ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure.

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

Rapid and simple identification of trace amounts of sodium azide in beverages and bodily fluids followed by derivatization and liquid chromatography-electrospray ionization tandem mass spectrometry.

RATIONALE: Sodium azide (NaN3 ) is a toxic chemical agent to humans by ingestion and inhalation with a growing number of intentional exposures and accidental cases over the last few decades. Due to its low molecular weight and lack of any chromophore, its retention and detection by reverse-phase liquid chromatography-ultraviolet-mass spectrometry methods are a challenging task. METHODS: To be able to confirm azide exposure, we have developed a method to identify azide in both beverages and bodily fluids. The identification of azide (N3 - ) is based on derivatization with N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) at 25°C for 15 min followed by LC/ESI-MS/MS analysis, with no other sample preparation. RESULTS: The azide after derivatization (CAX-N3 ) was stable, retainable by LC and sensitively detected by selected reaction monitoring. The ESI-MS/MS fragmentation of the M+ precursor ion produced characteristic product ions at m/z 118, 100, 91 and 86. The calibration curves for CAX-N3 showed linearity over two orders of magnitude with R2 value of 0.99. Low limits of identification of 0.1-0.5 ng/mL were obtained in all investigated matrices (drinking water, tea, orange juice, plasma and urine). CONCLUSIONS: Compared with previously reported chromatography-based methods, this method that was based on derivatization and LC/ESI-MS/MS analysis was substantially more sensitive, simpler and faster. The method can be used for forensic investigation to confirm azide exposure from fatal use to much smaller intoxication dose.

A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s-1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

Asymmetric Azidation under Hydrogen Bonding Phase-Transfer Catalysis: A Combined Experimental and Computational Study.

Asymmetric catalytic azidation has increased in importance to access enantioenriched nitrogen containing molecules, but methods that employ inexpensive sodium azide remain scarce. This encouraged us to undertake a detailed study on the application of hydrogen bonding phase-transfer catalysis (HB-PTC) to enantioselective azidation with sodium azide. So far, this phase-transfer manifold has been applied exclusively to insoluble metal alkali fluorides for carbon-fluorine bond formation. Herein, we disclose the asymmetric ring opening of meso aziridinium electrophiles derived from ß-chloroamines with sodium azide in the presence of a chiral bisurea catalyst. The structure of novel hydrogen bonded azide complexes was analyzed computationally, in the solid state by X-ray diffraction, and in solution phase by 1H and 14N/15N NMR spectroscopy. With N-isopropylated BINAM-derived bisurea, end-on binding of azide in a tripodal fashion to all three NH bonds is energetically favorable, an arrangement reminiscent of the corresponding dynamically more rigid trifurcated hydrogen-bonded fluoride complex. Computational analysis informs that the most stable transition state leading to the major enantiomer displays attack from the hydrogen-bonded end of the azide anion. All three H-bonds are retained in the transition state; however, as seen in asymmetric HB-PTC fluorination, the H-bond between the nucleophile and the monodentate urea lengthens most noticeably along the reaction coordinate. Kinetic studies corroborate with the turnover rate limiting event resulting in a chiral ion pair containing an aziridinium cation and a catalyst-bound azide anion, along with catalyst inhibition incurred by accumulation of NaCl. This study demonstrates that HB-PTC can serve as an activation mode for inorganic salts other than metal alkali fluorides for applications in asymmetric synthesis.

Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.

Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering.

Cellular energy required for the maintenance of cellular life is stored in the form of adenosine triphosphate (ATP). Understanding cellular mechanisms, including ATP-dependent metabolisms, is crucial for disease diagnosis and treatment, including drug development and investigation of new therapeutic systems. As an ATP-dependent metabolism, endocytosis plays a key role not only in the internalization of molecules but also in processes including cell growth, differentiation, and signaling. To understand cellular mechanisms including endocytosis, many techniques ranging from molecular approaches to spectroscopy are used. Surface-enhanced Raman scattering (SERS) is shown to provide valuable label-free molecular information from living cells. In this study, receptor-mediated endocytosis was investigated with SERS by inhibiting endocytosis with ATP depletion agents: sodium azide (NaN3) and 2-deoxy-d-glucose (dG). Human lung bronchial epithelium (Beas-2b) cells, normal prostate epithelium (PNT1A) cells, and cervical cancer epithelium (HeLa) cells were used as models. First, the effect of NaN3 and dG on the cells were examined through cytotoxicity, apoptosis-necrosis, ATP assay, and uptake inhibition analysis. An attempt to relate the spectral changes in the cellular spectra to the studied cellular events, receptor-mediated endocytosis inhibition, was made. It was found that the effect of two different ATP depletion agents can be discriminated by SERS, and hence receptor-mediated endocytosis can be tracked from single living cells with the technique without using a label and with limited sample preparation.

Copper-Catalyzed Azide-Alkyne Cycloaddition of Hydrazoic Acid Formed In Situ from Sodium Azide Affords 4-Monosubstituted-1,2,3-Triazoles.

We report a copper-catalyzed cycloaddition of hydrogen azide (hydrazoic acid, HN3) with terminal alkynes to form 4-substituted-1H-1,2,3-triazoles in a sustainable manner. Hydrazoic acid was formed in situ from sodium azide under acidic conditions to react with terminal alkynes in a copper-catalyzed reaction. Using polydentate N-donor chelating ligands and mild organic acids, the reactions were realized to proceed at room temperature under aerobic conditions in a methanol-water mixture and with 5 mol % catalyst loadings to afford 4-substituted-1,2,3-triazoles in high yields. This method is amenable on a wide range of alkyne substrates, including unprotected peptides, showing diverse functional group tolerance. It is applicable for late-stage functionalization synthetic strategies, as demonstrated in the synthesis of the triazole analogue of losartan. The preparation of orthogonally protected azahistidine from Fmoc-l-propargylglycine was realized on a gram scale. The hazardous nature of hydrazoic acid has been diminished as it forms in situ in <6% concentrations at which it is safe to handle. Reactions of distilled solutions of hydrazoic acid indicated its role as a reactive species in the copper-catalyzed reaction.

Is In-Service Granular Activated Carbon Biologically Active? An Evaluation of Alternative Experimental Methods to Distinguish Adsorption and Biodegradation in GAC.

In-service granular activated carbon (GAC) may transform into biological activated carbon (BAC) and remove contaminants through both adsorption and biodegradation, but it is difficult to determine its biodegradative capacity. One approach to understand the GAC biodegradative capacity is to compare the performance between unsterilized and sterilized GAC, but the sterilization methods may not ensure effective microbial inhibition and may affect adsorption. This study identified the 14C-glucose respiration rate as the best metric to evaluate the effectiveness of three sterilization methods: sodium azide addition, autoclaving, and γ irradiation. The sterilization protocols were refined, including continuously feeding 300 mg/L of sodium azide, three cycles of autoclaving, and 10-12 kGy of γ irradiation. Parallel minicolumn tests were conducted to identify sodium azide addition as the most broadly effective sterilization method with an insignificant effect on adsorption in most cases, except for the adsorption of anionic compounds under certain conditions. Nevertheless, this problem was solved by decreasing the azide dosage as long as it is still sufficient to provide effective microbial inhibition. This study helps to develop an approach that differentiates adsorption and biodegradation in GAC, which could be used by future studies to advance our understanding of BAC filtration.

Mutagenic effects of sodium azide on in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.).

INTRODUCTION: Breeding studies are commonly conducted to develop new cultivars with high yield levels and improved quality traits. Chemically-induced mutations are used to create genetic variations in wheat genomes. Various physical and chemical mutagens are used to increase frequency of mutations and facilitate the selection processes. Sodium azide (SA) is largely employed to induce mutations of the genes regulating essential traits. Such mutations may also elucidate gene functions of the mutant phenotypes. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through SA for mature embryo culture in wheat. METHODS AND RESULTS: Sodium azide mutagenesis was experimented with 4 treatment durations (1, 2, 3 and 4 h) and 5 treatment concentrations (0, 1, 2, 3 and 4 mM). Mature embryos were subjected to experimental treatments to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. Primarily, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Based on LD50 criterion, the optimum value was achieved at 1 h duration of 4 mM SA concentration. Afterwards, inter-primer binding site markers were applied to investigate polymorphism and genomic instability in the regenerated plants. CONCLUSIONS: Present findings revealed that efficiency of chemical mutagenesis could be improved through the use of molecular technology and such mutations may assist plant breeders in developing high-yield cultivars.

The inactivation efficacy of plasma-activated acetic acid against Salmonella Typhimurium cells and biofilm.

AIM: This study aimed to examine the inactivation efficacy of plasma-activated acetic acid (PAAA) against Salmonella Typhimurium cells and biofilm and elucidate underlying chemical inactivation pathway. METHODS AND RESULTS: PAAA was prepared by discharging plasma to 20 ml of 0.2% (v/v) acetic acid (AA) for 20 min (2.2 kHz and 8.4 kVpp). The count of cells and biofilms decreased by 5.71 log CFU ml-1 and 4 log CFU/cm2 after 10 min of treatment with 0.2% PAAA and 0.4% PAAA compared with control group (without any treatment), respectively. In 0.2% PAAA, the concentrations of hydrogen peroxide (H2 O2 ) and nitrate anions were directly proportional to the plasma discharge time, whilst nitrite anion (NO2 - ) was not detected. However, the pH values of both 0.2% PAAA and plasma-activated water were inversely proportional to the plasma discharge time. Treatment with catalase, L-histidine, D-mannitol and sodium azide inhibited the antibacterial activity of PAAA. CONCLUSION: H2 O2 , singlet oxygen, hydroxyl radical and NO2 - are involved in the generation and decomposition of peroxynitrous acid generated from PAAA functioned as intermediate agent, which could diffuse through cell membranes of bacteria and induce cell injury. SIGNIFICANCE AND IMPACT OF STUDY: This study provides the understanding of efficacy and selectivity of PAAA which could be a novel decontamination agent.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

Bis(oxiranes) Containing Cyclooctane Core: Synthesis and Reactivity towards NaN3.

Reactions of oxirane ring opening provide a powerful tool for regio- and stereoselective synthesis of polyfunctional and heterocyclic compounds, widely used in organic chemistry and drug design. Cyclooctane, alongside other medium-sized rings, is of interest as a novel molecular platform for the construction of target-oriented leads. Additionally, cyclooctane derivatives are well known to be prone to transannular reactions, which makes them a promising object in the search for novel approaches to polycyclic structures. In the present work, a series of cyclooctanediones was studied in Corey-Chaykovsky reactions, and novel spirocyclic bis(oxiranes) containing cyclooctane core, namely, 1,5-dioxadispiro[2.0.2.6]dodecane and 1,8-dioxadispiro[2.3.2.3]dodecane, were synthesized. Ring opening of the obtained bis(oxiranes) upon treatment with sodium azide was investigated, and it was found that the reaction path is determined by the reciprocal orientation of oxygen atoms in the oxirane moieties. Diastereomers of the bis(oxiranes) with cis-orientation underwent independent ring opening, supplying corresponding diazidodiols, while in the case of stereoisomers with trans-orientation, domino-like reactions occurred, including intramolecular nucleophilic attack and the formation of a novel three- or six-membered O-containing ring. Summarily, a straightforward approach to polyfunctional compounds containing cyclooctane or oxabicyclo[3.3.1]nonane cores, employing bis(oxiranes), was elaborated.

Potential therapies for sodium azide intoxication; a case report and review of the literature.

Intoxications with sodium azide are rare and in almost all cases lethal in doses above 700 mg or 10mg/kg. We report a case of a patient who ingested 2 grams of sodium azide as a suicide attempt. Sodium azide irreversible blocks cytochrome C oxidase by inhibiting oxidative phosphorylation leading to cell death. There is currently no antidote available. Our patient was treated with a range of therapies, on site, in the emergency department and in the intensive care unit, such as sodium thiosulphate, methylene blue, intralipid, extensive gastric lavage, whole bowel irrigation combined with pro-kinetics, hydroxocobalamin and exchange transfusion. During the clinical course the patient developed cardiac failure, for which veno-arterial ECMO and an intra-aortic balloon pump was placed. However, cardiac function did not recover, leading to discontinuation of treatment after 7 days. As literature on sodium azide intoxication is scarce, we conducted a review to present potential treatment options.

Iron is a ligand of SecA-like metal-binding domains in vivo.

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.

Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.

Uric Acid as a Photosensitizer in the Reaction of Deoxyribonucleosides with UV Light of Wavelength Longer than 300 nm: Identification of Products from 2'-Deoxycytidine.

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

Elucidation of the RNA-granule inducing sodium azide stress response through transcriptome analysis.

Sodium azide is a commonly used cytochrome oxidase inhibitor that leads to translation repression and RNA granule assembly. The global changes in mRNA abundance in response to this stressor are unknown. RGG-motif proteins Scd6 and Sbp1 are translation-repressors and decapping-activators that localize to and affect the assembly of RNA granules in response to sodium azide stress. Transcriptome-wide effects of these proteins remain unknown. To address this, we have sequenced transcriptome of the: a) wild type strain under unstressed and sodium azide stress, b) Δscd6 and Δsbp1 strains under unstressed and sodium azide stress. Transcriptome analysis identified altered abundance of many transcripts belonging to stress-responsive pathways which were further validated by qRT-PCR results. Abundance of several transcripts was altered in Δscd6/Δsbp1 under normal conditions and upon stress. Overall, this study provides critical insights into transcriptome changes in response to sodium azide stress and the role of RGG-motif proteins in these changes.