微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。     

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 μg/ml mediating an anodic current of 15.1 μA/cm². On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 μA/cm². Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.     

生物工程强化微生物电合成转化CO_2的研究进展

微生物电合成(Microbial electrosynthesis,MES)可直接利用电能驱动微生物还原固定CO_2合成多碳化合物,为可再生新能源转化、精细化学品制备和生态环境保护提供新机遇。但是,微生物吸收胞外电极电子速率慢、产物合成效率低和产品品位不高,限制了MES实现工业化应用。在概述阴极电活性微生物吸收胞外电子的分子机制的基础上,重点综述近5年应用生物工程的理论和技术强化MES用于CO_2转化的策略与研究进展,包括改造和调控胞外电子传递通路和胞内代谢途径以及定向构建有限微生物混合培养菌群三方面,阐明了生物工程可有效突破MES中电子传递慢和可用代谢途径相对单一等瓶颈。针对目前生物工程在改进MES所面临的主要问题,从胞外电子传递机理研究、基因工具箱开发、组学技术与现代分析技术联用等角度展望了今后的研究方向。     

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member''s contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.     

Quantification of regional right ventricular strain in healthy rats using 3D spiral cine dense MRI

Statistical data from clinical studies suggests that right ventricular (RV) circumferential strain (E_cc) and longitudinal strain (E_ll) are significant biomarkers for many cardiovascular diseases. However, a detailed and regional characterization of these strains in the RV is very limited. In the current study, RV images were obtained with 3D spiral cine DENSE MRI in healthy rats. An algorithm for surface growing was proposed in order to fit irregular topology. Specifically, a new custom plugin for the DENSEanalysis program, called 3D DENSE Plugin for Crescent Organ, was developed for surface reconstruction and precise segmentation of organs with sharp curvature, such as the murine RV. The RV free wall (RVFW) was divided into three longitudinal thirds (i.e., basal, middle, and apical) with each one partitioned into circumferential fourths (i.e., anterior, anteriorlateral, inferiorlateral and inferior). Peak systolic strains were quantified for each segment and comparisons were performed statistically. The inclusion of a new plugin was able to generate global values for E_cc and E_ll that are in good agreement with previous findings using MRI. Despite no regional variation found in the peak E_cc, the peak E_ll exhibited regional variation at the anterior side of the RV, which is potentially due to differences in biventricular torsion at the RV insertion point and fiber architecture. These results provide fundamental insights into the regional contractile function of the RV in healthy rat and could act as a normative baseline for future studies on regional changes induced by disease or treatment.     

人工电活性微生物菌群的设计与应用

包括产电菌群和噬电菌群的人工电活性微生物菌群(synthetic electroactive microbial consortia)通过菌种间的物质能量级联反应介导化学能与(光)电能间的相互转化，其可利用底物来源广泛、双向电子传递速率快、环境稳定性强，在清洁电能开发、废水处理、环境修复、生物固碳固氮以及生物燃料、无机纳米材料、高聚物等高值化学品合成等多个领域具有广泛的应用前景。针对人工电活性微生物菌群设计、构建与应用，本文总结电活性微生物菌群界面电子传递和种间电子传递机制，概括基于“劳力分工”原理设计构建人工电活性微生物菌群物质能量级联反应基本架构，总结菌群关系与菌群生态位优化等人工电活性微生物菌群工程化策略，分类列举人工电活性微生物菌群在利用廉价生物质产电、生物光伏固碳产电，光驱噬电生物菌群固氮等相关应用。最后对人工电活性微生物菌群未来研究方向进行了展望。     

Electroactive mixed culture derived biofilms in microbial bioelectrochemical systems: the role of pH on biofilm formation, performance and composition

The pH-value played a crucial role for the development and current production of anodic microbial electroactive biofilms. It was demonstrated that only a narrow pH-window, ranging from pH 6 to 9, was suitable for growth and operation of biofilms derived from pH-neutral wastewater. Any stronger deviation from pH neutral conditions led to a substantial decrease in the biofilm performance. Thus, average current densities of 151, 821 and 730 μA cm(-2) were measured for anode biofilms grown and operated at pH 6, 7 and 9 respectively. The microbial diversity of the anode chamber community during the biofilm selection process was studied using the low cost method flow-cytometry. Thereby, it was demonstrated that the pH value as well as the microbial inocula had an impact on the resulting anode community structure. As shown by cyclic voltammetry the electron transfer thermodynamics of the biofilms was strongly depending on the solution's pH-value.     

Phototaxis index of Daphnia carinata as an indicator of joint toxicity of copper,cadmium, zinc,nitrogen and phosphorus in aqueous solutions

An orthogonal design method was used for examining the responses of Daphnia carinata to 16 combination mixtures of copper, cadmium, zinc, nitrogen and phosphorus in synthetic solutions. Phototaxis index (I_p), mobilization inhibition percentage (MIP) and mortality percentage (MP) were used as test endpoints for toxicity assessment. The results show that Cu and Cd were the dominant toxicants that affected the D. carinata in the test solutions. However, the contribution of nitrogen, relative to other components, to the overall toxicity to D. carinata could increase over time. It was found that three toxicity parameters (i.e. I_p, MIP and MP) used in this study were significantly interrelated (at p < 0.003 level). Cluster analysis also indicates that there was a general agreement between the toxicity indicated by I_p and the universally accepted test endpoints (MIP or MP); MIP and MP represent the universally accepted test endpoints, i.e. mobilization inhibition and mortality. These results suggest that the phototaxis index of D. carinata is an appropriate test endpoint for toxicity testing of environmental samples. This has implications for developing rapid, non-destructive bioassays for ecotoxicological assessment of aquatic environments.     

Comparative bioelectrochemical analysis of Pseudomonas aeruginosa and Escherichia coli with anaerobic consortia as anodic biocatalyst for biofuel cell application

Aims: To study the bioelectrochemical behaviour of Pseudomonas aeruginosa (MTCC 17702) and Escherichia coli (MTCC 10436) and to assess their potential to act as anodic biocatalyst with the function of anaerobic consortia for microbial (bio) fuel cell (BFC) application. Methods and Results: Three BFCs (single chamber; open‐air cathode; noncatalysed electrodes) were operated simultaneously in acidophilic microenvironments. Pseudomonas aeruginosa (BFC_P) showed higher current density (264 mA m^?2) followed by mixed culture (BFC_M; 166 mA m^?2) and E. coli (BFC_E; 147 mA m^?2). However, total operating period and substrate degradation were relatively found to be effective with mixed culture (58%; 72 h) followed by BFC_P (39%; 60 h) and BFC_E (31%; 48 h). Higher electron discharge (ED) was observed with Ps. aeruginosa while mixed culture showed the involvement of redox mediators in the ED process. Conclusions: Mixed culture showed to sustain biopotential for longer periods along with a stable ED. The presence of redox signals and high substrate degradation was also evidencing its performance compared to the pure strains studied. This supports the practical utility of mixed culture over the pure cultures for real‐field BFC applications especially while operating with wastewater. Significance and Impact of the Study: This study revealed the efficiency and viability of mixed consortia in comparison with pure strains for microbial (bio) fuel cell applications.     

Leg stiffness and joint stiffness while running to and jumping over an obstacle

During running, muscles of the lower limb act like a linear spring bouncing on the ground. When approaching an obstacle, the overall stiffness of this leg-spring system (k_leg) is modified during the two steps preceding the jump to enhance the movement of the center of mass of the body while leaping the obstacle. The aim of the present study is to understand how k_leg is modified during the running steps preceding the jump. Since k_leg depends on the joint torsional stiffness and on the leg geometry, we analyzed the changes in these two parameters in eight subjects approaching and leaping a 0.65 m-high barrier at 15 km h⁻¹. Ground reaction force (F) was measured during 5–6 steps preceding the obstacle using force platform and the lower limb movements were recorded by camera. From these data, the net muscular moment (M_j), the angular displacement (θ_j) and the lever arm of F were evaluated at the hip, knee and ankle. At the level of the hip, the M_j–θ_j relation shows that muscles are not acting like torsional springs. At the level of the knee and ankle, the M_j–θ_j relation shows that muscles are acting like torsional springs: as compared to steady-state running, the torsional stiffness k_j decreases from ~1/3 two contacts before the obstacle, and increases from ~2/3 during the last contact. These modifications in k_j reflect in changes in the magnitude of F but also to changes in the leg geometry, i.e. in the lever arms of F.     

Development of Enterobacter aerogenes fuel cells: From in situ biohydrogen oxidization to direct electroactive biofilm

This study described an Enterobacter aerogenes-catalyzed microbial fuel cell (MFC) with a carbon-based anode that exhibited a maximum power density of 2.51 W/m³ in the absence of artificial electron mediators. The MFC was started up rapidly, within hours, and the current generation in the early stage was demonstrated to result from in situ oxidation of biohydrogen produced by E. aerogenes during glucose fermentation. Over periodic replacement of substrate, both planktonic biomass in the culture liquid and hydrogen productivity decreased, while increased power density and coulombic efficiency and decreased internal resistance were unexpectedly observed. Using scanning electron microscopy and cyclic voltammetry, it was found that the enhanced MFC performance was associated with the development of electroactive biofilm on the anodic surface, proposed to involve an acclimation and selection process of E. aerogenes cells under electrochemical tension. The significant advantage of rapid start-up and the ability to develop an electroactive biofilm identifies E. aerogenes as a suitable biocatalyst for MFC applications.     

Modelling and analysis of the continuous affinity-recycle extraction process: a case of specific elution with low molecular weight competitive inhibitor

The continuous affinity-recycle extraction (CARE) process of specifie elution with low molecular weight competitive inhibitor is mathematically modelled taking into account the presence of membrane rejections to the components in a crude broth. The process performance, defined as purification factor (PF) and recovery yield (REC), is analyzed by computer simulations. The results show that for constant affinity systems (ligate and ligand as well as inhibitor) and operating conditions an optimal value of the inhibitor concentration exists to give maxima of REC and PF, and the optimal value decreases with the increase of the affinity binding constant of ligate and inhibitor. Although the increase in affinity-recycle flow rate results in the decrease of PF, an optimal value of the affinity-recycle flow rate exists to show a maximum of REC. Hence in the process design the selection of the affinity-recycle flow rate is also of importance to obtain higher REC and PF simultaneously. The consideration of membrane rejections will in practice be useful to analyse the separation of a binary broth using ultrafiltration membranes which reject to the components. For a multicomponent broth, however, membranes without rejection to all components should be employed to simplify the process design and optimization. In general, the model is useful to design a CARE process using nonporous microparticles or macromolecules as affinity supports.List of Symbols C _i mol/l contaminant concentration in feed - C ₀₁ mol/l contaminant concentration in waste stream - C ₀₂ mol/l contaminant concentration in product stream - e _i mol/l ligate concentration in feed - e _j mol/l free ligate concentration in Con. j - E _lj mol/l concentration of ligate bound to ligand in Con. - E _oj mol/l ligate concentration in waste (j=1) or product (j=2) stream - E _tj mol/l total ligate concentration in Con. j - E _xj mol/l concentration of ligate bound to inhibitor in Con. j - F _e l/s eluant flow rate - F _i l/s feed flow rate - F _oj l/s flow rate of waste (j=1) or product (j=2) stream - F _r l/s affinity-recycle flow rate - f _j affinity binding fraction of ligate to ligand in Con. - j index for container No., j=1 for Con. 1 and j=2 for Con. 2 - k _l l/mol affinity binding constant of ligate and ligand - K _x l/mol affinity binding constant of ligate and inhibitor - L _o mol/l total ligand concentration in both containers - L _j mol/l free ligand concentration in Con. j - PF purification factor - REC% recovery yield - R _j rejection coefficient of total ligate in Con. j - R _m membrane rejection coefficient of free ligate - R _mc membrane rejection coefficient of contaminant - r _e ratio of eluant to feed flow rate - r _r ratio of affinity-recycle to feed flow rate - X _oj mol/l inhibitor concentration in waste (j=1) or product (j=2) stream - X _ej mol/l concentration of inhibitor bound to ligate in Con. j - X _i mol/1 inhibitor concentration in eluant feed - X _j mol/l free inhibitor concentration in Con. j - X _tj mol/l total inhibitor concentration in Con. j The project is sponsored by the National Natural Science Foundation of China, 21st Century Science Foundation for Youth, Tianjin, and the Foundation of the State Education Commission of China.     

A facilitated electron transfer of copper–zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper–zinc superoxide dismutase (Cu₂Zn₂SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65±3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k_s is 1.2±0.2 s⁻¹ and the anodic (α_a) and cathodic (α_c) transfer coefficients are 0.39±0.02 and 0.61±0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu₂Zn₂SOD), its Cu- or Zn-free derivatives (E₂Zn₂SOD and Cu₂E₂SOD, E designates an empty site) and the SOD reconstituted from E₂Zn₂SOD and Cu²⁺. The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

Reduction of anion reversal potential subverts the inhibitory control of firing rate in spinal lamina I neurons: towards a biophysical basis for neuropathic pain

Background

Reduction of the transmembrane chloride gradient in spinal lamina I neurons contributes to the cellular hyperexcitability producing allodynia and hyperalgesia after peripheral nerve injury. The resultant decrease in anion reversal potential (i.e. shift in E _anion to less negative potentials) reduces glycine/GABA_A receptor-mediated hyperpolarization, but the large increase in membrane conductance caused by inhibitory input can nonetheless shunt concurrent excitatory input. Without knowing the relative contribution of hyperpolarization and shunting to inhibition's modulation of firing rate, it is difficult to predict how much net disinhibition results from reduction of E _anion. We therefore used a biophysically accurate lamina I neuron model to investigate quantitatively how changes in E _anion affect firing rate modulation.

Results

Simulations reveal that even a small reduction of E _anion compromises inhibitory control of firing rate because reduction of E _anion not only decreases glycine/GABA_A receptor-mediated hyperpolarization, but can also indirectly compromise the capacity of shunting to reduce spiking. The latter effect occurs because shunting-mediated modulation of firing rate depends on a competition between two biophysical phenomena: shunting reduces depolarization, which translates into reduced spiking, but shunting also shortens the membrane time constant, which translates into faster membrane charging and increased spiking; the latter effect predominates when average depolarization is suprathreshold. Disinhibition therefore occurs as both hyperpolarization- and shunting-mediated modulation of firing rate are subverted by reduction of E _anion. Small reductions may be compensated for by increased glycine/GABA_A receptor-mediated input, but the system decompensates (i.e. compensation fails) as reduction of E _anion exceeds a critical value. Hyperexcitability necessarily develops once disinhibition becomes incompensable. Furthermore, compensation by increased glycine/GABA_A receptor-mediated input introduces instability into the system, rendering it increasingly prone to abrupt decompensation and even paradoxical excitation.

Conclusion

Reduction of E _anion dramatically compromises the inhibitory control of firing rate and, if compensation fails, is likely to contribute to the allodynia and hyperalgesia associated with neuropathic pain. These data help explain the relative intractability of neuropathic pain and illustrate how it is important to choose therapies not only based on disease mechanism, but based on quantitative understanding of that mechanism.     

Effective Population Size,Genetic Variation,and Their Relevance for Conservation: The Bighorn Sheep in Tiburon Island and Comparisons with Managed Artiodactyls

The amount of genetic diversity in a finite biological population mostly depends on the interactions among evolutionary forces and the effective population size (N _e) as well as the time since population establishment. Because the N _e estimation helps to explore population demographic history, and allows one to predict the behavior of genetic diversity through time, N _e is a key parameter for the genetic management of small and isolated populations. Here, we explored an N _e-based approach using a bighorn sheep population on Tiburon Island, Mexico (TI) as a model. We estimated the current (N _crnt) and ancestral stable (N _stbl) inbreeding effective population sizes as well as summary statistics to assess genetic diversity and the demographic scenarios that could explain such diversity. Then, we evaluated the feasibility of using TI as a source population for reintroduction programs. We also included data from other bighorn sheep and artiodactyl populations in the analysis to compare their inbreeding effective size estimates. The TI population showed high levels of genetic diversity with respect to other managed populations. However, our analysis suggested that TI has been under a genetic bottleneck, indicating that using individuals from this population as the only source for reintroduction could lead to a severe genetic diversity reduction. Analyses of the published data did not show a strict correlation between H _E and N _crnt estimates. Moreover, we detected that ancient anthropogenic and climatic pressures affected all studied populations. We conclude that the estimation of N _crnt and N _stbl are informative genetic diversity estimators and should be used in addition to summary statistics for conservation and population management planning.     

Skeletal muscle fat oxidation is increased in African-American and white women after 10 days of endurance exercise training

Objective: Obesity is associated with lower rates of skeletal muscle fatty acid oxidation (FAO), which is linked to insulin resistance. FAO is reduced further in obese African‐American (AAW) vs. white women (CW) and may also be lower in lean AAW vs. CW. In lean CW, endurance exercise training (EET) elevates the oxidative capacity of skeletal muscle. Therefore, we determined whether EET would elevate skeletal muscle FAO similarly in AAW and CW with a lower lipid oxidative capacity. Research Methods and Procedures: In vitro rates of FAO were assessed in rectus abdominus muscle strips using [1‐¹⁴C] palmitate (Pal) from lean AAW [BMI = 24.2 ± 0.9 (standard error) kg/m²] and CW (23.6 ± 0.8 kg/m²) undergoing voluntary abdominal surgery. Lean AAW (22 ± 0.9 kg/m²) and CW (24 ± 0.8 kg/m²) and obese AAW (36 ± 1.2 kg/m²) and CW (40 ± 1.3 kg/m²) underwent 10 consecutive days of EET on a cycle ergometer (60 min/d, 75% peak oxygen uptake). FAO was measured in vastus lateralis homogenates as captured ¹⁴CO₂ using [1‐¹⁴C] Pal, palmitoyl‐CoA (Pal‐CoA), and palmityl‐carnitine (Pal‐Car). Results: Muscle strip experiments showed suppressed rates of FAO (p = 0.03) in lean AAW vs. CW. EET increased the rates of skeletal muscle Pal oxidation (p = 0.05) in both lean AAW and CW. In obese subjects, Pre‐EET Pal (but not Pal‐CoA or Pal‐Car) oxidation was lower (p = 0.05) in AAW vs. CW. EET increased Pal oxidation 100% in obese AAW (p < 0.05) and 59% (p < 0.05) in obese CW. Similar increases (p < 0.05) in post‐EET FAO were observed for Pal‐CoA and Pal‐Car in both groups. Discussion: Both lean and obese AAW possess a lower capacity for skeletal muscle FAO, but EET increases FAO similarly in both AAW and CW. These data suggest the use of EET for treatment against obesity and diabetes for both AAW and CW.     

Cerebral Perfusion Measurements in Elderly with Hypertension Using Arterial Spin Labeling

Purpose

The current study assesses the feasibility and value of crushed cerebral blood flow (CBF_crushed) and arterial transit time (ATT) estimations for large clinical imaging studies in elderly with hypertension.

Material and Methods

Two pseudo-continuous arterial spin labeling (ASL) scans with (CBF_crushed) and without flow crushers (CBF_non-crushed) were performed in 186 elderly with hypertension, from which CBF and ATT maps were calculated. Standard flow territory maps were subdivided into proximal, intermediate and distal flow territories, based on the measured ATT. The coefficient of variation (CV) and physiological correlations with age and gender were compared between the three perfusion parameters.

Results

There was no difference in CV between CBF_crushed and CBF_non-crushed (15–24%, p>0.4) but the CV of ATT (4–9%) was much smaller. The total gray matter correlations with age and gender were most significant with ATT (p = .016 and p<.001 respectively), in between for CBF_crushed (p = .206 and p = .019) and least significant for CBF_non-crushed (p = .236 and p = .100).

Conclusion

These data show the feasibility and added value of combined measurements of both crushed CBF and ATT for group analyses in elderly with hypertension. The obtained flow territories provide knowledge on vascular anatomy of elderly with hypertension and can be used in future studies to investigate regional vascular effects.     

The effects of external potassium and long duration voltage conditioning on the amplitude of sodium currents in the giant axon of the squid, Loligo pealei

Giant axons were voltage-clamped in solutions of constant sodium concentration (230 mM) and variable potassium concentrations (from 0 to 210 mM). The values of the peak initial transient current, I_p, were measured as a function of conditioning prepulse duration over the range from less than 1 msec to over 3 min. Prepulse amplitudes were varied from E _m = -20 mv to E _m = -160 mv. The attenuation of the I_p values in high [K_o] was found to vary as a function of time when long duration conditioning potentials were applied. In both high and low [K_o], I_p values which had reached a quasi-steady—state level within a few milliseconds following a few milliseconds of hyperpolarization were found to increase following longer hyperpolarization. A second plateau was reached with a time constant of about 100–500 msec and a third with a time constant in the range of 30 to 200 sec. The intermediate quasi-steady—state level was absent in K-free ASW solutions. Sodium inactivation curves, normalized to I _pmax values obtained at either the first or second plateaus, were significantly different in different [K_o]. The inactivation curves, however, tended to superpose after about 1 min of hyperpolarizing conditioning. The time courses and magnitudes of the intermediate and very slow sodium conductance restorations induced by long hyperpolarizing pulses are in agreement with those predicted from the calculated rates and magnitudes of [K⁺] depletion in the space between the axolemma and the Schwann layer.