Hybrid dynamic modeling of Escherichia coli central metabolic network combining Michaelis–Menten and approximate kinetic equations

The construction of dynamic metabolic models at reaction network level requires the use of mechanistic enzymatic rate equations that comprise a large number of parameters. The lack of knowledge on these equations and the difficulty in the experimental identification of their associated parameters, represent nowadays the limiting factor in the construction of such models. In this study, we compare four alternative modeling approaches based on Michaelis–Menten kinetics for the bi-molecular reactions and different types of simplified rate equations for the remaining reactions (generalized mass action, convenience kinetics, lin-log and power-law). Using the mechanistic model for Escherichia coli central carbon metabolism as a benchmark, we investigate the alternative modeling approaches through comparative simulations analyses. The good dynamic behavior and the powerful predictive capabilities obtained using the hybrid model composed of Michaelis–Menten and the approximate lin-log kinetics indicate that this is a possible suitable approach to model complex large-scale networks where the exact rate laws are unknown.     

Short-term bioaccumulation, circulation and metabolism of estradiol-17β in the oyster Crassostrea gigas

Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As estradiol-17β has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas. When dissolved in seawater, in less than 48 h estradiol-17β concentrated up to 31 times in the soft tissues of the suspension-feeder mollusc. Injected in the adductor muscle, estradiol-17β circulated from muscle to the gonad, the gills, the mantle, the labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland. Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17β-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain,and is consistent with Michaelis–Menten mechanism

Background

It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease.

Methods

Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis.

Results

The CHS4 domain comprised of sequence residues 116–263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis–Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94–170) within CHS4 is indispensable.

Conclusion

The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does.

Significance

Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.     

Stress–strain analysis of jejunal contractility in response to flow and ramp distension in type 2 diabetic GK rats: Effect of carbachol stimulation

Investigation of intestinal motility in a genetic model of GK rats abandons the possible neurotoxic effect of streptozotocin in streptozotocin-induced diabetic model. Seven GK male rats (GK group) and nine normal Wistar rats (Normal group) were used in the study. The motility experiments were carried out in an organ bath containing physiological Krebs solution. Before and after 10⁻⁵ M carbachol application, the pressure and diameter changes of jejunum were obtained in relation to (1) basic contraction, (2) flow-induced contraction with different outlet resistance pressures and (3) contractions induced by ramp distension. The frequency and amplitude of contractions were analyzed from pressure–diameter curves. Distension-induced contraction thresholds and maximum contraction amplitude of basic and flow-induced contractions were calculated in terms of stress and strain. (1) The contraction amplitude increased to the peak value in less than 10 s after adding carbachol. More than two peaks were observed in the GK group. (2) Carbachol decreased the pressure and stress threshold and Young's modulus in the GK group (P<0.01). (3) Carbachol increased the maximum pressure and stress of flow-induced contractions at most outlet pressure levels in both two groups (P<0.001). Furthermore, the flow-induced contractions were significantly bigger at low outlet pressure levels in GK group (P<0.05 and P<0.01). (4) The contraction frequency, the strain threshold and the maximum contraction strain did not differ between the two groups (P>0.05) and between before and after carbachol application (P>0.05). In GK diabetic rats, the jejunal contractility was hypersensitive to flow and distension stimulation after carbachol application.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n–3), to eicosapentaenoic acid, 20:5(n–3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C₁₈ to C₂₀ polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n–3), and its elongation product, 20:4(n–3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 μM), U-¹⁴C (1 μM) or deuterated (d5; 25 μM) fatty acids. Stearidonic acid, 18:4(n–3), was metabolised to 20:5(n–3) in both cells lines, but more so in AS than in TF cells. Δ5 desaturation was more active in TF cells than in AS cells, whereas C₁₈ to C₂₀ elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n–3)) were produced by both cell lines, although there was significant production of 22:5(n–3) in both cultures, especially when 20:4(n–3) was supplemented. We conclude that limited elongation of C₁₈ to C₂₀ fatty acids rather than limited fatty acyl Δ5 desaturation accounts for the limited rate of conversion of 18:3(n–3) to 20:5(n–3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C₁₈ to C₂₀ PUFA by the turbot and the Atlantic salmon in vivo.     

Shrinking body sizes in response to warming: explanations for the temperature–size rule with special emphasis on the role of oxygen

Body size is central to ecology at levels ranging from organismal fecundity to the functioning of communities and ecosystems. Understanding temperature-induced variations in body size is therefore of fundamental and applied interest, yet thermal responses of body size remain poorly understood. Temperature–size (T–S) responses tend to be negative (e.g. smaller body size at maturity when reared under warmer conditions), which has been termed the temperature–size rule (TSR). Explanations emphasize either physiological mechanisms (e.g. limitation of oxygen or other resources and temperature-dependent resource allocation) or the adaptive value of either a large body size (e.g. to increase fecundity) or a short development time (e.g. in response to increased mortality in warm conditions). Oxygen limitation could act as a proximate factor, but we suggest it more likely constitutes a selective pressure to reduce body size in the warm: risks of oxygen limitation will be reduced as a consequence of evolution eliminating genotypes more prone to oxygen limitation. Thus, T–S responses can be explained by the ‘Ghost of Oxygen-limitation Past’, whereby the resulting (evolved) T–S responses safeguard sufficient oxygen provisioning under warmer conditions, reflecting the balance between oxygen supply and demands experienced by ancestors. T–S responses vary considerably across species, but some of this variation is predictable. Body-size reductions with warming are stronger in aquatic taxa than in terrestrial taxa. We discuss whether larger aquatic taxa may especially face greater risks of oxygen limitation as they grow, which may be manifested at the cellular level, the level of the gills and the whole-organism level. In contrast to aquatic species, terrestrial ectotherms may be less prone to oxygen limitation and prioritize early maturity over large size, likely because overwintering is more challenging, with concomitant stronger end-of season time constraints. Mechanisms related to time constraints and oxygen limitation are not mutually exclusive explanations for the TSR. Rather, these and other mechanisms may operate in tandem. But their relative importance may vary depending on the ecology and physiology of the species in question, explaining not only the general tendency of negative T–S responses but also variation in T–S responses among animals differing in mode of respiration (e.g. water breathers versus air breathers), genome size, voltinism and thermally associated behaviour (e.g. heliotherms).     

Differential response of methionine metabolism in two grain legumes,soybean and azuki bean,expressing a mutated form of Arabidopsis cystathionine γ-synthase

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.     

The effect of hydrogen bonding in two crystal modifications of aquabis(N,N-dimethylglycinato-κN,κO)copper(II): Experimental and theoretical study

From the crystals of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) dihydrate (compound 1, space group P2₁2₁2₁) novel crystal structure of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) (compound 2, space group Pbca) was obtained and analysed by X-ray diffraction. In the crystal structure 1, the O-H?O hydrogen bonds form three-dimensional network. In the crystal structure 2, two-dimensional layers stacking to each other are formed, with non-polar N,N-dimethyl groups placed on the opposite sides of the layers, and with the polar part in the middle forming CO?O-H and C-H?O hydrogen bonds. Different hydrogen bonding patterns in 1 and 2 do not pronouncedly affect molecular geometry of the title compound. Molecular mechanics force field suited for studying the properties of bis(amino acidato)copper(II) complexes in the solid state can follow the differences between the experimental molecular structures in the two diverse crystalline surroundings. To make possible direct comparison between crystal lattices, the force field was applied to predict unit cell packing of supposed anhydrous bis(N,N-dimethylglycinato)copper(II) in space group Pbca. Relative intermolecular energies of hypothetic anhydrous crystal and simulated 1 and 2 crystals are discussed. On the basis of experimental and theoretical results we conclude that the main effect of two water molecules of crystallisation in 1 is to stabilise the crystal packing via hydrogen bonding, whilst similar pyramidal copper(II) coordination geometry in 1 and 2 is due to axially coordinated water molecule and its intermolecular interactions.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Changes in stream chemistry and biology in response to reduced levels of acid deposition during 1987–2003 in the Neversink River Basin,Catskill Mountains

Atmospheric acid deposition has decreased in the northeastern United States since the 1970s, resulting in modest increases in pH, acid-neutralizing capacity (ANC), and decreases in inorganic monomeric aluminum (Al_IM) concentrations since stream chemistry monitoring began in the 1980s in the acid-sensitive upper Neversink River basin in the Catskill Mountains of New York. Stream pH has increased by 0.01 units/year during 1987–2003 at three sites in the Neversink basin as determined by Seasonal Kendall trend analysis. In light of this observed decrease in stream acidity, we sampled 12 stream sites within the Neversink River watershed for water chemistry, macroinvertebrates, fish, and periphytic diatoms in 2003 to compare with a similar data set collected in 1987. Metrics and indices that reflect sensitivity to stream acidity were developed with these biological data to determine whether changes in stream biota over the intervening 16 years parallel those of stream chemistry. Statistical comparisons of data on stream chemistry and an acid biological assessment profile (Acid BAP) derived from invertebrate data showed no significant differences between the two years. For pH and ANC, however, values in 2003 were generally lower than those in 1987; this difference likely resulted from higher streamflow in summer 2003. Despite these likely flow-induced changes in summer 2003, an ordination and cluster analysis of macroinvertebrate taxa based on the Acid BAP indicated that the most acidic sites in the upstream half of the East Branch Neversink River form a statistically significant separate cluster consistent with less acidic stream conditions. This analysis is consistent with limited recovery of invertebrate species in the most acidic reaches of the river, but will require additional improvement in stream chemistry before a stronger conclusion can be drawn. Data on the fish and periphytic diatom communities in 2003 indicate that slimy sculpin had not extended their habitat to upstream reaches that previously were devoid of this acid-intolerant species in 1987; a diatom acid-tolerance index indicates continued high-acid impact throughout most of the East Branch and headwaters of the West Branch Neversink River.     

Is the critical role of loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase in catalysis due to loop-3 residues arginine-84 and tryptophan-89? Site-directed mutagenesis, biochemical, and crystallographic studies

Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.