Impacts of single‐walled carbon nanotubes on microbial community structure in activated sludge

Aims: Single‐walled carbon nanotubes (SWNTs) are likely to become increasingly widespread and yet their environmental impact is not well understood. The purpose of the current study was to evaluate the impact of SWNTs on microbial communities in a ‘sentinel’ environmental system, activated sludge batch‐scale reactors. Methods and Results: Triplicate batch reactors were exposed to SWNTs and compared to control reactors exposed to impurities associated with SWNTs. Automated ribosomal intergenic spacer analysis (ARISA) was used to assess bacterial community structure in each reactor. SWNT exposure was found to impact microbial community structure, while SWNT‐associated impurities had no effect, compared to controls. 16S rRNA gene sequence analysis indicated that dominant phylotypes detected by ARISA included members of the families Sphingomonadaceae and Cytophagacaceae and the genus Zoogloea. ARISA results indicated an adverse impact of SWNTs on the sphingomonad relative to other community members. Changes in community structure also occurred in both SWNT‐exposed and control reactors over the experimental time period and with the date on which activated sludge was obtained from a wastewater treatment facility. Conclusions: These results indicate that SWNTs differentially impact members of the activated sludge reactor bacterial community. Significance and Impact of the Study: The finding that community structure was affected by SWNTs indicates that this emerging contaminant differentially impacted members of the activated sludge bacterial community and raises the concern that SWNTs may also affect the services it provides.     

Preparation of a Fludarabine Intermediate via Selective Alkylation of 2-Fluoroadenine

The reaction between 2-fluoroadenine (3) and 1,3,5-tri-O-benzyl-1-α-d-chloroarabinofuranose (4) with potassium t-amylate was evaluated in various solvents to afford 9-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (5) and the corresponding α-anomer (6). In addition, 7-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (7) and an unusual “bis-fluoroadenine” nucleoside (8) were isolated as by-products. The highest anomeric ratio (β/α > 10) and conversion (>80%) were observed with the highly polar solvent sulfolane. This reaction was demonstrated on gram scale as a practical laboratory synthesis of 5, a known intermediate in the synthesis of fludarabine.     

Intermediate Conductances during Deactivation of Heteromultimeric Shaker Potassium Channels

A previous study of the T442S mutant Shaker channel revealed activation-coupled subconductance levels that apparently represent kinetic intermediates in channel activation (Zheng, J., and F.J. Sigworth. 1997. J. Gen. Physiol. 110:101–117). We have now extended the study to heteromultimeric channels consisting of various numbers of mutant subunits as well as channels without mutant subunits, all in the background of a chimeric Shaker channel having increased conductance. It has been found that activation-coupled sublevels exist in all these channel types, and are traversed in at least 80% of all deactivation time courses. In symmetric K⁺ solutions, the currents in the two sublevels have a linear voltage dependence, being 23–44% and 54–70% of the fully open conductance. Sublevels in different channel types share similar voltage dependence of the mean lifetime and similar ion selectivity properties. However, the mean lifetime of each current level depends approximately geometrically on the number of mutant subunits in the channel, becoming shorter in channels having fewer mutant subunits. Each mutant subunit appears to stabilize all of the conducting states by ∼0.5 kcal/mol. Consistent with previous results in the mutant channel, sublevels in channels with two or no mutant subunits also showed ion selectivities that differ from that of the fully open level, having relatively higher K⁺ than Rb⁺ conductances. A model is presented in which Shaker channels have two coupled activation gates, one associated with the selectivity filter and a second associated with the S6 helix bundle.     

Importance of the mitochondrial outer membrane channel as a model biological channel

The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.     

Review. Peering into an ATPase ion pump with single-channel recordings

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na+,K+-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na+,K+-ATPase is transformed into a gated cation channel. Millions of Na+ or K+ ions per second flow through such an open pump-channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na+,K+-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pump's transmembrane segments allows mapping and characterization of the route traversed by transported ions.     

Two-pore domain potassium channels: variation on a structural theme

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na(+) and Ca(2+) channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K(+) ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K(+) channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K(+) (K2P) channels also contribute to the steady outward leak of K(+) ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K(+) channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

Gating pore currents,a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na⁺ and Ca²⁺ channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K⁺ ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K⁺ channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K⁺ (K2P) channels also contribute to the steady outward leak of K⁺ ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K⁺ channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K(+)]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K(+)] modulate the rate of channel activation. Elevation of symmetrical [K(+)] or [Rb(+)] from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar [K(+)]-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and its Electrochemical Properties

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4–9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H₂O₂ but without an added mediator or a reducing substrate.     

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

Summary The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [³H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na⁺ and K⁺ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K _m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na⁺ and K⁺ binding sites. Na⁺ and K⁺ were partially replaceable, with the selectivity of the Na⁺ site being Na⁺ > Li⁺ > NH ₄ ⁺ ; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ also supported a small amount of specific binding when substituted for Na⁺. The selectivity of the K⁺ site was K⁺ Rb⁺ > NH ₄ ⁺ > Cs⁺; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li⁺ could partially substitute for Na⁺ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K⁺ site was NH ₄ ⁺ > K⁺ Rb⁺ > Cs⁺ other monovalent cations. The effect of Cl^- on bumetanide binding was biphasic, being stimulatory at low [Cl^-] but inhibitory at high [Cl^-]. As this implies the existence of two Cl^- binding sites (termed Cl _H and Cl _L for the high- and low- affinity sites, respectively) each phase was examined individually. Cl^- binding to Cl _H could be described by a rectangular hyperbola with a K _m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl^-] revealed that it was of a noncompetitive type (K _i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl _H was highly selective with Cl^- > SCN^- > Br^-; F^-, NO ₃ ^- , ClO ₄ ^- , MeSO ₄ ^- , gluconate^- and SO ₄ ^2- were inactive. The efficacy of anion inhibition of binding to Cl _L was ClO ₄ ^- > I^- > SCN^- > NO₃ > Cl^-; F^-, MeSO ₄ ^- , gluconate^-, and SO ₄ ^2- were inactive. Thus, Cl _H is much more selective than Cl _L and largely accounts for the specificity of the system with respect to anion transport. SO ₄ ^- , NO ₃ ^- , I^-, SCN^- and ClO ₄ ^- did not support cotransport when bound to Cl _L and the latter three anions were inhibitory. Mg²⁺ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.We thank Drs. Brad Pewitt, John Westley and Mrinalini Rao for discussion, Sara Leung and Artelia Watson for their excellent technical assistance, and Dr. R.J. Turner for his gift of [³H] bumetanide. This work was supported in part by Cystic Fibrosis Center grant #CF RO11 7-04.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.     

The functional properties of Ompβ, the regularly arrayed porin of the hyperthermophilic bacterium Thermotoga maritima

Abstract The regularly arrayed outer membrane protein, Ompβ, of Thermotoga maritima was purified to homogeneity and was characterized functionally by incorporation into artificial lipid bilayers. The polypeptide has an apparent molecular mass ( M _r) of approx. 40 000 and forms stable trimers in the presence of 1% octyl-polyoxyethylene or 2% SDS which dissociate when boiling the sample. The protein has a secondary structure (predominantly β-sheet) and an amino acid composition characteristic for porins. Pore-forming activity was demonstrated by porin incorporation into artificial bilayers proving that Ompβ is a true porin: selectivity measurements showed a 4.4-fold selectivity for cations over anions. Conductivity of the porin is influenced by surface charges and also depends on the applied voltage.     

The effect of porosity of seiving particles on the romoval efficiency of organic substances via biofilter in the fixed bed

This paper was investigated to clarify the possibility of a biodegradation of materials adsorbed on different porous granular-activated carbons (GACs) such as coal-& coconut-based GAC. Total organic carbon, humic substance and ammonia were used to compare their removal efficiencies. The objective of this study is to determine the adsorption capacity of bioregenerated GAC. When raw water reacted with chloride, the yield of THMs increased as a function of the input amount of chloride. The formation of trihalomethanes (THMs) was investigated in water treated with chlorine when humic acid was used as THM precursor. As the input amount of chloride in raw water increased by two or five-fold to remove the NH₃, the chloroform of the THMs significantly increased also five or ten-fold. It was found that the chloroform was significantly removed by the treatment of biological activated carbon (BAC) in comparison with the ozone treatment, and the removal efficiency of THMs in coal-typed GAC was 10–30% better than coconut-typed GAC due to the biological degradation on the surface of the activated carbons.     

Lithium-selective permeation through lipid bilayer membranes mediated by a di-imide ionophore with nonsymmetrical imide substituents (ETH1810)

Summary The neutral, noncyclic Li^–-selective ionophore ETH1810, which is a di-imide, differs structurally from previous similar ionophores by removal of the intramolecular symmetry of the N-imide substituents. Properties of this ionophore, as a potential carrier of lithium, were probed through studies of ionophore-induced changes in electrical properties of lipid bilayer membranes. ETH1810 was found capable of transporting lithium and other monovalent cations, across lipid bilayer membranes, forming 21 ionophore: ion membrane-permeating species. It was found to be 10-fold more potent than ETH1644, which was the previous best ionophore of this type. The selectivity sequence among alkali cations was found to be: Li⁺(1)>Na⁺ (0,009)>K⁺ (0.004)>Cs⁺(0.0035). Among the physiological alkali cations, it constitutes a 40 (vs. Na⁺) to 160% (vs. K⁺) improvement over ETH1644. ETH1810 was also found to be capable of acting as a carrier of biogenic amines and related molecules, with the following selectivity sequence: tryptamine (20)>phenylethylamine (7.8)>tyramine (4.3)>serotonin (2.5)>Li⁺ (1)>NH ₄ ⁺ (0.013)>dopamine (0.012). It was found that protons, at physiological concentrations, do not interfere with the lithium transport mediated by ETH1810. The relationship between the improvements in ionic selectivity and potencyvs. the differences in structural features is discussed.