Analysis of arsenic induced physiological and biochemical responses in a medicinal plant,Withania somnifera

Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India. However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources. In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 μM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes. Plants showed more As accumulation upon exposure to AsIII (320 μg g⁻¹ DW in roots and 161 μg g⁻¹ DW in leaves) than to AsV (173 μg g⁻¹ DW in roots and 100 μg g⁻¹ DW in leaves) after 10 days of treatment. Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations. Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1–5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols. In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses.     

The Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca2+ Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H2O2-Induced Apoptosis with Ca2+ Overload

Intracellular calcium concentration ([Ca²⁺]_i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca²⁺]_i and its control mechanisms in process of hydrogen peroxide (H₂O₂)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca²⁺ indicator to detect [Ca²⁺]_i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H₂O₂-induced retinal cell apoptosis occurred at 4 h after H₂O₂ stress and increased in a time-dependent manner, but [Ca²⁺]_i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H₂O₂ stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca²⁺]_i, which appeared only at 0.5 h after βE2 application; c) increased [Ca²⁺]_i under 100 µM H₂O₂ treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca²⁺ stores; d) importantly, the transiently increased [Ca²⁺]_i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca²⁺ channels (L-VGCC), but the increased [Ca²⁺]_i induced by 100 µM H₂O₂ treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H₂O₂, which was also associated with its transient [Ca²⁺]_i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.     

Exogenous 5-Aminolevulenic Acid Promotes Seed Germination in Elymus nutans against Oxidative Damage Induced by Cold Stress

The protective effects of 5-aminolevulenic acid (ALA) on germination of Elymus nutans Griseb. seeds under cold stress were investigated. Seeds of E. nutans (Damxung, DX and Zhengdao, ZD) were pre-soaked with various concentrations (0, 0.1, 0.5, 1, 5, 10 and 25 mg l⁻¹) of ALA for 24 h before germination under cold stress (5°C). Seeds of ZD were more susceptible to cold stress than DX seeds. Both seeds treated with ALA at low concentrations (0.1–1 mg l⁻¹) had higher final germination percentage (FGP) and dry weight at 5°C than non-ALA-treated seeds, whereas exposure to higher ALA concentrations (5–25 mg l⁻¹) brought about a dose dependent decrease. The highest FGP and dry weight of germinating seeds were obtained from seeds pre-soaked with 1 mg l⁻¹ ALA. After 5 d of cold stress, pretreatment with ALA provided significant protection against cold stress in the germinating seeds, significantly enhancing seed respiration rate and ATP synthesis. ALA pre-treatment also increased reduced glutathione (GSH), ascorbic acid (AsA), total glutathione, and total ascorbate concentrations, and the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), whereas decreased the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and superoxide radical (O₂ ^•−) release in both germinating seeds under cold stress. In addition, application of ALA increased H⁺-ATPase activity and endogenous ALA concentration compared with cold stress alone. Results indicate that ALA considered as an endogenous plant growth regulator could effectively protect E. nutans seeds from cold-induced oxidative damage during germination without any adverse effect.     

Selection and characterization of tobacco plants with novel o(2)-resistant photosynthesis

Plants were obtained with novel O₂-resistant photosynthetic characteristics. At low CO₂ (250-350 μL CO₂ L⁻¹) and 30°C when O₂ was increased from 1% to 21% to 42%, the ratio of net CO₂ uptake in O₂-resistant whole plants or leaf discs compared to wild type increased progressively, and this was not related to stomatal opening. Dihaploid plantlets regenerated from anther culture were initially screened and selected for O₂-resistant growth in 42% O₂/160 μL CO₂ L⁻¹ and 0.18% of the plantlets showed O₂-resistant photosynthesis. About 30% of the progeny (6 of 19 plants) of the first selfing of a fertile plant derived from a resistant dihaploid plant had O₂-resistant photosynthesis, and after a second selfing this increased to 50% (6 of 12 plants). In 21% O₂ and low CO₂, net photosynthesis of the resistant plants was about 15% greater on a leaf area basis than wild type. Net photosynthesis was compared in leaf discs at 30 and 38°C in 21% O₂, and at the higher temperature O₂-resistant plants showed still greater photosynthesis than wild type. The results suggest that the O₂-resistant photosynthesis described here is associated with a decreased stoichiometry of CO₂ release under conditions of rapid photorespiration. This view was supported by the finding that leaves of O₂-resistant plants averaged 40% greater catalase activity than wild type.     

Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: Implications for paleoclimate reconstruction

Stable oxygen isotope (δ¹⁸O) compositions from vertebrate tooth enamel are widely used as biogeochemical proxies for paleoclimate. However, the utility of enamel oxygen isotope values for environmental reconstruction varies among species. Herein, we evaluate the use of stable oxygen isotope compositions from pronghorn (Antilocapra americana Gray, 1866) enamel for reconstructing paleoclimate seasonality, an elusive but important parameter for understanding past ecosystems. We serially sampled the lower third molars of recent adult pronghorn from Wyoming for δ¹⁸O in phosphate (δ¹⁸O_PO4) and compared patterns to interpolated and measured yearly variation in environmental waters as well as from sagebrush leaves, lakes, and rivers (δ¹⁸O_w). As expected, the oxygen isotope compositions of phosphate from pronghorn enamel are enriched in ¹⁸O relative to environmental waters. For a more direct comparison, we converted δ¹⁸O_w values into expected δ¹⁸O_PO4* values (δ¹⁸O_W‐_PO4*). Pronghorn δ¹⁸O_PO4 values from tooth enamel record nearly the full amplitude of seasonal variation from Wyoming δ¹⁸O_W‐PO4* values. Furthermore, pronghorn enamel δ¹⁸O_PO4 values are more similar to modeled δ¹⁸O_W‐PO4* values from plant leaf waters than meteoric waters, suggesting that they obtain much of their water from evaporated plant waters. Collectively, our findings establish that seasonality in source water is reliably reflected in pronghorn enamel, providing the basis for exploring changes in the amplitude of seasonality of ancient climates. As a preliminary test, we sampled historical pronghorn specimens (1720 ± 100 AD), which show a mean decrease (a shift to lower values) of 1–2‰ in δ¹⁸O_PO4 compared to the modern specimens. They also exhibit an increase in the δ¹⁸O amplitude, representing an increase in seasonality. We suggest that the cooler mean annual and summer temperatures typical of the 18th century, as well as enhanced periods of drought, drove differences among the modern and historical pronghorn, further establishing pronghorn enamel as excellent sources of paleoclimate proxy data.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Thermal thresholds as predictors of seed dormancy release and germination timing: altitude-related risks from climate warming for the wild grapevine Vitis vinifera subsp. sylvestris

Background and Aims

The importance of thermal thresholds for predicting seed dormancy release and germination timing under the present climate conditions and simulated climate change scenarios was investigated. In particular, Vitis vinifera subsp. sylvestris was investigated in four Sardinian populations over the full altitudinal range of the species (from approx. 100 to 800 m a.s.l).

Methods

Dried and fresh seeds from each population were incubated in the light at a range of temperatures (10–25 and 25/10 °C), without any pre-treatment and after a warm (3 months at 25 °C) or a cold (3 months at 5 °C) stratification. A thermal time approach was then applied to the germination results for dried seeds and the seed responses were modelled according to the present climate conditions and two simulated scenarios of the Intergovernmental Panel on Climate Change (IPCC): B1 (+1·8 °C) and A2 (+3·4 °C).

Key Results

Cold stratification released physiological dormancy, while very few seeds germinated without treatments or after warm stratification. Fresh, cold-stratified seeds germinated significantly better (>80 %) at temperatures ≥20 °C than at lower temperatures. A base temperature for germination (T_b) of 9·0–11·3 °C and a thermal time requirement for 50 % of germination (θ₅₀) ranging from 33·6 °Cd to 68·6 °Cd were identified for non-dormant cold-stratified seeds, depending on the populations. This complex combination of thermal requirements for dormancy release and germination allowed prediction of field emergence from March to May under the present climatic conditions for the investigated populations.

Conclusions

The thermal thresholds for seed germination identified in this study (T_b and θ₅₀) explained the differences in seed germination detected among populations. Under the two simulated IPCC scenarios, an altitude-related risk from climate warming is identified, with lowland populations being more threatened due to a compromised seed dormancy release and a narrowed seed germination window.     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

Flavonoid constituents,cytotoxic and antioxidant activities of Gleditsia triacanthos L. leaves

Gleditsia triacanthos L. is a deciduous tree belonging to the family Fabaceae. It possesses important biological activities as anti-mutagenic, anticancer, cytotoxic and treating rheumatoid arthritis. The total ethanol extract (EtOHE) and successive extracts (petroleum ether, chloroform, ethyl acetate, and aqueous ethanol) were prepared from the leaves. Eight flavone glycosides and two flavone aglycones named vicenin-I (1), vitexin (2), isovitexin (3), orientin (4), isoorientin (5), luteolin-7-O-ß-glucopyranoside (6), luteolin-7-O-ß-galactopyranoside (7), apigenin-7-O-ß-glucopyranoside (8), luteolin (9) and apigenin (10) were isolated from the aqueous ethanol extract of G. triacanthos L. leaves. Potent cytotoxic activity of the EtOHE extract was observed against the liver (IC₅₀ = 1.68 μg), breast (IC₅₀ = 0.74 μg), cervix (IC₅₀ = 1.28 μg), larynx (IC₅₀ = 0.67 μg) and colon (IC₅₀ = 2.50 μg) cancer cell lines. Cytotoxic activity of compounds 2, 4, 6 and 8 against, the liver, breast and colon cancer cell lines was also proved. Evaluation of the in-vivo antioxidant activity of the EtOHE and successive extracts revealed that the highest activity was exhibited by 100 mg of EtOHE (97.89% potency) as compared with vitamin E (100% potency). Compound 6 showed 91.8% free radical scavenging activity.     

Effects of temperature on the phase behavior and permeability of thylakoid lipid vesicles : relevance to chilling stress

Large unilamellar vesicles composed of thylakoid glycolipids, phosphatidylglycerol, and varying proportions of dipalmitoylphosphatidylglycerol (DPPG) have been examined for the temperature dependence of their permeability to ⁸⁶Rb⁺ and for the occurrence of liquid-crystalline to gel (L_α-to-L_β) phase separations. In vesicles in which the normal 12 mole percent of moderately unsaturated thylakoid phosphatidylglycerol was partially or completely replaced by DPPG, analysis by differential scanning calorimetry indicated that an L_α-to-L_β phase separation did not occur between 0 and 60°C. However, in similar vesicle dispersions that were first subjected to a freeze-thaw cycle, L_α-to-L_β phase separations were observed to occur between 17 and 53°C. The temperature and enthalpy of these phase separations were closely related to the proportion of DPPG in the original lipid mixture. In parallel experiments, large unilamellar vesicles were measured for their permeability to ⁸⁶Rb⁺ between 7 and 30°C. There were no systematic increases in permeability to ⁸⁶Rb⁺ as a function of DPPG content at the temperatures relevant to chilling stress in higher plants. It is concluded that (a) L_α-to-L_β phase separations do not occur in well-defined galactolipid vesicles containing ≤12 mole percent DPPG between 0 and 60°C and (b) these vesicles show no alterations in permeability to ⁸⁶Rb⁺ between 7 and 30°C that are relevant to chilling stress in higher plants.     

Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii,Tibetan peony

Background and Aims

Epicotyl dormancy break in seeds that have deep simple epicotyl morphophysiological dormancy (MPD) requires radicle emergence and even a certain root length in some species. However, the mechanisms by which root length affects epicotyl dormancy break are not clear at present. This study aims to explore the relationship between root length and epicotyl dormancy release in radicle-emerged seeds of Tibetan peony, Paeonia ludlowii, with discussion of the possible mechanisms.

Methods

Radicle-emerged seeds (radicle length 1·5, 3·0, 4·5 and 6·0 cm) were incubated at 5, 10 and 15 °C. During the stratification, some seeds were transferred to 15 °C and monitored for epicotyl–plumule growth. Hormone content was determined by ELISA, and the role of hormones in epicotyl dormancy release was tested by exogenous hormone and embryo culture.

Key Results

Cold stratification did not break the epicotyl dormancy until the root length was ≥6 cm. The indole-3-actic acid (IAA) and GA₃ contents of seeds having 6 cm roots were significantly higher than those of seeds with other root lengths, but the abscisic acid (ABA) content was lowest among radicle-emerged seeds. GA₃ (400 mg L⁻¹) could break epicotyl dormancy of all radicle-emerged seeds, while IAA (200 mg L⁻¹) had little or no effect. When grown on MS medium, radicles of naked embryos grew and cotyledons turned green, but epicotyls did not elongate. Naked embryos developed into seedlings on a mixed medium of MS + 100 mg L⁻¹ GA₃.

Conclusions

A root length of ≥6·0 cm is necessary for epicotyl dormancy release by cold stratification. The underlying reason for root length affecting epicotyl dormancy release is a difference in the GA₃/ABA ratio in the epicotyl within radicle-emerged seeds, which is mainly as a result of a difference in ABA accumulation before cold stratification.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Randomised,double-blind,placebo-controlled crossover study to investigate different dosing regimens of olodaterol delivered via Respimat? in patients with moderate to severe persistent asthma

Background

A Phase II, multicentre, randomised, double-blind, placebo-controlled, crossover trial comparing the 24-h forced expiratory volume in 1 s (FEV₁) time profile after 3 weeks’ treatment with once-daily (QD) or twice-daily (BID) olodaterol (at the same total daily dose) versus placebo delivered via Respimat® in patients with moderate to severe asthma.

Methods

Patients were randomised to different sequences of olodaterol with 2-week washout, either as a total daily dose of 5 μg (5 μg QD [AM] or 2.5 μg BID) or placebo, or 10 μg (10 μg QD [AM] or 5 μg BID) or placebo. Primary end point was FEV₁ area under the curve from 0 to 24 h (AUC_0–24) response (defined as change from study baseline FEV₁) after 3 weeks. Key secondary end points were FEV₁ AUC_0–12 and AUC_12–24 responses.

Results

Two hundred and six patients received treatment. All olodaterol treatments demonstrated statistically significant improvements in FEV₁ AUC_0–24 response at 3 weeks versus placebo (p < 0.0001); adjusted mean treatment difference versus placebo was 0.191 L for olodaterol 2.5 μg BID (95 % confidence interval [CI] 0.152, 0.229), 0.150 L for 5 μg QD (95 % CI 0.111, 0.189), 0.228 L for 5 μg BID (95 % CI 0.190, 0.266) and 0.209 L for 10 μg QD (95 % CI 0.170, 0.247). These results were supported by the key secondary end points. Olodaterol 5 μg QD provided numerically lower mean values for 24-h bronchodilation than olodaterol 2.5 μg BID (p = 0.0465), with no statistically significant difference between treatment with olodaterol 10 μg QD and 5 μg BID. No relevant differences in morning and evening peak expiratory flow or Asthma Control Questionnaire scores at 3 weeks were observed between different doses and regimens. Adverse events were generally mild to moderate and comparable between groups.

Conclusions

All doses and dose frequencies provided adequate 24-h bronchodilation superior to placebo. Based on the results of this study, it would be reasonable to include both posologies of 5 μg olodaterol daily (5 μg QD or 2.5 μg BID, both delivered in two puffs per dose from the Respimat® inhaler) in subsequent studies. Further studies are necessary to confirm the optimum dosing regimen in asthma. No safety concerns were identified.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01311661","term_id":"NCT01311661"}}NCT01311661

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0243-1) contains supplementary material, which is available to authorized users.     

Adaptational changes in the lipids and fatty acid profile of the cell and thylakoid membrane of rice plants exposed to sunlight

Adaptational changes occurring in the lipids and fatty acids of the cell and the thylakoid membrane in response to high light treatment, was studied in 30 days old rice (Oryza sativa L. cv. Jyothi) plants grown under low (150–200 μmol m⁻² s⁻¹) or moderate (600–800 μmol m⁻² s⁻¹) light conditions. Results were compared with rice plants grown in high (1200–2200 μmol m⁻² s⁻¹) light conditions. Exposure of rice plants and isolated chloroplast to high light, resulted in an increase in the amount of malonaldehyde, indicating oxidation of membrane lipids. Qualitative and quantitative changes in the phosphoglycolipids and quantitative changes in neutral lipids were observed in rice plants grown under the different growth conditions. A few of the phosphoglycolipids and neutral lipids were present exclusively in plants grown at low or moderate or high light, indicating requirement of different type of lipid composition of rice plants in response to their different growth irradiances. However, no significant quantitative changes were observed in the different saturated and unsaturated fatty acid groups of total lipids in low, moderate and high light grown rice plants, as a result of exposure to high light. No qualitative changes in the fatty acid composition due to difference in growth irradiance or high light treatment were seen. The changes observed in the phosphoglycolipids and neutral lipid composition of cell and thylakoid membrane of low, moderate and high light grown rice plants in response to high light, are probably the result of physiological changes in the rice plants, to sustain optimum structure and function of the cell and thylakoid membrane to maintain active physiological functions to endure high light conditions.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

Small airway dysfunction is associated to excessive bronchoconstriction in asthmatic patients

Background

We investigated whether a relationship between small airways dysfunction and bronchial hyperresponsiveness (BHR), expressed both in terms of ease of airway narrowing and of excessive bronchoconstriction, could be demonstrated in asthma.

Methods

63 (36 F; mean age 42 yr ± 14) stable, mild-to-moderate asthmatic patients (FEV₁ 92% pred ±14; FEV₁/FVC 75% ± 8) underwent the methacholine challenge test (MCT). The degree of BHR was expressed as PD₂₀ (in μg) and as ∆FVC%. Peripheral airway resistance was measured pre- and post-MCT by impulse oscillometry system (IOS) and expressed as R5-R20 (in kPa sL⁻¹).

Results

All patients showed BHR to methacholine (PD₂₀ < 1600 μg) with a PD₂₀ geometric (95% CI) mean value of 181(132–249) μg and a ∆FVC% mean value of 13.6% ± 5.1, ranging 2.5 to 29.5%. 30 out of 63 patients had R5-R20 > 0.03 kPa sL⁻¹ (>upper normal limit) and showed ∆FVC%, but not PD₂₀ values significantly different from the 33 patients who had R5-R20 ≤ 0.03 kPa sL⁻¹ (15.8% ± 4.6 vs 11.5% ± 4.8, p < 0.01 and 156(96–254) μg vs 207 (134–322) μg, p = 0.382). In addition, ∆FVC% values were significantly related to the corresponding pre- (r = 0.451, p < 0.001) and post-MCT (r = 0.376, p < 0.01) R5-R20 values.

Conclusions

Our results show that in asthmatic patients, small airway dysfunction, as assessed by IOS, is strictly associated to BHR, expressed as excessive bronchoconstriction, but not as ease of airway narrowing.     

The combined effect of water deficit stress and TiO2 nanoparticles on cell membrane and antioxidant enzymes in Helianthus annuus L.

Nanotechnology has become one of the several approaches attempting to ameliorate the severe effect of drought on plant''s production and to increase the plants tolerance against water deficit for the water economy. In this research, the effect of foliar application of TiO₂, nanoparticles or ordinary TiO₂, on Helianthus annuus subjected to different levels of water deficit was studied. Cell membrane injury increased by increasing the level of water deficit and TiO₂ concentration, and both types of TiO₂ affected the leaves in analogous manner. Ord-TiO₂ increased H₂O₂ generation by 67–240% and lipid peroxidation by 4–67% in leaves. These increases were more than that induced by Nano-TiO₂ and the effect was concentration dependent. Proline significantly increased in leaves by water deficit stress, reaching at 25% field capacity (FC) to more than fivefold compared to that in plants grown on full FC. Spraying plants with water significantly decreased the activities of enzymes in the water deficit stressed roots. The water deficit stress exerted the highest magnitude of effect on the changes of cell membrane injury, MDA, proline content, and activities of CAT and GPX. Nano-TiO₂ was having the highest effect on contents of H₂O₂ and GPX activity. In roots, the level of water deficit causes highest effect on enzyme activities, but TiO₂ influenced more on the changes of MDA and H₂O₂ contents. GPX activity increased by 283% in leaves of plants treated with 50 and 150 ppm Nano-TiO₂, while increased by 170% in those treated with Ord-TiO₂, but APX and CAT activities increased by 17–197%, in average, with Ord-TiO₂. This study concluded that Nano-TiO₂ didn’t ameliorate the effects of drought stress on H. annuus but additively increased the stress, so its use in nano-phytotechnology mustn’t be expanded without extensive studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-022-01153-z.     

Analysis of the Relative Increase in Photosynthetic O2 Uptake When Photosynthesis in Grapevine Leaves Is Inhibited following Low Night Temperatures and/or Water Stress

We found similarities between the effects of low night temperatures (5°C–10°C) and slowly imposed water stress on photosynthesis in grapevine (Vitis vinifera L.) leaves. Exposure of plants growing outdoors to successive chilling nights caused light- and CO₂-saturated photosynthetic O₂ evolution to decline to zero within 5 d. Plants recovered after four warm nights. These photosynthetic responses were confirmed in potted plants, even when roots were heated. The inhibitory effects of chilling were greater after a period of illumination, probably because transpiration induced higher water deficit. Stomatal closure only accounted for part of the inhibition of photosynthesis. Fluorescence measurements showed no evidence of photoinhibition, but nonphotochemical quenching increased in stressed plants. The most characteristic response to both stresses was an increase in the ratio of electron transport to net O₂ evolution, even at high external CO₂ concentrations. Oxygen isotope exchange revealed that this imbalance was due to increased O₂ uptake, which probably has two components: photorespiration and the Mehler reaction. Chilling- and drought-induced water stress enhanced both O₂ uptake processes, and both processes maintained relatively high rates of electron flow as CO₂ exchange approached zero in stressed leaves. Presumably, high electron transport associated with O₂ uptake processes also maintained a high ΔpH, thus affording photoprotection.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.