Silicon reduces aluminum accumulation and mitigates toxic effects in cowpea plants

Aluminum (Al) is the third most abundant metal in the Earth’s surface, and Al toxicity promotes several negative effects in plant metabolism. Silicon (Si) is the second most common mineral in soil and is considered a beneficial element for plants, improving their tolerance to biotic and abiotic stresses. The aim of this study is to determine whether Si can reduce the accumulation of Al, explain the possible contribution of Si in mitigating Al toxicity, and indicate the better Si dose–response for cowpea plants. The experiment had a factorial design with two levels of aluminum (0 and 10 mM Al) and three levels of silicon (0, 1.25 and 2.50 mM Si). The utilization of Si in plants exposed to Al toxicity contributed to significant reductions in the Al contents of all tissues, corresponding to reductions of 51, 29 and 41% in roots, stems and leaves, respectively, upon treatment with 2.50 mM Si + 10 mM Al compared to the control treatment (0 mM Si + 10 mM Al). Al toxicity promoted decreases in Φ_PSII, q_P and ETR, whereas 2.50 mM Si induced increases of 54, 185 and 29%, respectively. Plants exposed to Al had lower values of P _N, WUE and P _N/C _i, whereas Si application at a concentration of 2.50 mM yielded improvements of 53, 32 and 67%, respectively. Al exposure increased SOD, CAT, APX and POX activities, whereas treatment with 2.50 mM Si + 10 mM Al produced significant variations of 72, 97, 48 and 32%, respectively, compared to 0 mM Si + 10 mM Al. Our results proved that Si reduced the Al contents in all tissues. Si also improved the photochemical efficiency of PSII, gas exchange, pigments and antioxidant enzymes, contributing to a reduction in the accumulation of oxidative compounds. These benefits corroborate the multiple roles exercised by Si in metabolism and reveal that Si immobilizes the Al in roots and reduce the accumulation of this metal in other organs, mitigating the damage caused by Al in cowpea plants. In relation to dose–response, plants exposed to 1.25 mM Si without Al presented better results in terms of growth, whereas the toxic effects of plants exposed to Al were mitigated with 2.50 mM Si.     

Silicon ameliorates manganese toxicity by regulating manganese transport and antioxidant reactions in rice (Oryza sativa L.)

Background and aims

This study aimed to investigate the roles of silicon (Si) in ameliorating manganese (Mn) toxicity in two rice (Oryza sativa L.) cultivars: i.e. cv. Xinxiangyou 640 (XXY), a Mn-sensitive cultivar and cv. Zhuliangyou 99 (ZLY), a Mn-tolerant cultivar.

Methods

Plants were cultured in nutrient solution containing normal Mn (6.7 μM) or high Mn (2.0 mM), both with or without Si supply at 1.5 mM Si.

Results

Plant growth was severely inhibited by high Mn in cv. XXY, but was enhanced by Si supply. In cv. XXY, Si-enhanced tolerance resulted from a restriction of Mn transport, whereas in cv. ZLY Mn uptake was depressed. In cv. XXY, high Mn significantly increased superoxide dismutase (SOD), catalase and ascorbate peroxidase activities but decreased non-protein thiols and glutathione concentrations, leading to accumulation of H₂O₂ and malondialdehyde. The addition of Si significantly counteracted high Mn-elevated malondialdehyde and H₂O₂ concentrations and enhanced plant growth. In cv. ZLY, high Mn considerably raised SOD activities and glutathione concentrations, thus leading to relatively low oxidative damage.

Conclusions

Si-enhanced Mn tolerance was attributed mainly to restricted Mn transport in cv. XXY but to depressed Mn uptake in cv. ZLY. Silicon mainly influenced non-enzymatic antioxidants in these two rice cultivars under high Mn stress.     

Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells

Fibroblast growth factors (FGFs) comprise a large family of signaling molecules that involve cell patterning, mobilization, differentiation, and proliferation. Various FGFs, including FGF-1, FGF-2, and FGF-5, have been shown to play a role in cytoprotection during adverse cardiac events; however, whether FGF-8 is a cytoprotective remains unclear. The current study was designed to evaluate the effect of FGF-8 treatment on oxidative stress-induced apoptosis in H9c2 cells. Cells were divided into three groups: control, H₂O₂ (400 µm H₂O₂), and H₂O₂ + FGF-8 (4 ng/ml FGF-8). Our results suggest apoptosis was significantly (p < 0.05) enhanced in the H₂O₂ group relative to control. Moreover, a significant (p < 0.05) decline in apoptosis was observed in the H₂O₂ + FGF-8 group compared to H₂O₂-treated cells as evidenced by TUNEL staining, a cell death detection ELISA, and cell viability. Levels of downstream apoptotic mediators, caspase-3 and caspase-9, were significantly (p < 0.05) upregulated following H₂O₂ treatment but were abrogated following FGF-8 application. Expression levels of Forkhead box protein O1 (FoxO-1), MnSOD, catalase, pAKT, and p-mTOR were significantly (p < 0.05) reduced in the H₂O₂ group (p < 0.05). Notably, these levels were significantly (p < 0.05) reversed following FGF-8 treatment. Our data, for the first time, suggest FGF-8 is an anti-apoptotic mediator in oxidative-stressed H9c2 cells. Furthermore, our data demonstrate that apoptotic inhibition by FGF-8 is consequent to FoxO-1 oxidative detoxification as well as augmentation to the PI3K/AKT cell survival pathway.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition

Key message

Cell growth medium composition has profound impacts on the O -glycosylation of a “designer” arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein.

Abstract

Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)₃₂ tag (32 tandem repeats of “Ser-Pro” motif). The (SP)₃₂ tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)₃₂ tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

     

Stress influenced the aerotolerance of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> hsryfm 1301

Objective

To investigate the aerotolerance of Lactobacillus rhamnosus hsryfm 1301 and its influencing factors.

Results

The growth rate of L. rhamnosus hsryfm 1301 weakened noticeably when the concentration of supplemented H₂O₂ reached 1 mM, and only 2% of all L. rhamnosus hsryfm 1301 cells survived in MRS broth supplemented with 2 mM H₂O₂ for 1 h. After pretreatment with 0.5 mM H₂O₂, the surviving cells of L. rhamnosus hsryfm 1301 in the presence of 5 mM H₂O₂ for 1 h increased from 3.7 to 7.8 log CFU. Acid stress, osmotic stress, and heat stress at 46 °C also enhanced its aerotolerance, while heat stress at 50 °C reduced the tolerance of L. rhamnosus hsryfm 1301 to oxidative stress. Moreover, treatment with 0.5 mM H₂O₂ increased the heat stress tolerance of L. rhamnosus hsryfm 1301 by approximately 150-fold.

Conclusions

Lactobacillus rhamnosus hsryfm 1301 possesses a stress-inducible defense system against oxidative stress, and the cross-adaptation to different stresses is a promising target to increase the stress tolerance of L. rhamnosus hsryfm 1301 during probiotic food and starter culture production.

     

Asymmetric effects of litter removal and litter addition on the structure and function of soil microbial communities in a managed pine forest

Aims

The effect of different MeJA doses applied prior to or simultaneously with toxic Al on biochemical and physiological properties of Vaccinium corymbosum cultivars with contrasting Al resistance was studied.

Methods

Legacy (Al-resistant) and Bluegold (Al-sensitive) plants were treated with and without toxic Al under controlled conditions: a) without Al and MeJA, b) 100 μM Al, c) 100 μM Al + 5 μM MeJA, d) 100 μM Al + 10 μM MeJA and e) 100 μM Al + 50 μM MeJA. MeJA was applied to leaves 24 h prior to or simultaneously with Al in nutrient solution. After 48 h, Al-concentration, lipid peroxidation (LP), H₂O₂, antioxidant activity, total phenols, total flavonoids, phenolic compounds and superoxide dismutase activity (SOD) of plant organs were analyzed.

Results

Al-concentrations increased with Al-treatment in both cultivars, being Al, LP and H₂O₂ concentrations reduced with low simultaneous MeJA application. Higher MeJA doses induced more oxidative damage than the lowest. Legacy increased mainly non-enzymatic compounds, whereas Bluegold increased SOD activity to counteract Al³⁺.

Conclusions

Low MeJA doses applied simultaneously with Al³⁺ increased Al-resistance in Legacy by increasing phenolic compounds, while Bluegold reduced oxidative damage through increment of SOD activity, suggesting a diminution of its Al-sensitivity. Higher MeJA doses could be potentially toxic. Studies are needed to determine the molecular mechanisms involved in the protective MeJA effect against Al-toxicity.

     

Selenium Modulates Oxidative Stress-Induced Cell Apoptosis in Human Myeloid HL-60 Cells Through Regulation of Calcium Release and Caspase-3 and -9 Activities

Selenium is an essential chemopreventive antioxidant element to oxidative stress, although high concentrations of selenium induce toxic and oxidative effects on the human body. However, the mechanisms behind these effects remain elusive. We investigated toxic effects of different selenium concentrations in human promyelocytic leukemia HL-60 cells by evaluating Ca²⁺ mobilization, cell viability and caspase-3 and -9 activities at different sample times. We found the toxic concentration and toxic time of H₂O₂ as 100 μm and 10 h on cell viability in the cells using four different concentrations of H₂O₂ (1 μm–1 mm) and six different incubation times (30 min, 1, 2, 5, 10, 24 h). Then, we found the therapeutic concentration of selenium to be 200 nm by cells incubated in eight different concentrations of selenium (10 nm–1 mm) for 1 h. We measured Ca²⁺ release, cell viability and caspase-3 and -9 activities in cells incubated with high and low selenium concentrations at 30 min and 1, 2, 5, 10 and 24 h. Selenium (200 nm) elicited mild endoplasmic reticulum stress and mediated cell survival by modulating Ca²⁺ release, the caspases and cell apoptosis, whereas selenium concentrations as high as 1 mm induced severe endoplasmic reticulum stress and caused cell death by activating modulating Ca²⁺ release, the caspases and cell apoptosis. In conclusion, these results explained the molecular mechanisms of the chemoprotective effect of different concentrations of selenium on oxidative stress-induced apoptosis.     

Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation

Background

Volatile fatty acids (VFA) are building blocks for the chemical industry. Sustainable, biological production is constrained by production and recovery costs, including the need for intensive pH correction. Membrane electrolysis has been developed as an in situ extraction technology tailored to the direct recovery of VFA from fermentation while stabilizing acidogenesis without caustic addition. A current applied across an anion exchange membrane reduces the fermentation broth (catholyte, water reduction: H₂O + e^? → ½ H₂ + OH^?) and drives carboxylate ions into a clean, concentrated VFA stream (anolyte, water oxidation: H₂O → 2e^? + 2 H⁺ + O₂).

Results

In this study, we fermented thin stillage to generate a mixed VFA extract without chemical pH control. Membrane electrolysis (0.1 A, 3.22 ± 0.60 V) extracted 28 ± 6 % of carboxylates generated per day (on a carbon basis) and completely replaced caustic control of pH, with no impact on the total carboxylate production amount or rate. Hydrogen generated from the applied current shifted the fermentation outcome from predominantly C2 and C3 VFA (64 ± 3 % of the total VFA present in the control) to majority of C4 to C6 (70 ± 12 % in the experiment), with identical proportions in the VFA acid extract. A strain related to Megasphaera elsdenii (maximum abundance of 57 %), a bacteria capable of producing mid-chain VFA at a high rate, was enriched by the applied current, alongside a stable community of Lactobacillus spp. (10 %), enabling chain elongation of VFA through lactic acid. A conversion of 30 ± 5 % VFA produced per sCOD fed (60 ± 10 % of the reactive fraction) was achieved, with a 50 ± 6 % reduction in suspended solids likely by electro-coagulation.

Conclusions

VFA can be extracted directly from a fermentation broth by membrane electrolysis. The electrolytic water reduction products are utilized in the fermentation: OH^? is used for pH control without added chemicals, and H₂ is metabolized by species such as Megasphaera elsdenii to produce greater value, more reduced VFA. Electro-fermentation displays promise for generating added value chemical co-products from biorefinery sidestreams and wastes.

     

Adiponectin and colon cancer: evidence for inhibitory effects on viability and migration of human colorectal cell lines

Exogenous hydrogen peroxide (H₂O₂) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H₂O₂-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H₂O₂ inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H₂O₂-treated lung cancer cells. H₂O₂ increased intracellular ROS levels, including that of O ₂ ^·? , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H₂O₂ triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O ₂ ^·? levels in H₂O₂-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O ₂ ^·? , in H₂O₂-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H₂O₂-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H₂O₂ induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H₂O₂-induced oxidative stress and GSH depletion.     

Desulfurization with <Emphasis Type="Italic">Thialkalivibrio versutus</Emphasis> immobilized on magnetic nanoparticles modified with 3-aminopropyltriethoxysilane

Objective

Thialkalivibrio versutus D301 cells were immobilized on Fe₃O₄ nanoparticles (NPs) synthesized by an improved chemical coprecipitation method and modified with 3-aminopropyltriethoxysilane (APTES), then the immobilized cells were used in sulfur oxidation.

Results

The prepared Fe₃O₄–APTES NPs had a narrow size distribution (10 ± 2 nm) and were superparamagnetic, with a saturation magnetization of 60.69 emu/g. Immobilized cells had a saturation magnetization of 34.95 emu/g and retained superparamagnetism. The optimum conditions for cell immobilization were obtained at pH 9.5 and 1 M Na⁺. The immobilization capacity of Fe₃O₄–APTES NPs was 7.15 g DCW/g-NPs that was 2.3-fold higher than that of Fe₃O₄ NPs. The desulfurization efficiency of the immobilized cells was close to 100%, having the same sulfur oxidation capacity as free cells. Further, the immobilized cells could be reused at least eight times, retaining more than 85% of their desulfurization efficiency.

Conclusion

Immobilization of cells with the modified magnetic NPs efficiently increased cell controllability, have no effect on their desulfurization activity and could be effectively used in large-scale industrial applications.

     

Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice

Objectives

To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine.

Results

C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209 ± 21 μg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4⁺ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458 ± 98 pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8⁺ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization.

Conclusions

A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.

     

[(B3O3H3)nM]+(n = 1, 2;M = Cu, Ag, Au): a new class of metal-cation complexes

A density functional theory (DFT) investigation into the structures and bonding characteristics of [(B₃O₃H₃)_nM]⁺(n?=?1, 2;M?=?Cu, Ag, Au) complexes was performed. DFT calculations and natural bond orbital (NBO) analyses indicate that the ΙB metal complexes of boroxine exhibit intriguing bonding characteristics, different from the typical cation–π interactions between ΙB metal-cations and benzene. The complexes of [B₃O₃H₃M]⁺ and [(B₃O₃H₃)₂?M]⁺ (M?=?Cu, Ag, and Au) favor the conformation of perfectly planar structures with the C_2v and D_2h symmetry along one of the threefold molecular axes of boroxine, respectively. Detailed natural resonance theory (NRT) and canonical molecular orbitals (CMOs) analyses show that interaction between the metal cation and the boroxine in [B₃O₃H₃M]⁺ (M?=?Cu, Ag, and Au) is mainly ionic, while the ΙB metal-cations←π donation effect is responsible for the binding site. In these complexes, boroxine serves as terminals η¹-B₃O₃H₃ with one O atom of the B₃O₃ ring. The infra-red (IR) spectra of [B₃O₃H₃M]⁺ were simulated to facilitate their future experimental characterization. The complexes all give two IR active modes at about 1,300 and 2,700 cm^?1, which are inactive in pure boroxine. Simultaneously, the B–H stretching modes of the complexes are red-shifted due to the interaction between the metal-cation and boroxine. To explore the possibility of the structural pattern developed in this work forming mesoporous materials, complexes [(B₃O₃H₃M)₆]⁶⁺ (M?=?Cu, Ag, and Au) were also studied, which appear to be unique and particular interesting: they are all true minima with D_6h symmetries and pore sizes ranging from 12.04 Å to 13.65 Å.

The cantharidin-induced oxidative burst in tobacco BY-2 cell suspension cultures

Summary The in vivo induction of H₂O₂ production was tested on tobacco cell suspension cultures (Nicotiana tabacum cv. Bright Yellow-2). The measurement of H₂O₂ was based on the oxidation of 3,5-dichloro-2-hydroxybenzensulfonic acid by endogenous peroxidases and spectrophotometric detection after reaction with 4-aminoanti-pyrine. The phosphatase inhibitor cantharidin induced a transient increase in H₂O₂ synthesis. The timing of the H₂O₂ production, the level of induction by cantharidin and the background H₂O₂ production were dependent on the tobacco cell concentration used. A concentration curve of cantharidin revealed saturating kinetics for the H₂O₂ detection (E₅₀=46 to 70 M, E_max=101 to 128 mol/h·g fresh weight). An inhibitor study with the tobacco BY-2 cells showed high inhibitions of the H₂O₂ induction with the flavin analogues diphenylene iodonium (I₅₀=1.26M) and acridine orange and with membrane-permeative thiol reagents (N-ethyl maleimide, N-pyrene maleimide, iodoacetate); whereas the nonpermeative thiol reagentp-chloromercuribenzoic acid was ineffective. Therefore, the induction of H₂O₂ production with phosphatase inhibitors (cantharidin) showed comparable properties to the elicitor-induced oxidative-burst response in other plant cells.Abbreviations AcOr acridine orange - AOS active-oxygen species - BY-2 Bright Yellow-2 - pCMBS p-chloromercuribenzoic acid - DHBS 3,5-dichloro-2-hydroxybenzenesulfonic acid - DMSO dimethylsulfoxide - DPI diphenylene iodonium - EtOH ethanol - H₂O₂ hydrogen peroxide - HRP horseradish peroxidase - MS Murashige and Skoog - NEM N-ethyl maleimide - NPM N-pyrene maleimide - O ₂ ^– superoxide - SOD superoxide dismutase     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Increased prevalence of tumor-infiltrating regulatory T cells is closely related to their lower sensitivity to H2O2-induced apoptosis in gastric and esophageal cancer

Purpose and experimental design

Although an increase in regulatory T cells (Tregs) is observed in tumor microenvironments, the underlying mechanism is not fully clarified. Since it was suggested that Tregs showed a lower sensitivity toward oxidative stress in comparison with conventional T cells, in the present study, we investigated the H₂O₂ production and apoptosis of Tregs in gastric and esophageal cancer tissues, employing flow cytometric analysis using fresh samples (n = 93) and immunohistochemical analysis (n = 203).

Results

The increased tumor-infiltrating Tregs coexisted with elevated H₂O₂ production according to disease progression. The grade of apoptosis in Tregs was less pronounced than that in conventional T cells, and there was a positive correlation between H₂O₂ production and the grade of apoptosis in conventional T cells, while there was no correlation between H₂O₂ production and the grade of apoptosis in Tregs. Moreover, Tregs were less sensitive to H₂O₂-induced apoptosis compared with conventional T cells in vitro.

Conclusions

We have demonstrated that the increased prevalence of tumor-infiltrating Tregs closely related to their lower sensitivity to H₂O₂-induced apoptosis.     

Characterization of H<Subscript>2</Subscript> photoproduction by marine green alga <Emphasis Type="Italic">Tetraselmis subcordiformis</Emphasis> integrated with an alkaline fuel cell

Objectives

To investigate the feasibility of coupling carbonyl cyanide m-chlorophenylhydrazone-regulated photohydrogen production by Tetraselmis subcordiformis in a photobioreactor to an alkaline fuel cell (AFC).

Results

H₂ evolution kinetics in the AFC integrated process was characterized. The duration of H₂ evolution was prolonged and its yield was improved about 1.5-fold (to 78 ± 5 ml l^?1) compared with that of the process without AFC. Improved H₂ yield was possibly caused by removal of H₂ feedback inhibition by H₂ consumption in situ. Decreases in the H₂ production rate correlated with the gradual deactivation of PSII and hydrogenase activities. The H₂ yield was closely associated with catabolism of starch and protein.

Conclusion

A marine green algal CO₂-supplemented culture integrated with in situ H₂-consumption by an AFC system was developed as a viable protocol for the H₂ production.