Photoenzymatic enantioselective intermolecular radical hydroalkylation.

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Modular Access to Chiral Amines via Imine Reductase-Based Photoenzymatic Catalysis.

The development of catalysts serves as the cornerstone of innovation in synthesis, as exemplified by the recent discovery of photoenzymes. However, the repertoire of naturally occurring enzymes repurposed by direct light excitation to catalyze new-to-nature photobiotransformations is currently limited to flavoproteins and keto-reductases. Herein, we shed light on imine reductases (IREDs) that catalyze the remote C(sp3)-C(sp3) bond formation, providing a previously elusive radical hydroalkylation of enamides for accessing chiral amines (45 examples with up to 99% enantiomeric excess). Beyond their natural function in catalyzing two-electron reductive amination reactions, upon direct visible-light excitation or in synergy with a synthetic photoredox catalyst, IREDs are repurposed to tune the non-natural photoinduced single-electron radical processes. By conducting wet mechanistic experiments and computational simulations, we unravel how engineered IREDs direct radical intermediates toward the productive and enantioselective pathway. This work represents a promising paradigm for harnessing nature's catalysts for new-to-nature asymmetric transformations that remain challenging through traditional chemocatalytic methods.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Hydrochar from corn stalk used as bio-asphalt modifier: High-temperature performance improvement.

Biomass utilization, even for conversion products like hydrochar or biochar, has an increasing demand because improper disposal can cause intensive pollution. In this study, hydrochar obtained by hydrothermal treatment of corn stalk was added to virgin asphalt as a novel modifier by manual stirring and high-speed shearing. This hydrochar-modified asphalt (HCMA) showed a better high-temperature performance compared to unmodified asphalt, and the optimized dosage was 6 wt% with Rutting Index reaching 76 °C, and its penetration and softening point reaching 31.70 (0.1 mm) and 54.70 °C, respectively. The macroscopic representation of modified asphalt was conducted by microscopic characterization methods such as Fourier Transform Infrared Spectroscopy (FTIR) and Gel Permeation Chromatography (GPC). It was demonstrated that the performance was improved by the good blending state between hydrochar and asphalt. The application of hydrochar in modifying asphalt can reduce pollution and enhance its high-temperature performance, which has a potentially extensive application prospect in pavement engineering in subtropical and tropical climate.

Spin-Regulated Electron Transfer and Exchange-Enhanced Reactivity in Fe4 S4 -Mediated Redox Reaction of the Dph2 Enzyme During the Biosynthesis of Diphthamide.

The [4Fe-4S]-dependent radical S-adenosylmethionine (SAM) proteins is one of large families of redox enzymes that are able to carry a panoply of challenging transformations. Despite the extensive studies of structure-function relationships of radical SAM (RS) enzymes, the electronic state-dependent reactivity of the [4Fe-4S] cluster in these enzymes remains elusive. Using combined MD simulations and QM/MM calculations, we deciphered the electronic state-dependent reactivity of the [4Fe-4S] cluster in Dph2, a key enzyme involved in the biosynthesis of diphthamide. Our calculations show that the reductive cleavage of the S-C(γ) bond is highly dependent on the electronic structure of [4Fe-4S]. Interestingly, the six electronic states can be classified into a low-energy and a high-energy groups, which are correlated with the net spin of Fe4 atom ligated to SAM. Due to the driving force of Fe4-C(γ) bonding, the net spin on the Fe4 moiety dictate the shift of the opposite spin electron from the Fe1-Fe2-Fe3 block to SAM. Such spin-regulated electron transfer results in the exchange-enhanced reactivity in the lower-energy group compared with those in the higher-energy group. This reactivity principle provides fundamental mechanistic insights into reactivities of [4Fe-4S] cluster in RS enzymes.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway.

BACKGROUND/AIMS: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS), toll-like receptor 4 (TLR4), receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis), which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP) channel opening against high glucose-induced cardiac injury and inflammation. METHODS: H9c2 cardiac cells were treated with 35 mM glucose (HG) to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP) and secretion of inflammatory cytokines were measured as injury indexes. RESULTS: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (an inhibitor of TLR4) co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener) or pinacidil (Pin, a non-selective KATP channel opener) or N-acetyl-L-cysteine (NAC, a ROS scavenger) pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker) or glibenclamide (Gli, a non-selective KATP channel blocker) pre-treatment did not aggravate HG-induced injury and inflammation. CONCLUSION: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations.

P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (-)-naseseazine C (2) through intramolecular C-N bond formation and intermolecular C-C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C-N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7-H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π-π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3-C6' bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3-C6' bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C-N cyclization and intermolecular C-C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.

Mechanistic Insights from the Crystal Structure and Computational Analysis of the Radical SAM Deaminase DesII.

Radical S-adenosyl-L-methionine (SAM) enzymes couple the reductive cleavage of SAM to radical-mediated transformations that have proven to be quite broad in scope. DesII is one such enzyme from the biosynthetic pathway of TDP-desosamine where it catalyzes a radical-mediated deamination. Previous studies have suggested that this reaction proceeds via direct elimination of ammonia from an α-hydroxyalkyl radical or its conjugate base (i.e., a ketyl radical) rather than 1,2-migration of the amino group to form a carbinolamine radical intermediate. However, without a crystal structure, the active site features responsible for this chemistry have remained largely unknown. The crystallographic studies described herein help to fill this gap by providing a structural description of the DesII active site. Computational analyses based on the solved crystal structure are consistent with direct elimination and indicate that an active site glutamate residue likely serves as a general base to promote deprotonation of the α-hydroxyalkyl radical intermediate and elimination of the ammonia group.

Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase.

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.

Hydrogen sulfide inhibits abnormal proliferation of lymphocytes via AKT/GSK3ß signal pathway in systemic lupus erythematosus patients.

BACKGROUND/AIM: The abnormal activation of the AKT/GSK3ß signal pathway in lymphocytes from systemic lupus erythematosus (SLE) patients plays an important role in the pathogenesis of the disease. Recently Hydrogen sulfide (H2S) has been recognized as a crucial gaseous signaling molecule, involved in regulation of cell proliferation. However, the role of H2S in regulating the abnormal activation of lymphocytes from SLE patients has not been established. This study was conducted to investigate the effect of H2S on lymphocytes and to explore the mechanisms involved. METHODS: The lymphocytes were isolated from SLE patients with or without renal disease and healthy controls. The cells were treated as indicated in each experiment. Cell viability was analyzed by CCK-8. Cell cycle distribution was determined by flow cytometry. Western blot was used to detect the expression of phosphorylated AKT (ser473), GSK3ß (ser9) and CDK2, p27(Kip1) and p21(WAF1/CIP1). RESULTS: Our findings showed that proliferation of lymphocytes was stimulated following treatment with NaHS (a H2S donor) at low NaHS concentrations (<1mM) but inhibited at high NaHS concentrations (>2mM). Similar results were observed using GYY4137, which is a slow-releasing H2S donor. Pretreatment of lymphocytes from SLE patients with NaHS at high concentrations prior to exposure to phytohemagglutinin (PHA) significantly attenuated proliferation, evidenced by decrease in cell viability and S phase distribution of cell cycle. Pretreatment with NaHS decreased PHA-induced expression of CDK2, phosphorylation levels of AKT (ser473) and GSK3ß (ser9) and increased the expression of p27(Kip1) and p21(WAF1/CIP1). Moreover, pretreatment with NaHS blunted the stimulation of SLE lymphocyte proliferation by GSK3ß inhibitor lithium chloride. CONCLUSION: These results demonstrate that H2S inhibits the abnormal activation of lymphocytes from SLE patients throuqh the AKT/GSK3ß signal pathway.

Exogenous hydrogen sulfide protects against doxorubicin-induced inflammation and cytotoxicity by inhibiting p38MAPK/NFκB pathway in H9c2 cardiac cells.

BACKGROUND/AIM: We have demonstrated that exogenous hydrogen sulfide (H2S) protects H9c2 cardiac cells against the doxorubicin (DOX)-induced injuries by inhibiting p38 mitogen-activated protein kinase (MAPK) pathway and that the p38 MAPK/nuclear factor-κB (NF-κB) pathway is involved in the DOX-induced inflammatory response and cytotoxicity. The present study attempts to test the hypothesis that exogenous H2S might protect cardiomyocytes against the DOX-induced inflammation and cytotoxicity through inhibiting p38 MAPK/NF-κB pathway. METHODS: H9c2 cardiac cells were exposed to 5µM DOX for 24 h to establish a model of DOX cardiotoxicity. The cells were pretreated with NaHS( a donor of H2S) or other drugs before exposure to DOX. Cell viability was analyzed by cell counter kit 8 ( CCK-8), The expression of NF-κB p65 and inducible nitric oxide synthase (iNOS) was detected by Western blot assay. The levels of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our findings demonstrated that pretreatment of H9c2 cardiac cells with NaHS for 30 min before exposure to DOX markedly ameliorated the DOX-induced phosphorylation and nuclear translocation of NF-κB p65 subunit. Importantly, the pretreatment with NaHS significantly attenuated the p38 MAPK/NF-κB pathway-mediated inflammatory responses induced by DOX, as evidenced by decreases in the levels of IL-1ß, IL-6 and TNF-α. In addition, application of NaHS or IL-1ß receptor antagonist (IL-1Ra) or PDTC (an inhibitor of NF-κB) attenuated the DOX-induced expression of iNOS and production of nitric oxide (NO), respectively. Furthermore, IL-1Ra also dramatically reduced the DOX-induced cytotoxicity and phosphorylation of NF-κB p65. The pretreatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS) prior to exposure to DOX depressed the phosphorylation of NF-κB p65 induced by DOX. CONCLUSION: The present study has demonstrated the new mechanistic evidence that exogenous H2S attenuates the DOX-induced inflammation and cytotoxicity by inhibiting p38 MAPK/NF-κB pathway in H9c2 cardiac cells. We also provide novel data that the interaction between NF-κB pathway and IL-1ß is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac cells.

Inhibition of ROS-activated p38MAPK pathway is involved in the protective effect of H2S against chemical hypoxia-induced inflammation in PC12 cells.

We have demonstrated the neuroprotection of hydrogen sulfide (H2S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H2S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl2, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl2-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl2-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl2-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl2-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H2S donor, 400 µmol/l) for 30 min before exposure to CoCl2 markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H2S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.

Photoenzymatic Enantioselective Intermolecular Radical Hydroamination.

Since the discovery of Hofmann-Löffler-Freytag reaction more than 130 years ago, nitrogen-centered radicals have been widely studied in both structures and reactivities1-2. Nevertheless, catalytic enantioselective intermolecular radical hydroamination remains a challenge due to the existence of side reactions, short lifetime of nitrogen-centered radicals, and lack of understanding of the fundamental catalytic steps. In chemistry, nitrogen-centered radicals are produced with radical initiators, photocatalysts, or electrocatalysts. On the other hand, the generation and reaction of nitrogen-centered radicals are unknown in nature. Here we report a pure biocatalytic system by successfully repurposing an ene-reductase through directed evolution for the photoenzymatic production of nitrogen-centered radicals and enantioselective intermolecular radical hydroaminations. These reactions progress efficiently at room temperature under visible light without any external photocatalysts and exhibit excellent enantioselectivities. Detailed mechanistic study reveals that the enantioselectivity originates from the radical-addition step while the reactivity originates from the ultrafast photoinduced electron transfer (ET) from reduced flavin mononucleotide (FMNH-) to nitrogen-containing substrates.

Reduced expression of Krüppel-like factor 17 is related to tumor growth and poor prognosis in lung adenocarcinoma.

Krüppel-like factor 17 (KLF17), a new member of the Krüppel-like factors (KLFs), has been reported to be a negative regulator of epithelial-mesenchymal transition (EMT) and metastasis in breast cancer. However, the biological role and clinical significance of KLF17 in lung adenocarcinoma has been less clear. In the present study, we showed that KLF17 expression was decreased in lung adenocarcinoma. Reduced expression of KLF17 was correlated significantly with a short survival time in patients with lung adenocarcinoma (P<0.0001). Moreover, KLF17 expression was an independent prognostic indicator for patients with lung adenocarcinoma. KLF17 expression level was correlated with the tumor stage (P=0.016) and tumor size (P=0.001) in lung adenocarcinoma. Overexpression of KLF17 inhibited cell growth in A549 and PC-9 cell lines. In conclusion, it is possible that KLF17 inhibits tumor growth in lung adenocarcinoma. The reduced expression of KLF17 is a valuable prognostic indicator for patients with lung adenocarcinoma, and KLF17 could be a novel target for treatment of lung adenocarcinoma.

Inhibition of ROS-activated ERK1/2 pathway contributes to the protection of H2S against chemical hypoxia-induced injury in H9c2 cells.

Hydrogen sulfide (H(2)S) has been shown to exert cardioprotective effects. However, the roles of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in H(2)S-induced cardioprotection have not been completely elucidated. In this study, cobalt chloride (CoCl(2)), a chemical hypoxia mimetic agent, was applied to treat H9c2 cells to establish a chemical hypoxia-induced cardiomyocyte injury model. The results showed that pretreatment with NaHS (a donor of H(2)S) before exposure to CoCl(2) attenuated the decreased cell viability, the increased apoptosis rate, the loss of mitochondrial membrane potential (ΔΨm), and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cells. Exposure of H9c2 cells to CoCl(2) or hydrogen peroxide (H(2)O(2)) upregulated expression of phosphorylated (p) ERK1/2, which was reduced by pretreatment with NaHS or N-acetyl-L-cysteine, a ROS scavenger. More importantly, U0126, a selective inhibitor of ERK1/2, mimicked the above cytoprotection of H(2)S against CoCl(2)-induced injury in H9c2 cells. In conclusion, these results indicate that H(2)S protects H9c2 cells against chemical hypoxia-induced injury partially by inhibiting ROS-mediated activation of ERK1/2.

Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress.

The roles of hydrogen sulfide (H(2)S) and endoplasmic reticulum (ER) stress in doxorubicin (DOX)-induced cardiotoxicity are still unclear. This study aimed to dissect the hypothesis that H(2)S could protect H9c2 cells against DOX-induced cardiotoxicity by inhibiting ER stress. Our results showed that exposure of H9c2 cells to DOX significantly inhibited the expression and activity of cystathionine-γ-lyase (CSE), a synthetase of H(2)S, accompanied by the decreased cell viability and the increased reactive oxygen species (ROS) accumulation. In addition, exposure of cells to H(2)O(2) (an exogenous ROS) mimicked the inhibitory effect of DOX on the expression and activity of CSE. Pretreatment with N-acetyl-L: -cysteine (NAC) (a ROS scavenger) attenuated intracellular ROS accumulation, cytotoxicity, and the inhibition of expression and activity of CSE induced by DOX. Notably, the ER stress-related proteins, including glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were obviously upregulated in DOX-treated H9c2 cells. Pretreatment with sodium hydrosulfide (NaHS, a H(2)S donor) before DOX exposure markedly suppressed DOX-induced overexpressions of GRP78 and CHOP, cytotoxicity and oxidative stress. In conclusion, we have demonstrated that ROS-mediated inhibition of CSE is involved in DOX-induced cytotoxicity in H9c2 cells, and that exogenous H(2)S can confer protection against DOX-induced cardiotoxicity partly through inhibition of ER stress.

Heat shock protein 90 mediates cytoprotection by H2S against chemical hypoxia-induced injury in PC12 cells.

1. Increasing evidence indicates that hydrogen sulphide (H2S) may serve as an important biological cytoprotective agent. Heat shock protein (Hsp) 90 can attenuate stress-induced injury. However, whether Hsp90 mediates the cytoprotective effect of H2S against chemical hypoxia-induced injury in PC12 cells is not known. 2. In the present study, CoCl2 (a chemical hypoxia mimetic) was used to treat PC12 cells to create a model of chemical hypoxia. To explore the role of Hsp90 in the cytoprotection afforded by H2S against chemical hypoxia-induced injury, 2 µmol/L 17-allylaminogeldanamycin (17-AAG), a selective inhibitor of Hsp90, was administered for 30 min prior to preconditioning with 400 µmol/L NaHS, followed by chemical hypoxia. 3. Cobalt chloride reduced cell viability (by 52.7 ± 1.5%), increased PC12 cell apoptosis (by 42.1 ± 1.5%), induced reactive oxygen species (ROS) by 3.79% compared with control and induced the dissipation of mitochondrial membrane potential (MMP) by 2.56% compared with control. 4. Pretreatment of PC12 cells with 100-400 µmol/L sodium hydrosulphide (NaHS), an H2S donor, for 3 h prior to exposure to 600 µmol/L CoCl2 provided significant, concentration-dependant protection to PC12 cells against CoCl2-induced cytotoxicity. Specifically, pretreatment of PC12 cells with 400 µmol/L NaHS decreased apoptosis to 16.77 ± 1.77% and blocked the CoCl2-induced increase in ROS production and loss of MMP. 5. At 400 µmol/L, NaHS upregulated Hsp90 in a time-dependant manner (over the period 0-180 min). In addition to its effects on Hsp90 expression, NaHS pretreatment of PC12 cells augmented the overexpression of Hsp90 induced by 600 µmol/L CoCl2 by 1.38-fold (P < 0.01). 6. Treatment of PC12 cells with 2 µmol/L 17-AAG for 30 min prior to NaHS pretreatment blocked the overexpression of Hsp90 induced by NaHS preconditioning, as evidenced by decreased cell viability (by 54.2 + 1.2%; P < 0.01), increased PC12 cell apoptosis (by 36.6 ± 1.2%; P < 0.01) and increasing ROS production. 7. The findings of the present study provide novel evidence that Hsp90 mediates H2S-induced neuroprotection against chemical hypoxia-induced injury via anti-oxidant and anti-apoptotic effects.

Protective effects of early hypoxic post-conditioning in cultured cortical neurons.

PRIMARY OBJECTIVE: Recent evidence suggests that delayed hypoxic post-conditioning is neuroprotective. The aim of the present study was to test whether early post-conditioning applied immediately after hypoxia could protect cultured neurons from hypoxia/reoxygenation (H/R)-induced injuries. METHODS: Primary cortical neuronal culture depleted of microglia was exposed to H/R. Post-conditioning started immediately after hypoxia and consisted of three cycles of 15-minutes of reoxygenation and 15-minutes of hypoxia. Cell viability assay was performed using Cell Counting Kit-8 (CCK-8). Apoptosis was evaluated by Hoechst 33258 staining, FITC-Annexin V/PI double staining and Western blot assay (testing the cleaved caspase-3 expression). Reactive oxygen species (ROS), intracellular Ca(2+) and mitochondrial membrane potential (MMP) were examined using confocal laser-scanning microscopy. MAIN RESULTS: H/R significantly reduced cell viability and increased neuronal apoptosis and necrosis. Furthermore, the expression of cleaved caspase-3, ROS production and intracellular Ca(2+) were increased. MMP was attenuated. Injuries induced by H/R were substantially attenuated by early hypoxic post-conditioning. Changes in cleaved caspase-3 expression, ROS production, intracellular Ca(2+) level and MMP in response to H/R were significantly decreased by the post-conditioning. CONCLUSIONS: The findings demonstrated that early hypoxic post-conditioning could protect neurons against H/R-induced injuries independent of microglial cells, possibly by inhibiting ROS over-production and intracellular Ca(2+) accumulation and maintaining MMP.

Morphine treatment enhances extracellular ATP enzymolysis and adenosine generation in rat astrocytes.

Recent studies have shown that astrocytes play important roles in ATP degradation and adenosine (a well known analgesic molecule) generation, which are closely related to pain signaling pathway. The aim of this study was to investigate whether morphine, a well known analgesic drug, could affect the speeds of ATP enzymolysis and adenosine generation in rat astrocytes. Intracellular calcium concentration ([Ca(2+)](i)) of astrocyte was measured by flow cytometry, and the time points that morphine exerted notable effects were determined for subsequent experiments. Cultured astrocytes were pre-incubated with morphine (1 µmol/L) and then were incubated with substrates, ATP and AMP, for 30 min. The speeds of ATP enzymolysis and adenosine generation were measured by high performance liquid chromatography (HPLC). The results showed that both 1.5 and 48 h of morphine pre-incubation induced maximal ATP enzymolysis speed in astrocytes among all the time points, and there was no statistical difference of ATP enzymolysis speed between morphine treatments for 1.5 and 48 h. As to adenosine, morphine pre-incubation for 1.5 h statistically increased adenosine generation, which was degraded from AMP, in cultured astrocytes compared with control group. However, no difference of adenosine generation was observed after 48 h of morphine pre-incubation. These results indicate that treatment of morphine in vitro dynamically changes the concentrations of ATP and adenosine in extracellular milieu of astrocytic cells. In addition, astrocyte can be regarded as at least one of the target cells of morphine to induce changes of ATP and adenosine levels in central nervous system.

[Corrigendum] Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

Following the publication of this article, an interested reader drew to the authors' attention that, in Fig. 4 on p. 1913, the t-Akt panel in Fig. 4A looked unexpectedly similar to the ß-actin panel in Fig. 4C. The authors were able to refer back to their original data, and realized that the Figure had been compiled incorrectly; essentially, the data for the t-Akt panel had been duplicated, and the data for the ß-actin panel in Fig. 4C had not been included in the Figure as intended. The revised version of Fig. 4, showing the correct data for the ß-actin panel in Fig. 4C, is shown opposite. This error did not have a significant impact on the results or the conclusions reported in this study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and all of the authors agree to the publication of this Corrigendum. The authors sincerely apologize for this mistake, and regret any inconvenience this mistake has caused. [the original article was published in Oncology Reports 36: 1909-1916, 2016; DOI: 10.3892/or.2016.5014].