RIG1 suppresses Ras activation and induces cellular apoptosis at the Golgi apparatus

Retinoid-inducible gene 1 encodes RIG1 is a growth regulator, which inhibits the pathways of the RAS/mitogen-activated protein kinases by suppressing the activation of RAS. Confocal microscopic analysis demonstrated that RIG1 is localized in the endoplasmic reticulum (ER) and Golgi apparatus in HtTA cervical cancer cells. Carboxyterminal-deleted RIG1 targeted to the Golgi or ER was constructed and validated. The activation of HRAS was inhibited by 25.1% or 81.4% in cells cotransfected with wild-type or Golgi-targeted RIG1, respectively. Expression of wild-type or Golgi-targeted RIG1 for 24 h induced cellular apoptosis in HtTA cells, as assessed by MTT assay, the release of lactate dehydrogenase, and chromatin condensation. In contrast, ER-targeted RIG1 and carboxyterminal-deleted RIG1 (RIG1DeltaC) exhibited no activity. Caspase-2, -3, and -9 were activated following the expression of wild-type and Golgi-targeted RIG1. Although the caspase-3 inhibitor Z-DEVD-FMK partially or completely reversed the cell death induced by wild-type or Golgi-targeted RIG1, it did not prevent the anti-RAS effect of RIG1. In conclusion, the proapoptotic and anti-RAS activities of RIG1 are primarily associated with the Golgi localization of the protein. The proapoptotic activities of RIG1 are mediated through the activation of caspase-2 and -3 and are independent of its effect on RAS.     

Spin-flip reactions of Zr + C2H6 researched by relativistic density functional theory

Density functional theory (DFT) with relativistic corrections of zero-order regular approximation (ZORA) has been applied to explore the reaction mechanisms of ethane dehydrogenation by Zr atom with triplet and singlet spin-states. Among the complicated minimum energy reaction path, the available states involves three transition states (TS), and four stationary states (1) to (4) and one intersystem crossing with spin-flip (marked by ?): ³ Zr + C ₂ H ₆ → ³ Zr-CH ₃ -CH ₃ ( ³ 1) → ³ TS _1/2 → ³ ZrH-CH ₂ -CH ₃ ( ³ 2) → ³ TS _2/3 ? ¹ ZrH₂-CH₂ = CH₂ ( ¹ 3) → ¹ TS _3/4 → ¹ ZrH ₃ -CH = CH ₂ ( ¹ 4). The minimum energy crossing point is determined with the help of the DFT fractional-occupation-number (FON) approach. The spin inversion leads the reaction pathway transferring from the triplet potential energy surface (PES) to the singlet’s accompanying with the activation of the second C-H bond. The overall reaction is calculated to be exothermic by about 231 kJ mol^?1. Frequency and NBO analysis are also applied to confirm with the experimental observed data.

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.     

Effects of nitrate concentration on the denitrification potential of a calcic cambisol and its fractions of N2, N2O and NO

Background and aims

The direct measurement of denitrification dynamics and its product fractions is important for parameterizing process-oriented model(s) for nitrogen cycling in various soils. The aims of this study are to a) directly measure the denitrification potential and the fractions of nitrogenous gases as products of the process in laboratory, b) investigate the effects of the nitrate (NO ₃ ^? ) concentration on emissions of denitrification gases, and c) test the hypothesis that denitrification can be a major pathway of nitrous oxide (N₂O) and nitric oxide (NO) production in calcic cambisols under conditions of simultaneously sufficient supplies of carbon and nitrogen substrates and anaerobiosis as to be found to occur commonly in agricultural lands.

Methods

Using the helium atmosphere (with or without oxygen) gas-flow-soil-core technique in laboratory, we directly measured the denitrification potential of a silt clay calcic cambisol and the production of nitrogen gas (N₂), N₂O and NO during denitrification under the conditions of seven levels of NO ₃ ^? concentrations (ranging from 10 to 250 mg N kg^?1 dry soil) and an almost constant initial dissolved organic carbon concentration (300 mg C kg^?1 dry soil).

Results

Almost all the soil NO ₃ ^? was consumed during anaerobic incubation, with 80–88 % of the consumed NO ₃ ^? recovered by measuring nitrogenous gases. The results showed that the increases in initial NO ₃ ^? concentrations significantly enhanced the denitrification potential and the emissions of N₂ and N₂O as products of this process. Despite the wide range of initial NO ₃ ^? concentrations, the ratios of N₂, N₂O and NO products to denitrification potential showed much narrower ranges of 51–78 % for N₂, 14–36 % for N₂O and 5–22 % for NO.

Conclusions

These results well support the above hypothesis and provide some parameters for simulating effects of variable soil NO ₃ ^? concentrations on denitrification process as needed for biogeochemical models.     

Determination of the critical soil mineral nitrogen concentration for maximizing maize grain yield

Background and aims

A critical soil mineral nitrogen concentration (N_min) for guiding fertilizer application and maximizing maize grain yield is needed.

Methods

A three-year field experiment with three N regimes, unfertilized (N0), optimized N management (Opt.) and conventional N practice (Con.) was performed in maize.

Results

The mean soil N_min in 0–60 cm soil profile for N0, Opt. and Con. treatments was 2.0, 6.7 and 8.9 mg?kg^–1 at V8–VT growth stages and 2.2, 6.1 and 11.2 mg?kg^–1 on average over the whole growth season, respectively. Correspondingly, the soil N supplying capacity (soil N_min content?+?fertilizer N) of the three N treatments was smaller, identical or greater than the plant N accumulation at different growth stages. The Opt. treatment had significantly higher N use efficiency, N recovery efficiency and N partial factor productivity compared with the Con. treatment, while it did not cause maize yield loss.

Conclusions

Compared with the insensitivity of the critical shoot N dilution curve to excessive N application, soil N_min showed strong response to all treatments. We propose a minimum of soil N_min of 6.1 mg?kg^–1 at the sowing–V8, 6.7 mg?kg^–1 at the V8–VT, and 5.5 mg?kg^–1 at the VT–R6 growing stages with an average of about 6 mg?kg^–1 of soil N_min in the 0–60 soil depth for maximizing maize yield and N use efficiency in northern China. To maintain this critical N_min value over the whole growth period, N topdressing at V8 and V12 stages was recommended.     

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Background

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.

Methods

In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨ_m) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.

Results

Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨ_m and the activation of caspase-3.

Conclusion

These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.     

Nitrous oxide emissions and nitrate leaching from a rain-fed wheat-maize rotation in the Sichuan Basin, China

Aims

A 3-year field experiment (October 2004–October 2007) was conducted to quantify N₂O fluxes and determine the regulating factors from rain-fed, N fertilized wheat-maize rotation in the Sichuan Basin, China.

Methods

Static chamber-GC techniques were used to measure soil N₂O fluxes in three treatments (three replicates per treatment): CK (no fertilizer); N150 (300 kg N fertilizer ha^?1 yr^?1 or 150 kg N?ha^?1 per crop); N250 (500 kg N fertilizer ha^?1 yr^?1 kg or 250 kg N?ha^?1 per crop). Nitrate (NO ₃ ^? ) leaching losses were measured at nearby sites using free-drained lysimeters.

Results

The annual N₂O fluxes from the N fertilized treatments were in the range of 1.9 to 6.7 kg N?ha^?1 yr^?1 corresponding to an N₂O emission factor ranging from 0.12 % to 1.06 % (mean value: 0.61 %). The relationship between monthly soil N₂O fluxes and NO ₃ ^- leaching losses can be described by a significant exponential decaying function.

Conclusions

The N₂O emission factor obtained in our study was somewhat lower than the current IPCC default emission factor (1 %). Nitrate leaching, through removal of topsoil NO ₃ ^? , is an underrated regulating factor of soil N₂O fluxes from cropland, especially in the regions where high NO ₃ ^- leaching losses occur.     

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and risk of arsenic-induced urothelial carcinoma in residents of southwestern Taiwan

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Does radial oxygen loss and iron plaque formation on roots alter Cd and Pb uptake and distribution in rice plant tissues?

Background and Aims

Metal (e.g. Cd and Pb) pollution in agricultural soils and crops have aroused considerable attention in recent years. This study aimed to evaluate the effects of ROL and Fe plaque on Cd and Pb accumulation and distribution in the rice plant.

Methods

A rhizobag experiment was employed to investigate the correlations among radial oxygen loss (ROL), Fe plaque formation and uptake and distribution of Cd and Pb in 25 rice cultivars.

Results

Large differences between the cultivars were found in rates of ROL (1.55 to 6.88 mmol O₂ kg^?1 root d.w. h^?1), Fe plaque formation (Fe: 6,117–48,167 mg kg^?1; Mn: 127–1,089 mg kg^?1), heavy metals in shoot (Cd: 0.13–0.35 mg kg^?1; Pb: 4.8–8.1 mg kg^?1) and root tissues (Cd: 1.1–3.5 mg kg^?1; Pb: 45–199 mg kg^?1), and in Fe plaque (Cd: 0.54–2.6 mg kg^?1; Pb: 102–708 mg kg^?1). Rates of ROL were positively correlated with Fe plaque formation and metal deposition on root surfaces, but negatively correlated with metal transfer factors of root/plaque and distributions in shoot and root tissues.

Conclusions

ROL-induced Fe plaque promotes metal deposition on to root surfaces, leading to a limitation of Cd and Pb transfer and distribution in rice plant tissues.     

Responses of CO2 efflux from an alpine meadow soil on the Qinghai Tibetan Plateau to multi-form and low-level N addition

Aims

To assess the effects of atmospheric N deposition on the C budget of an alpine meadow ecosystem on the Qinghai–Tibetan Plateau, it is necessary to explore the responses of soil-atmosphere carbon dioxide (CO₂) exchange to N addition.

Methods

Based on a multi-form, low-level N addition experiment, soil CO₂ effluxes were monitored weekly using the static chamber and gas chromatograph technique. Soil variables and aboveground biomass were measured monthly to examine the key driving factors of soil CO₂ efflux.

Results

The results showed that low-level N input tended to decrease soil moisture, whereas medium-level N input maintained soil moisture. Three-year N additions slightly increased soil inorganic N pools, especially the soil NH ₄ ⁺ -N pool. N applications significantly increased aboveground biomass and soil CO₂ efflux; moreover, this effect was more significant from NH ₄ ⁺ -N than from NO ₃ ^? -N fertilizer. In addition, the soil CO₂ efflux was mainly driven by soil temperature, followed by aboveground biomass and NH ₄ ⁺ -N pool.

Conclusions

These results suggest that chronic atmospheric N deposition will stimulate soil CO₂ efflux in the alpine meadow on the Qinghai–Tibetan Plateau by increasing available N content and promoting plant growth.     

Coarse woody debris modifies surface soils of degraded temperate eucalypt woodlands

Aims

Reintroductions of coarse woody debris (CWD) to Australia’s temperate eucalypt woodlands have been proposed to address the paucity of CWD in these landscapes. This study aimed to quantify the effects of CWD on surface soils.

Methods

Values of C, N, C:N, P, NO₃ ^?, NH₄ ⁺, pH and electrical conductivity (EC) were measured adjacent to, and at reference distances from CWD. Soils were measured at depths of 0–1 cm, 1–3 cm and 3–5 cm for 12 individual CWD samples of varying decay classes and diameters. A linear mixed model was used to test the effects of the presence of CWD, soil depth and CWD decay class and diameter.

Results

Significantly larger values for C, N, C:N, P, NO₃ ^?, EC, and significantly smaller values for pH were found adjacent to CWD. The greatest impact of CWD was on the upper most surface soil. CWD decay class and diameter had little influence on the measured soil characteristics.

Conclusion

This is the first quantitative determination of the effects of eucalypt CWD on woodland soils in Australia. The effect of added CWD is rapid, occurring after just 2 years. The results suggest that the effects are due to the structural properties of CWD.     

Beyond DNA binding - a review of the potential mechanisms mediating quinacrine's therapeutic activities in parasitic infections,inflammation, and cancers

Background

Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/- and Src/Yes/Fyn^-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake.

Results

Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.

Conclusion

Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.     

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions

Background

The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133⁺/α₂β₁ ^hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133^-/α₂β₁ ^low) counterparts, resulting in an informative cancer stem cell gene-expression signature.

Results

Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches.

Conclusion

We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.     

Contribution of 15N-labelled leaf litter to N turnover, nitrous oxide emissions and N sequestration in a beech forest during eleven years

Aims

Decomposition of leaf litterfall plays a major role for nitrogen (N) dynamics in soils. However, little is known as to which extent beech leaf litter contributes to N turnover and nitrous oxide (N₂O) emissions within one decade after litterfall.

Methods

In 1997, we exchanged recently fallen leaf litter by ¹⁵N-labelled litter in a beech stand (Fagus sylvatica) at the Solling, Germany. Measurements were conducted 2–3 and 10–11 years after litter exchange.

Results

Two years after litter exchange, 92 % of added ¹⁵N was recovered in the surface 10 cm of the soil. The labelled N was primarily found in the upper part of the F layer of the moder type humus. Eleven years after litter exchange, 73 % of the added ¹⁵N was lost and the remaining 27 % was mainly recovered in the lower part of the F layer indicating N sequestration. The remaining leaf litter N was subject to measurable N mineralisation (2–3 % of litter N) and N₂O production (0.02 %). Between 0.3 % (eleventh year) and 0.6 % (second year) of total annual N₂O emissions were attributed to beech leaf litter of a single year.

Conclusions

Most of the annual N₂O emissions (1.33–1.54 kg N ha^?1 yr^?1) were probably derived from older soil N pools.     

Increased photosynthetic capacity in response to nitrate is correlated with enhanced cytokinin levels in rice cultivar with high responsiveness to nitrogen nutrients

Background and aims

Ammonium (NH₄ ⁺) is the preferred nitrogen nutrient over nitrate (NO₃ ^–) in Oryza sativa L. (rice), but photosynthetic capacity is enhanced by partial NO₃ ^– nutrition (PNN). The role of cytokinin in the effects of PNN on photosynthetic capacity is unknown.

Methods

We investigated effects of PNN on six cytokinin fractions in roots, xylem sap, and leaves and on the expression of eight cytokinin synthesis genes in the roots of Nanguang and Elio rice cultivars. The effect of exogenous cytokinin (6-BA) on leaf growth and photosynthetic activity was examined.

Results

Cell expansion and CO₂ assimilation in the first fully expanded leaf were enhanced by PNN in Nanguang but not in Elio. The concentrations of cytokinins in roots, xylem sap, and leaves of Nanguang increased approximately 25–34 % with PNN compared with sole NH₄ ⁺, but no difference was observed in Elio. Exogenous 6-BA counteracted the effects of sole NH₄ ⁺ on leaf growth and photosynthetic activity in both cultivars. OsIPT3 was the key NO₃ ^–-responsive cytokinin synthesis gene in cv. Nanguang.

Conclusions

High NO₃ ^– responsiveness is associated with increased cytokinin synthesis and transport from the root to the leaf and is strongly related to a higher photosynthetic capacity in cv. Nanguang.     

Phenotypic seedling responses of a metal-tolerant mutant line of sunflower growing on a Cu-contaminated soil series: potential uses for biomonitoring of Cu exposure and phytoremediation

Background and aims

The potential use of a metal-tolerant sunflower mutant line for both biomonitoring and phytoremediating a Cu-contaminated soil series was investigated.

Methods

The soil series (21–1,170 mg Cu kg^?1) was sampled in field plots at control and wood preservation sites. Sunflowers were cultivated 1 month in potted soils under controlled conditions.

Results

pH and dissolved organic matter influenced Cu concentration in the soil pore water. Leaf chlorophyll content and root growth decreased as Cu exposure rose. Their EC₁₀ values corresponded to 104 and 118 μg Cu L^?1 in the soil pore water, 138 and 155 mg Cu kg^?1 for total soil Cu, and 16–18 mg Cu kg^?1 DW shoot. Biomass of plant organs as well as leaf area, length and asymmetry were well correlated with Cu exposure, contrary to the maximum stem height and leaf water content.

Conclusions

Physiological parameters were more sensitive to soil Cu exposure than the morphological ones. Bioconcentration and translocation factors and distribution of mineral masses for Cu highlighted this mutant as a secondary Cu accumulator. Free Cu²⁺ concentration in soil pore water best predicted Cu phytoavailability. The usefulness of this sunflower mutant line for biomonitoring and Cu phytoextraction was discussed.     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Degradation kinetics of biochar from pyrolysis and hydrothermal carbonization in temperate soils

Background and Aims

Estimates of biochar residence times in soils range over three orders of magnitude. We present the first direct comparison between the biodegradation of a char from hydrothermal carbonization (htcBC) and pyrolysis (pyrBC) with high temporal resolution.

Methods

Mineralization of the biochars and their shared Miscanthus feedstock in three soils was determined directly by the ¹³CO₂ efflux using a novel method incorporating wavelength scanned cavity ring-down spectroscopy. Biochar half-life (t_1/2) was estimated with three empirical models.

Results

(1) The htcBC was readily biodegradable, whereas pyrBC was more recalcitrant. (2) Cumulative degradation of both biochars increased with soil organic carbon and nitrogen content. (3) The corrected Akaike information criterion (AIC_C) showed an overall preference for the double exponential model (DEM) reflecting a labile and a recalcitrant C-pool, over the first-order degradation model (FODM) and a logarithmic model. (4) The DEM resulted in t_1/2 ranging from 19.7–44.5, 0.7–2.1 and 0.8–1.3 years for pyrBC, htcBC and feedstock, respectively.

Conclusion

The degradation was rather similar between feedstock and htcBC but one order of magnitude slower for pyrBC. The AIC_C preferred FODM in two cases, where the DEM parameters indicated no distinction between a labile and recalcitrant carbon pool.     

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Background

The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.

Results

Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the K_M for NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_M for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1 as compared to 4.2 mg × (L × h)^-1 for the wild type strain.

Conclusion

Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.     

PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins

Background

The hallmark of HIV-1 pathogenesis is the progressive CD4⁺ T cell depletion and high propensity of CD4⁺ T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr_71-82 mitochondriotoxic domain containing the conserved sequence _71-HFRIGCRHSRIG_-82 with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A₁ is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal Findings

Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A₁ binding sequence from Vpr_77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR_83–85 and T89A substitutions in the Vpr_77–92 sequence prevent PP2A₁ binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr_71–82 domain, has no effect on the biological properties of the Vpr_77–92 domain.

Conclusion

Together our data provide evidence for the first time that the Vpr_77–92 sequence delineates a biological active domain of Vpr with PP2A₁ binding and pro-apopototic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr_77–92 domain in full length Vpr protein and also in entire HIV provirus.