Ion binding constants for gramicidin a obtained from water permeability measurements

Gramicidin A pores are permeable to water and small monovalent cations. For K, Rb, and Cs there is good evidence from conductances and permeability ratios that a second ion can enter a pore already occupied by another, but for Na this evidence is inconclusive and comparison of tracer fluxes and single channel conductances suggests that second ion entries are prohibited. Partly as a result of the complications of second ion entry there have been widely differing estimates for the dissociation constants for the first ion in the channel. Dani and Levitt (1981, Biophys. J. 35: 485–499) introduced a method for calculating ion binding constants from simultaneous measurements of water fluxes and membrane conductance. They found no evidence for second ion binding and calculated dissociation constants of 115 mm for Li, 69 mm for K, and 2 mm for Tl. It is shown here that the two-ion, four-state model predicts a dependence of water permeability on ion concentration that is difficult to distinguish from the predictions of block by a single ion. Using a modified technique that allows measurement of higher conductances, the first ion dissociation constants have been determined as 80 mm for Na, 40 mm for Rb and 15 mm for Cs. These values and those of Dani and Levitt fall in a smooth sequence. The dissociation constant for Cs is consistent with single channel conductances and flux ratios. There is a discrepancy between this constant for Na and the value, 370 mm, calculated from the single channel conductances and the assumption that a second ion cannot enter or affect an occupied pore. The dissociation constant for Rb is intermediate between those for K and Cs whereas tracer flux measurements (Schagina, Grinfeldt & Lev, 1983. J. Membrane Biol. 73: 203–216) have suggested that Rb interacts much more strongly with the channel than Cs.We should like to thank the National Grid plc, for the grant which supported K.-W.W., the Wellcome Trust for a visiting Fellowship for S.T. in Cambridge, and the Cambridge Society of Bombay which supported S.B.H. in Bombay.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.     

Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints

Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H₂ production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design “fine-tuned” metabolic engineering strategies in silico that can be implemented directly with available genomic tools.     

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at or . Further, community input related to this document can also be provided via this electronic forum. The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration Sponsor: Metabolomics Society http://www.metabolomicssociety.org/ Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/ http://msi-workgroups.sourceforge.net/chemical-analysis/ Version: Revision: 5.1 Date: 09 January, 2007     

冬作季节土地管理对水稻土CH_4排放季节变化的影响

通过温室盆栽试验对水稻土CH4 排放的季节变化及冬作季节土地管理的影响进行了研究 .结果表明 ,冬作季节种植紫云英、淹水休闲及干燥休闲但泡水前施用稻草处理泡水后 3 0dCH4 排放量分别高达 13 3d观测期总排放量的 67.5 %、3 5 .5 %及 3 3 .3 % ,且在泡水后第 13天及水稻移栽后第 7、40、91天分别出现 4个CH4 排放高峰 ;而种植小麦和干燥休闲但冬作前施用稻草处理泡水后 5 5dCH4 排放量才占观测期总排放量的 6.74%和 0 .2 7% ,随后至水稻收获CH4 排放通量也不高 .冬作季节土地管理引起的水稻生长期土壤Eh季节变化的差异是造成CH4 排放通量季节变化差异的主要原因     

Benthic shear stress gradient defines three mutually exclusive modes of non-biological internal nutrient loading in shallow lakes

Assessment of the importance of internal nutrient loading is essential for managing and restoring eutrophic shallow lakes. To date, studies of internal loads have tended to focus on one of two abiotic processes, either molecular diffusion or sediment/nutrient entrainment (resuspension). This study presents a new approach to determining the non-biological fluxes of nitrogen (N) and phosphorus (P) from the sediment to the water column of shallow lakes. Three mutually exclusive flux processes: (i) molecular diffusion, (ii) turbulent diffusion (eddy diffusivity) and (iii) wind-induced resuspension of N and P, were related to a gradient of benthic shear stress. A model presented here allowed the durations and magnitudes of different non-biological fluxes to be calculated over time, based on benthic shear stress. Two site-specific critical shear stress thresholds determined which of the three flux processes dominated for any benthic shear stress value. The model was calibrated for a shallow lake and the continuous flux of nutrient from the sediment to the overlying water generated by each process during that period was calculated, enabling the estimation of the relative importance of each of the three flux processes over a one-year period. Wind-induced resuspension dominated the internal nutrient flux, operating for 38% of the time and contributing 0.9 T P year⁻¹ and 10.2 T N year⁻¹ to the internal nutrient load. In contrast, molecular diffusion only contributed 0.01–0.02 T P year⁻¹ and 0.12–0.20 T N year⁻¹ to the water column, while turbulent diffusion provided up to 0.6 T P year⁻¹ and 6.2 T N year⁻¹. Our model suggests that turbulent diffusion is a neglected and potentially important process contributing to internal nutrient loading in shallow lakes, whereas molecular diffusion appears to be relatively unimportant in lakes that experience turbulence at the sediment–water interface. Handling editor: L. Naselli-Flores     

Simultaneous Monitoring of Phosphine and of Phosphorus Species in Taihu Lake Sediments and Phosphine Emission from Lake Sediments

Phosphine (PH₃) was monitored in the Taihu Lake in China by a GC/NPD method, coupled with cryo-trapping enrichment technology. Results showed that PH₃ was universally detected in sediments, lake water and atmosphere of the Taihu Lake area. Total phosphorus (TP_s) and fractions of different phosphorus species in lake sediments were separately measured as dissolved phosphate (DP), phosphorus bound to aluminum (Al-P), iron (Fe-P) and calcium (Ca-P), occluded phosphorus (OP), and organic phosphorus (Org-P) by sequential chemical extraction. High PH₃ levels were correlated with high TP_s values in sediments and with eutrophication at different sites. In addition, a positive linear correlation equation was obtained between the concentrations of PH₃ in lake sediments and of the phosphorus fractions. The resulting multiple linear regression equation is PH₃ = −165 + 63.3 DP + 0.736 Al-P + 2.33 Ca-P + 2.29 Org-P. The flux of PH₃ across the sediment–water interface was estimated from sediment core incubation in May and October 2002. The annual average sediment–water flux of PH₃ was estimated at ca. 0.0138±0.005 pg dm⁻² h⁻¹, the average yearly emission value of PH₃ from Taihu Lake sediments to water was calculated to be 28.3±10.2 g year⁻¹, which causes a water PH₃ concentration of up to 0.178±0.064 pmol dm⁻³. The real importance of PH₃ could be higher, because PH₃ could be consumed in the oxic sediment–water boundary layer and in the water column. Spatial and temporal distributions of total phosphorus (TP_w) and chlorophyll a (Chl-a) in the water column of Taihu Lake were measured over the study period. Higher water PH₃ has also been found where the TP_w content was high. Similarly, high Chl-a was consistent with higher water PH₃. Positive relationships between PH₃ and TP_w (average R² = 0.47±0.26) and Chl-a (average R² = 0.23±0.31) were observed in Taihu Lake water.     

Dimethyl sulfide metabolism in salt marsh sediments

Abstract Anoxic sediment slurries prepared from Spartina salt marsh soils contained dimethyl sulfide (DMS) at concentrations ranging from 1 to 10 μM. DMS was produced in slurries over the initial 1–24 h incubation. After the initial period of production, DMS decreased to undetectable levels and methane thiol (MSH) was produced. Inhibition of methanogenesis caused a 20% decrease in the rate of DMS consumption, while inhibition of sulfate reduction caused a 80% decrease in DMS consumption. When sulfate reduction and methanogenesis were simultaneously inhibited, DMS did not decrease. DMS contributed about 28% to the methane production rate, while DMS probably contributed only 1% or less to the sulfate reduction rate. Incubation of the sediment slurries under an atmosphere of air resulted in similar DMS consumption compared to anaerobic incubations, but MSH and CH₄ were not evolved.
Sediments from the marsh released significant quantities of DMS when treated with cold alkali, indicating that potentially significant sources of DMS existed in the sediments. Values of base-hydrolyzable DMS as high as 190 μmol per liter of sediment were observed near the sediment surface, and values always decreased with depth in the sediment. Simple flux experiments with small intact sediment cores, showed that DMS was emitted from the marsh surface when cores were injected with glutaraldehyde or molybdate and 2-bromoethanesulfonate (BES), but nit when cores were left uninhibited. These results showed that DMS was readily metabolized by microbes in marsh sediments and that this metabolism may be responsible for reducing the emission of DMS from the marsh surface.