Insights into plant metabolic networks from steady-state metabolic flux analysis

Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways.     

Life cycle assessment of primary magnesium production using the Pidgeon process in China

Background, aims, and scope  China has been the largest primary magnesium producer in the world since year 2000 and is an important part of the global magnesium supply chain. Almost all of the primary magnesium in China is produced using the Pidgeon process invented in the 1940s in Canada. The environmental problems of the primary magnesium production with the Pidgeon process have already attracted much attention of the local government and enterprises. The main purposes of this research are to investigate the environmental impacts of magnesium production and to determine the accumulative environmental performances of three different scenarios. System boundary included the cradle-to-gate life cycle of magnesium production, including dolomite ore extraction, ferrosilicon production, the Pidgeon process, transportation of materials, and emissions from thermal power plant. The life cycle assessment (LCA) case study was performed on three different fuel use scenarios from coal as the overall fuel to two kinds of gaseous fuels, the producer gas and coke oven gas. The burden use of gaseous fuels was also considered. Methods  The procedures, details, and results obtained are based on the application of the existing international standards of LCA, i.e., the ISO 14040. Depletion of abiotic resources, global warming, acidification, and human toxicity were adopted as the midpoint impact categories developed by the problem-oriented approach of CML to estimate the characterized results of the case study. The local characterization and normalization factors of abiotic resources were used to calculate abiotic depletion potential (ADP). The analytic hierarchy process was used to determine the weight factors. Using the Umberto version 4.0, the emissions of dolomite ore extraction were estimated and the transportation models of the three scenarios were designed. Results and conclusions  The emissions inventory showed that both the Pidgeon process of magnesium production and the Fe–Si production were mainly to blame for the total pollutant emissions in the life cycle of magnesium production. The characterized results indicated that ADP, acidification potential, and human toxicity potential decreased cumulatively from scenarios 1 to 3, with the exception of global warming potential. The final single scores indicated that the accumulative environmental performance of scenario 3 was the best compared with scenarios 1 and 2. The impact of abiotic resources depletion deserves more attention although the types and the amount of mineral resources for Mg production are abundant in China. This study suggested that producer gas was an alternative fuel for magnesium production rather than the coal burned directly in areas where the cost of oven gas-produced coke is high. The utilization of “clean” energy and the reduction of greenhouse gases and acidic gases emission were the main goals of the technological improvements and cleaner production of the magnesium industry in China. Recommendation and perspective  This paper has demonstrated that the theory and method of LCA are actually helpful for the research on the accumulative environmental performance of primary magnesium production. Further studies with “cradle-to-cradle” scheme are recommended. Furthermore, other energy sources used in magnesium production and the cost of energy production could be treated in further research.     

Generation of primary amide glucosides from cyanogenic glucosides

The cyanogenic glucoside-related compound prunasinamide, (2R)-β-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H₂O₂ has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Differential methotrexate hepatotoxicity on rat hepatocytes in 2-D monolayer culture and 3-D gel entrapment culture

It has been reported that 3-D cultures of hepatocytes or HepG2 cells were less susceptible to methotrexate (MTX) than their 2-D counterparts. Such a mechanism was addressed in this study by investigation of MTX hepatotoxicity in gel entrapped (3-D) rat hepatocytes vs. traditional monolayer culture (2-D). Similarly, gel entrapped hepatocytes showed higher drug resistance to MTX than hepatocyte monolayers in whatever culture medium with or without modification by hormone supplements (dexamethasone, glucagon and insulin). It was also found that medium modification by hormones greatly increased drug resistance of hepatocyte monolayers but has only a slight effect on 3-D cultured hepatocytes. These differential MTX toxicities regarding culture medium and culture models were assumed to correlate with multidrug resistance associated protein 2 (Mrp2). The involvement of Mrp2 was confirmed directly by the fact that MTX intracellularly accumulated less in gel entrapped hepatocytes than in hepatocyte monolayer but could be enhanced by Mrp2 inhibitors accompanied by reduced drug resistance. Furthermore, the expression of Mrp2 on gene level and transportation activity together with bile-duct-like structure were more significantly evidenced in 3-D gel entrapment culture than in 2-D monolayer culture. In conclusion, the highly preserved Mrp2 in 3-D gel entrapped hepatocytes determines its high drug resistance to MTX. Gel entrapped hepatocytes could be useful for investigation of hepatic transportation and hepatotoxicity.     

Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde

Cholesterol secoaldehyde (ChSeco or 3β-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Aβ aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 μM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-l-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Aβ42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Aβ42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species.     

Sonic hedgehog mediates BDNF-induced neuroprotection against mitochondrial inhibitor 3-nitropropionic acid

Sonic hedgehog (SHH), a morphogen critical for embryogenesis, has also been shown to be neuroprotective. We have recently reported that pretreatment of rat cortical neurons for 8 h with brain-derived neurotrophic factor (BDNF; 100 ng/ml) affords protection against neurotoxicity of 3-nitropropionic acid (3-NP; 2.5 mM for 24 h), a mitochondrial complex II inhibitor. However, whether SHH is involved in BDNF-mediated neuroprotection remains unknown. Herein we tested whether BDNF induces SHH expression and if so, whether BDNF induction of SHH contributes to the observed neuroprotective effects. We found BDNF (100 ng/ml) increased SHH expression at both mRNA and protein levels. BDNF protection against 3-NP was abolished by cyclopamine (CPM; 5 μM), the SHH pathway inhibitor. Preconditioning of cortical neurons with N-terminal fragment of SHH (SHH-N; 0.1-1 ng/ml) was sufficient to confer resistance. These results indicate that BDNF induces SHH expression, which contributes to neuroprotection against 3-NP toxicity in rat cortical neurons.     

Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: The case of Euglena gracilis

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A_498, non fluorescent and B₄₆₂, fluorescent.Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A₄₉₈ becomes B₄₆₂), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B₄₆₂ form acts as donor for the non-fluorescent A₄₉₈ form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.     

Differences in sensitivity of cultured rat brain neuronal and glial cytochrome P450 2E1 to ethanol

The expression of the cytochrome P450s (CYPs) may vary in the different brain cells depending on their specialization and the presence of different endogenous factors. The present study was initiated to investigate the expression and catalytic activity of the constitutive and inducible forms of CYP2E1, the major ethanol inducible CYP, in cultured rat brain neuronal and glial cells. These cells exhibited relatively two-fold higher activity of N-nitrosodimethylamine demethylase (NDMA-d) when compared with the liver enzyme. Pretreatment with ethanol revealed a significant time and concentration dependent induction in NDMA-d activity in both cell types. Western blot, immunocytochemistry and RT-PCR also indicated significant induction of CYP2E1 in the cultured brain cells. Interestingly, the neuronal cells exhibited greater magnitude of induction than the glial cells. The relatively higher degree of induction in cultures of neurons has indicated enhanced sensitivity of neurons to the inductive effects of ethanol. This enhanced induction of CYP2E1 in neuronal cells has indicated that like regional specificity, cell specificity also exists in the induction of CYP2E1 and other CYPs.