Network-level analysis of metabolic regulation in the human red blood cell using random sampling and singular value decomposition

Background  

Extreme pathways (ExPas) have been shown to be valuable for studying the functions and capabilities of metabolic networks through characterization of the null space of the stoichiometric matrix (S). Singular value decomposition (SVD) of the ExPa matrix P has previously been used to characterize the metabolic regulatory problem in the human red blood cell (hRBC) from a network perspective. The calculation of ExPas is NP-hard, and for genome-scale networks the computation of ExPas has proven to be infeasible. Therefore an alternative approach is needed to reveal regulatory properties of steady state solution spaces of genome-scale stoichiometric matrices.     

Growth,secondary metabolite production and antioxidant enzyme response of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> adventitious root as affected by auxin and cytokinin

Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l⁻¹ indole butyric acid (IBA) and at 7 and 9 mg l⁻¹ naphthalene acetic acid (NAA). On the other hand, 9 mg l⁻¹ NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l⁻¹ IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l⁻¹) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l⁻¹) in combination with 5 mg l⁻¹ IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l⁻¹ IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.     

Determination of Fecal Glucocorticoid Metabolites to Evaluate Stress Response in <Emphasis Type="Italic">Alouatta pigra</Emphasis>

Measuring fecal glucocorticoid metabolites is now a common practice to assess the stress response in primates. Nevertheless, it is important to validate the utilized immunoassay for each primate species before the technique is applied to populations in the wild. We determined the stress response of black howlers (Alouatta pigra) via 2 different group-specific enzyme immunoassays (EIAs). 11-oxoetiocholanolone EIAs are suited to assess the stress response of black howlers via fecal glucocorticoid metabolites. Levels of fecal glucocorticoid metabolites increased after we applied a stressor, i.e. anesthesia, reaching peak concentrations 24–96 h poststressor. Both basal and stress-induced fecal glucocorticoid metabolite levels showed individual variations. The increase of fecal glucocorticoid metabolites after the stressor (paralleling increases in serum) indicates that one can effectively measure adrenocortical activity in Alouatta pigra via these 2 enzyme immunoassays. However, it is important to consider individual variations in the excretion of fecal glucocorticoid metabolites when planning field endocrinological research on Alouatta pigra. Fecal glucocorticoid metabolite excretion takes 1–3 d poststressor depending on the individual. Further, there is an important individual variability in the concentrations of glucocorticoid metabolites, which might reflect differences in stress reactivity or fecal glucocorticoid metabolite metabolism and excretion.     

Robust detection and verification of linear relationships to generate metabolic networks using estimates of technical errors

Background  

The size and magnitude of the metabolome, the ratio between individual metabolites and the response of metabolic networks is controlled by multiple cellular factors. A tight control over metabolite ratios will be reflected by a linear relationship of pairs of metabolite due to the flexibility of metabolic pathways. Hence, unbiased detection and validation of linear metabolic variance can be interpreted in terms of biological control. For robust analyses, criteria for rejecting or accepting linearities need to be developed despite technical measurement errors. The entirety of all pair wise linear metabolic relationships then yields insights into the network of cellular regulation.     

METANNOGEN: compiling features of biochemical reactions needed for the reconstruction of metabolic networks

Background  

One central goal of computational systems biology is the mathematical modelling of complex metabolic reaction networks. The first and most time-consuming step in the development of such models consists in the stoichiometric reconstruction of the network, i. e. compilation of all metabolites, reactions and transport processes relevant to the considered network and their assignment to the various cellular compartments. Therefore an information system is required to collect and manage data from different databases and scientific literature in order to generate a metabolic network of biochemical reactions that can be subjected to further computational analyses.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Stable isotope resolved metabolomics of lung cancer in a SCID mouse model

We have determined the time course of [U-¹³C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of ¹³C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC–MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal ¹³C incorporation into tissue was 15–20 min but with substantial differences in ¹³C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the ¹³C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The ¹³C metabolite profile shows considerable differences in [U-¹³C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model.     

Mechanistic insight from thermal activation parameters for oxygenation reactions of different substrates with biomimetic iron porphyrin models for compounds I and II

Compound I, an oxo–iron(IV) porphyrin π-cation radical species, and its one-electron-reduced form compound II are regarded as key intermediates in reactions catalyzed by cytochrome P450. Although both reactive intermediates can be easily produced from model systems such as iron(III) meso-tetra(2,4,6-trimethylphenyl)porphyrin hydroxide by selecting appropriate reaction conditions, there are only a few thermal activation parameters reported for the reactions of compound I analogues, whereas such parameters for the reactions of compound II analogues have not been investigated so far. Our study demonstrates that ΔH ^≠ and ΔS ^≠ are closely related to the chemical nature of the substrate and the reactive intermediate (viz., compounds I and II) in epoxidation and C–H abstraction reactions. Although most studied reactions appear to be enthalpy-controlled (i.e., ΔH ^≠ > −TΔS ^≠), different results were found for C–H abstractions catalyzed by compound I. Whereas the reaction with 9,10-dihydroanthracene as a substrate is also dominated by the activation enthalpy (ΔH ^≠ = 42 kJ/mol, ΔS ^≠ = 41 J/Kmol), the same reaction with xanthene shows a large contribution from the activation entropy (ΔH ^≠ = 24 kJ/mol, ΔS ^≠ = −100 J/kmol). This is of special interest since the activation barrier for entropy-controlled reactions shows a significant dependence on temperature, which can have an important impact on the relative reaction rates. As a consequence, a close correlation between bond strength and reaction rate—as commonly assumed for C–H abstraction reactions—no longer exists. In this way, this study can contribute to a proper evaluation of experimental and computational data, and to a deeper understanding of mechanistic aspects that account for differences in the reactivity of compounds I and II.     

A novel stable isotope labelling assisted workflow for improved untargeted LC–HRMS based metabolomics research

Many untargeted LC–ESI–HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly ¹³C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-¹³C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC–HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87–135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-¹³C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.     

Influence of stand density on soil CO2 efflux for a Pinus densiflora forest in Korea

We investigated the influence of stand density [938 tree ha⁻¹ for high stand density (HD), 600 tree ha⁻¹ for medium stand density (MD), and 375 tree ha⁻¹ for low stand density (LD)] on soil CO₂ efflux (R _S) in a 70-year-old natural Pinus densiflora S. et Z. forest in central Korea. Concurrent with R _S measurements, we measured litterfall, total belowground carbon allocation (TBCA), leaf area index (LAI), soil temperature (ST), soil water content (SWC), and soil nitrogen (N) concentration over a 2-year period. The R _S (t C ha⁻¹ year⁻¹) and leaf litterfall (t C ha⁻¹ year⁻¹) values varied with stand density: 6.21 and 2.03 for HD, 7.45 and 2.37 for MD, and 6.96 and 2.23 for LD, respectively. In addition, R _S was correlated with ST (R ² = 0.77–0.80, P < 0.001) and SWC (R ² = 0.31–0.35, P < 0.001). It appeared that stand density influenced R _S via changes in leaf litterfall, LAI and SWC. Leaf litterfall (R ² = 0.71), TBCA (R ² = 0.64–0.87), and total soil N contents in 2007 (R ² = 0.94) explained a significant amount of the variance in R _S (P < 0.01). The current study showed that stand density is one of the key factors influencing R _S due to the changing biophysical and environmental factors in P. densiflora.     

The control of mitochondrial succinate-dependent H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

In brain mitochondria succinate activates H₂O₂ release, concentration dependently (starting at 15 μM), and in the presence of NAD dependent substrates (glutamate, pyruvate, β-hydroxybutyrate). We report that TCA cycle metabolites (citrate, isocitrate, α-ketoglutarate, fumarate, malate) individually and quickly inhibit H₂O₂ release. When they are present together at physiological concentration (0.2, 0.01, 0.15, 0.12, 0.2 mM respectively) they decrease H₂O₂ production by over 60% at 0.1–0.2 mM succinate. The degree of inhibition depends on the concentration of each metabolite. Acetoacetate is a strong inhibitor of H₂O₂ release, starting at 10 μM and acting quickly. It potentiates the inhibition induced by TCA cycle metabolites. The action of acetoacetate is partially removed by β-hydroxybutyrate. Removal is minimal at 0.1 mM acetoacetate, and is higher at 0.5 mM acetoacetate. We conclude that several inhibitors of H₂O₂ release act jointly and concentration dependently to rapidly set the required level of H₂O₂ generation at each succinate concentration.     

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

Background  

The L-type Ca²⁺ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca²⁺ signaling but poorly understood. We characterized a single-base frame-shift mutant of α_1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca²⁺ channel fragments.     

Estimation of the number of extreme pathways for metabolic networks

Background  

The set of extreme pathways (ExPa), {p _i }, defines the convex basis vectors used for the mathematical characterization of the null space of the stoichiometric matrix for biochemical reaction networks. ExPa analysis has been used for a number of studies to determine properties of metabolic networks as well as to obtain insight into their physiological and functional states in silico. However, the number of ExPas, p = |{p _i }|, grows with the size and complexity of the network being studied, and this poses a computational challenge. For this study, we investigated the relationship between the number of extreme pathways and simple network properties.     

Production of ginsenoside and polysaccharide by two-stage cultivation of Panax quinquefolium L. cells

Based on the results of carbon source consumption in cell suspension culture of Panax quinquefolium L., 30 g L⁻¹ sucrose was fed into a 5-L stirred tank bioreactor on day 16 of culture to enhance cell density and metabolite production. Using a fed-batch cultivation strategy, polysaccharide production was enhanced to 1.608 g L⁻¹, which was 1.96-fold greater than with batch cultivation. The maximum saponin yield (7.828 mg L⁻¹) was obtained on day 24 and was about 36% higher than the yields obtained using batch cultivation. In a two-stage culture process, a combined treatment with sucrose, lactoalbumin hydrolysate, and methyl jasmonate caused a significant increase in total saponin yield (31.52 mg L⁻¹) in cell cultures after 27 d. This value represents an increase of 4.03-fold compared with the total saponin yield in fed-batch cultivation. The two-stage culture mode provided the best method for the in vitro production of secondary metabolites from P. quinquefolium.     

Evidence Group I mGluR Drugs Modulate the Activation Profile of Lipopolysaccharide-Exposed Microglia in Culture

Metabotropic glutamate receptors (mGluRs) may play a role in modulating microglial activation, but group I mGluRs have received little attention. This study aimed to investigate the effects of group I mGluR selective ligands, (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), in lipopolysaccharide (LPS; 50 ng/ml)-activated rat microglial cultures. (S)-3,5-DHPG (150 μM) significantly reduced (approximately 20–60%) the LPS-mediated production of nitrite (NO₂ ⁻), tumour necrosis factor alpha (TNF-α), and l-glutamate (Glu) at 24 and 72 h. Image analysis revealed increases in both cell area and number, with larger amoeboid microglia (with retracted processes) formed following 2 h LPS exposure. This cellular population was absent after addition of (S)-3,5-DHPG, an effect antagonised by AIDA, and a concomitant reduction in cell area was also found. Taken together, these biochemical and morphological observations suggest that (S)-3,5-DHPG reduces microglial activation, indicating a role for group I mGluRs in modulating microglial function.     

Autotrophic and heterotrophic respiration in needle fir and Quercus-dominated stands in a cool-temperate forest, central Korea

To investigate annual variation in soil respiration (R _S) and its components [autotrophic (R _A) and heterotrophic (R _H)] in relation to seasonal changes in soil temperature (ST) and soil water content (SWC) in an Abies holophylla stand (stand A) and a Quercus-dominated stand (stand Q), we set up trenched plots and measured R _S, ST and SWC for 2 years. The mean annual rate of R _S was 436 mg CO₂ m⁻² h⁻¹, ranging from 76 to 1,170 mg CO₂ m⁻² h⁻¹, in stand A and 376 mg CO₂ m⁻² h⁻¹, ranging from 82 to 1,133 mg CO₂ m⁻² h⁻¹, in stand Q. A significant relationship between R _S and its components and ST was observed over the 2 years in both stands, whereas a significant correlation between R _A and SWC was detected only in stand Q. On average over the 2 years, R _A accounted for approximately 34% (range 17–67%) and 31% (15–82%) of the variation in R _S in stands A and Q, respectively. Our results suggested that vegetation type did not significantly affect the annual mean contributions of R _A or R _H, but did affect the pattern of seasonal change in the contribution of R _A to R _S.     

Synthesis, characterization, and reaction pathways for the formation of a GMP adduct of a cytotoxic thiocyanato ruthenium arene complex

The organoruthenium complex [(η⁶-hmb)Ru(en)(Cl)][PF₆] (hmb is hexamethylbenzene, en is ethylenediamine) undergoes facile aquation and then reacts with KSCN in unbuffered solution to give the S-coordinated thiocyanato product [(η⁶-hmb)Ru(en)(S-SCN)]⁺ which slowly converts to the thermodynamically favored N-bound complex [(η⁶-hmb)Ru(en)(N-NCS)]⁺ (1 ⁺). Complex 1 was synthesized and characterized by X-ray crystallography and mass spectrometry. Despite its lack of hydrolysis over 24 h, complex 1 exhibits moderate cytotoxicity (IC₅₀ 24 μM) towards the human ovarian cancer cell line A2780, comparable with that of the chlorido analogue which is thought to be activated (towards potential target DNA) via a rapid aquation (Wang et. al. in Proc Natl Acad Sci USA 102:18269–18274, 2005). Detailed kinetic studies suggest that complex 1 binds to guanosine 5′-monophosphate (GMP) through direct N7 substitution of the N-bound SCN ligand. In the presence of a high concentration of chloride (104 mM), however, complex 1 may bind partly to GMP via Cl substitution.     

Kinetic study of DNA binding of cisplatin and of a bicycloalkyl-substituted (ethylenediamine)dichloroplatinum(II) complex

 Salmon sperm DNA platination has been conducted under strictly pseudo-first-order conditions with cisplatin (1) and rac-{(1S,2S,4S)-exo-2-(aminomethyl)-2-amino-7-bicyclo[2.2.1]heptane}dichloroplatinum(II) (2). An aquation step first occurs for both complexes, with the rate constants k ₁ = 1.12(0.02)×10^–4 s^–1 and 1.47(0.02)×10^–4 s^–1 respectively for 1 and 2 at 37  °C, values in agreement with those previously reported. It is followed by the actual platination step whose second-order rate constant has been determined for the first time by physicochemical techniques. The values for 1 and 2 respectively are: k ₂ = 2.08(0.07) M^–1 s^–1 and 3.9(0.4) M^–1 s^–1. These kinetic data are discussed in the context of a comparison of several biological properties of the two complexes. Received: 15 May 1998 / Accepted: 26 June 1998     

Some new aspects of the metabolism of phenacetin in the rat

1. Four new metabolites of phenacetin in the urine of the rat are described; these are (i) N-acetyl-S-ethylcysteine, (ii) quinol, (iii) acetamide and (iv) probably N-acetyl-S-2-(4-ethoxyacetanilido)cysteine S-oxide. 2. Metabolites (i), (iii) and (iv) were characterized and estimated by g.l.c., by t.l.c., by paper chromatography, by chemical reactions or by radioactive techniques after administration to rats of [ethyl-¹⁴C]phenacetin and [acetyl-³H]phenacetin; metabolite (ii), which was excreted mainly as conjugates of sulphuric acid and glucosiduronic acid, was measured by paper chromatography and characteristic colour reactions after enzymic and chemical hydrolysis of the conjugates. 3. Small amounts of azoxy-4-[ethyl-¹⁴C]ethoxybenzene and an unknown metabolite were also found in the urine of rats after administration of [ethyl-¹⁴C]phenacetin. 4. The likely mechanisms and some biological implications of these metabolic reactions are discussed.