy, RBP-A was found to be distributed to multiple compartments in the cell. In addition to the nucleus, RBP-A co-localizes with microtubules and is associated with cortical ER membranes. Collectively, these results indicate that employing a combination of in vitro binding and in vivo binding and localization studies is a valid strategy for the identification of putative prolamine mRNA binding proteins, such as RBP-A, which play a role in controlling expression of storage protein mRNAs in the cytoplasm.

Photodynamic therapy is an effective treatment for tumors that involves the administration of light-activated photosensitizers. However, most photosensitizers are insoluble and non-specific. To target the acid environment of tumor sites, we synthesized three poly(ethylene glycol) methacrylate-co-2-(diisopropylamino)ethyl methacrylate (PEGMA-co-DPA) copolymers capable of self-assembly to form pH sensitive nanoparticles in an aqueous environment, as a means of encapsulating the water-insoluble photosensitizer, meso-tetra(hydroxyphenyl)chlorin (m-THPC). The critical aggregation pH of the PEGMA-co-DPA polymers was 5.8-6.6 and the critical aggregation concentration was 0.0045-0.0089 wt% at pH 7.4. Using solvent evaporation, m-THPC loaded nanoparticles were prepared with a high drug encapsulation efficiency (approximately 89%). Dynamic light scattering and transmission electron microscopy revealed the spherical shape and 132 nm diameter of the nanoparticles. The in vitro release rate of m-THPC at pH 5.0 was faster than at pH 7.0 (58% versus 10% m-THPC released within 48 h, respectively). The in vitro photodynamic therapy efficiency was tested with the HT-29 cell line. m-THPC loaded PEGMA-co-DPA nanoparticles exhibited obvious phototoxicity in HT-29 colon cancer cells after light irradiation. The results indicate that these pH sensitive nanoparticles are potential carriers for tumor targeting and photodynamic therapy.

Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein previously implicated in various signaling pathways, but whose function remains enigmatic. Here, we provide evidence that RanBPM functions as an activator of apoptotic pathways induced by DNA damage. First, transient expression of RanBPM in HeLa cells induced cell death through caspase activation, and in the long-term, forced expression of RanBPM impaired cell viability. RanBPM COOH-terminal domain stimulated the ability of RanBPM to induce caspase activation, whereas this activity was negatively regulated by the central SPRY domain. Second, small interfering RNA-directed knockdown of RanBPM prevented DNA damage-induced apoptosis, as evidenced by the marked reduction in caspase-3 and caspase-2 activation. This correlated with a magnitude fold increase in the survival of RanBPM-depleted cells. Following ionizing radiation treatment, we observed a progressive relocalization of RanBPM from the nucleus to the cytoplasm, suggesting that the activation of apoptotic pathways by RanBPM in response to ionizing radiation may be regulated by nucleocytoplasmic trafficking. Finally, RanBPM downregulation was associated with a marked decrease of mitochondria-associated Bax, whereas Bcl-2 overall levels were dramatically upregulated. Overall, our results reveal a novel proapoptotic function for RanBPM in DNA damage-induced apoptosis through the regulation of factors involved in the mitochondrial apoptotic pathway.

The stress-activated protein kinases (SAPKs), namely, p38 and JNK, are members of the mitogen-activated protein kinase family and are important determinants of cell fate when cells are exposed to environmental stresses such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks), which are in turn activated by various SAP2K kinases (SAP3Ks). Because conventional methods, such as immunoblotting using phospho-specific antibodies, measure the average activity of SAP3Ks in a cell population, the intracellular dynamics of SAP3K activity are largely unknown. Here, we developed a reporter of SAP3K activity toward the MKK6 SAP2K, based on fluorescence resonance energy transfer, that can uncover the dynamic behavior of SAP3K activation in cells. Using this reporter, we demonstrated that SAP3K activation occurs either synchronously or asynchronously among a cell population and in different cellular compartments in single cells, depending on the type of stress applied. In particular, SAP3Ks are activated by epidermal growth factor and osmostress on the plasma membrane, by anisomycin and UV in the cytoplasm, and by etoposide in the nucleus. These observations revealed previously unknown heterogeneity in SAPK responses and supplied answers to the question of the cellular location in which various stresses induce stimulus-specific SAPK responses.

Changes in the level of oxidative damage to proteins in CD1 outbred mice gamma irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to gamma radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.

Holoparasitic plants such as Orobanche spp. have lost their photosynthetic ability, so photoresponses to optimize photosynthesis are not necessary in these plants. Photoresponses are also involved in the regulation of plant development but the photoresponses of holoparasites have not been characterized in detail. In this study, the phytochrome (phy)-related photoresponse of Orobanche minor was investigated. Its photoreceptor, phytochrome A (OmphyA), was also characterized. Light effects on germination, shoot elongation, anthocyanin biosynthesis, and OmphyA expression and subcellular localization were analyzed. Red light (R):far-red light (FR) reversible inhibition of O. minor seed germination demonstrated that phy-mediated responses are retained in this holoparasite. Shoot elongation was inhibited by FR but not by R. This pattern is unique among known patterns of plant photoresponses. Additionally, molecular analysis showed that OmphyA is able to respond to the light signals. Interestingly, the unique pattern of photoresponses in O. minor seems to have been modified for adaptation to its parasitic life cycle. We hypothesize that this alteration has resulted from the loss or alteration of some phy-signaling components. Elucidation of altered components in phy signaling in this parasite will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.

Corticotropin releasing factor receptor type 1 (CRF1), a coordinator of the body responses to stress, is also expressed in human skin, where it undergoes alternative splicing. Since the epidermis is continuously exposed to the environmental stress, human keratinocytes were chosen to study the biological role of CRF1 alternative splicing. The expression pattern of CRF1 isoforms depended on cell density, presence or absence of serum, and exposure to ultraviolet irradiation (UVR). Only two isoforms alpha and c were predominantly localized to the cell membrane, with only CRF1alpha being efficient in stimulating cAMP responding element (CRE). CRF1d, f and g had intracellular localization, showing no or very low (g) activation of CRE. The co-expression of CRF1alpha with d, f or g resulted in intracellular retention of both isoforms suggesting dimerization confirmed by detection of high molecular weight complexes. The soluble isoforms e and h were diffusely distributed in the cytoplasm or localized to the ER, respectively, and additionally found in culture medium. These findings suggest that alternatively spliced CRF1 isoforms can interact and modify CRF1alpha subcellular localization, thus affecting its activity. We suggest that alternative splicing of CRF1 may play an important role in the regulation of skin cell phenotype with potential implications in pathology.