Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid-Supported Self-Sufficient Biocatalyst.

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2 ) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization.

Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field. One clear reason is that many evaluate immobilization based on only a few experiments that are not always well-designed. In contrast to many other reviews on the subject, here we highlight the pitfalls of using incorrectly designed immobilization protocols and explain why in many cases sub-optimal results are obtained. We also describe solutions to overcome these challenges and come to the conclusion that recent developments in material science, bioprocess engineering and protein science continue to open new opportunities for the future. In this way, enzyme immobilization, far from being a mature discipline, remains as a subject of high interest and where intense research is still necessary to take full advantage of the possibilities.

Monitoring and control of the release of soluble O2 from H2 O2 inside porous enzyme carrier for O2 supply to an immobilized d-amino acid oxidase.

While O2 substrate for bio-transformations in bulk liquid is routinely provided from entrained air or O2 gas, tailored solutions of O2 supply are required when the bio-catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O2 from H2 O2 by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O2 formed by an immobilized catalase upon feeding of H2 O2 . The internally released O2 is used to drive the reaction of d-amino acid oxidase (oxidation of d-methionine) that is co-immobilized with the catalase in the same carrier. The H2 O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100-500 µM. Thus, enzyme inactivation by excess H2 O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co-immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air-saturated level. Conversions at a 200 ml scale using varied H2 O2 feed rate (0.04-0.18 mmol/min) give the equivalent production rate from d-methionine (200 mM) and achieve rate enhancement by ∼1.55-fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O2 substrate from H2 O2 into carrier-immobilized enzymes. By addressing limitations of O2 supply via gas-liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2 -dependent biocatalytic reactions.

A simple and precise methodology to determine particulate matter mass in atmospheric filters; validation and application cases.

In the past decades, particulate matter (PM) measurements have been used extensively in atmospheric sciences, as it allows studying the evolution of tracers for different atmospheric processes and the effects of atmospheric pollution on human health. However, measuring PM mass requires a constant control of the laboratory conditions due to its capacity to absorb humidity. For this reason, this study was focused on developing a novel, simple and precise methodology to determine the corrections of the filter mass due to humidity changes. The control and corrections are possible using a "control filter", which is always adapted to the environmental conditions of the laboratory. To check the consistency of this method, it was proved that the mass of any problem filter and that of the control filter behave in a very similar way. This allows quantifying the mass changes of any problem filter by using the control filter, where the problem filters and the control filter must have the same chemical composition and dimensions. To validate this methodology, a comparison was made between the methodology proposed in this study (Method-1) and the one proposed by the EPA (Method-2), which is generally applied. The particulate matter mass (m) was obtained for a problem filter for different weights, achieving similar values using both methods. However, Method-1 still provided reliable mass measurements for relative humidities very different from 50%, even as low as 18%. It was also proved that the adsorption or loss of water by the particulate matter can be neglected, since m is much smaller than the blank filter mass. Method-1 was also employed in several samplings carried out using three PM10 samplers to determine contaminants, such as 7Be and 210Pb, obtaining a good agreement between all particulate masses and activities measured by the three samplers for all samplings.

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process.

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.

Combining a Genetically Engineered Oxidase with Hydrogen-Bonded Organic Frameworks (HOFs) for Highly Efficient Biocomposites.

Enzymes incorporated into hydrogen-bonded organic frameworks (HOFs) via bottom-up synthesis are promising biocomposites for applications in catalysis and sensing. Here, we explored synthetic incorporation of d-amino acid oxidase (DAAO) with the metal-free tetraamidine/tetracarboxylate-based BioHOF-1 in water. N-terminal enzyme fusion with the positively charged module Zbasic2 strongly boosted the loading (2.5-fold; ≈500 mg enzyme gmaterial-1 ) and the specific activity (6.5-fold; 23 U mg-1 ). The DAAO@BioHOF-1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during catalyst recycling. Further, extension to other enzymes, including cytochrome P450 BM3 (used in the production of high-value oxyfunctionalized compounds), points to the versatility of genetic engineering as a strategy for the preparation of biohybrid systems with unprecedented properties.

Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered ß-hexosaminidase immobilized on solid support.

The D746E variant of Bifidobacterium bifidum ß-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective ß-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145-200 mM) at quantitative yield from the donor substrate. The wild-type ß-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80-100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.

Process intensification for O2 -dependent enzymatic transformations in continuous single-phase pressurized flow.

Oxidative O2 -dependent biotransformations are promising for chemical synthesis, but their development to an efficiency required in fine chemical manufacturing has proven difficult. General problem for process engineering of these systems is that thermodynamic and kinetic limitations on supplying O2 to the enzymatic reaction combine to create a complex bottleneck on conversion efficiency. We show here that continuous-flow microreactor technology offers a comprehensive solution. It does so by expanding the process window to the medium pressure range (here, ≤34 bar) and thus enables biotransformations to be conducted in a single liquid phase at boosted concentrations of the dissolved O2 (here, up to 43 mM). We take reactions of glucose oxidase and d-amino acid oxidase as exemplary cases to demonstrate that the pressurized microreactor presents a powerful engineering tool uniquely apt to overcome restrictions inherent to the individual O2 -dependent transformation considered. Using soluble enzymes in liquid flow, we show reaction rate enhancement (up to six-fold) due to the effect of elevated O2 concentrations on the oxidase kinetics. When additional catalase was used to recycle dissolved O2 from the H2 O2 released in the oxidase reaction, product formation was doubled compared to the O2 supplied, in the absence of transfer from a gas phase. A packed-bed reactor containing oxidase and catalase coimmobilized on porous beads was implemented to demonstrate catalyst recyclability and operational stability during continuous high-pressure conversion. Product concentrations of up to 80 mM were obtained at low residence times (1-4 min). Up to 360 reactor cycles were performed at constant product release and near-theoretical utilization of the O2 supplied. Therefore, we show that the pressurized microreactor is practical embodiment of a general reaction-engineering concept for process intensification in enzymatic conversions requiring O2 as the cosubstrate.

The Microenvironment in Immobilized Enzymes: Methods of Characterization and Its Role in Determining Enzyme Performance.

The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product concentration but also in pH and ionic strength. The internal milieu for immobilized enzymes is affected by the chemical properties of the solid material and by the interplay of reaction and diffusion. Enzyme performance is influenced by the internal milieu in terms of catalytic rate ("activity") and stability. Elucidation, through direct measurement of differences in the internal as compared to the bulk milieu is, therefore, fundamentally important in the mechanistic characterization of immobilized enzymes. The deepened understanding thus acquired is critical for the rational development of immobilized enzyme preparations with optimized properties. Herein we review approaches by opto-chemical sensing to determine the internal milieu of enzymes immobilized in porous particles. We describe analytical principles applied to immobilized enzymes and focus on the determination of pH and the O2 concentration. We show measurements of pH and [O2] with spatiotemporal resolution, using in operando analysis for immobilized preparations of industrially important enzymes. The effect of concentration gradients between solid particle and liquid bulk on enzyme performance is made evident and quantified. Besides its use in enzyme characterization, the method can be applied to the development of process control strategies.

A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase.

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C-H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole-cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor-made, self-sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2 , the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl-activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/gcarrier ) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole-cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

Interaction of lipids with the neurotensin receptor 1.

Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 µmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Confocal Luminescence Lifetime Imaging with Variable Scan Velocity and Its Application to Oxygen Sensing.

The dependence of the luminescence lifetime on the probe environment is the basis of a range of sensing techniques. The major advantage of using the lifetime as the sensitive parameter is its independence on the probe concentration. However, the instrumentation for lifetime measurements is complex, generally requiring time-resolved excitation and detection. Here, we present a simple method for the measurement of luminescence lifetimes on the microsecond scale based on variable excitation time determined by the scanning velocity. The technique is implemented in a confocal laser scanning microscope (CLSM), thus allowing not only simple lifetime measurement but also phosphorescence lifetime imaging. Since the method exploits the spatiotemporal dependence of sample excitation in a CLSM, there is no need for a pulsed or modulated light source or for additional time-resolved detection. The method can be realized in a standard CLSM without any modifications. The principle is demonstrated on oxygen sensing by collisional quenching of an oxygen-sensitive ruthenium(II) complex.

Simultaneous Determination of Oxygen and pH Inside Microfluidic Devices Using Core-Shell Nanosensors.

A powerful online analysis setup for the simultaneous detection of oxygen and pH is presented. It features core-shell nanosensors, which enable contactless and inexpensive read-out using adapted oxygen meters via modified dual lifetime referencing in the frequency domain (phase shift measurements). Lipophilic indicator dyes were incorporated into core-shell structured poly(styrene-block-vinylpyrrolidone) nanoparticles (average diameter = 180 nm) yielding oxygen nanosensors and pH nanosensors by applying different preparation protocols. The oxygen indicator platinum(II) meso-tetra(4-fluorophenyl) tetrabenzoporphyrin (PtTPTBPF) was entrapped into the polystyrene core (oxygen nanosensors) and a pH sensitive BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPY) was incorporated into the polyvinylpyrrolidone shell (pH nanosensors). The brightness of the pH nanoparticles was increased by more than 3 times using a light harvesting system. The nanosensors have several advantages such as being excitable with red light, emitting in the near-infrared spectral region, showing a high stability in aqueous media even at high particle concentrations, high ionic strength, or high protein concentrations and are spectrally compatible with the used read-out device. The resolution for oxygen of the setup is 0.5-2.0 hPa (approximately 0.02-0.08 mg/L of dissolved oxygen) at low oxygen concentrations (<50 hPa) and 4-8 hPa (approximately 0.16-0.32 mg/L of dissolved oxygen) at ambient air oxygen concentrations (approximately 200 hPa at 980 mbar air pressure) at room temperature. The pH resolution is 0.03-0.1 pH units within the dynamic range (apparent pKa 7.23 ± 1.0) of the nanosensors. The sensors were used for online monitoring of pH changes during the enzymatic transformation of Penicillin G to 6-aminopenicillanic acid catalyzed by Penicillin G acylase in miniaturized stirred batch reactors or continuous flow microreactors.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5) nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc.

Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O2 -dependent enzyme.

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O2 -dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas-liquid phase contacting in a multi-channel microstructured reaction plate. Advanced non-invasive optical sensing is applied to measure liquid-phase oxygen concentrations in both in- and out-flow as well as directly in the microchannels (width: 600 µm; depth: 200 µm). Protein-surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d-amino acid oxidase) is fused to the positively charged mini-protein Zbasic2 and the channel surface contains a negatively charged γ-Al2 O3 wash-coat layer. Non-covalent wall attachment of the chimeric Zbasic2 _oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5-1.3 × 10(-4) m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL ) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · s(-1) ) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5) s(-1) was determined for the overall mass transfer coefficient KL a, comprising a KL of about 7 × 10(-5) m · s(-1) and a specific surface area of up to 10(5) m(-1) . Biotechnol. Bioeng. 2016;113: 1862-1872. © 2016 Wiley Periodicals, Inc.

Preoperative Intubation and Lack of Enteral Nutrition are Associated with Prolonged Stay After Arterial Switch Operation.

Mortality for the arterial switch operation (ASO) has diminished significantly over the past few decades. Some patients do, however, continue to have protracted and complicated courses after surgery. We attempted to determine which preoperative factors were best associated with prolonged hospital stay after ASO. We retrospectively reviewed all patients that underwent an ASO over a 10-year period. Outcomes of patients with postoperative stays (POS) >14 days (long stay group-LS) were compared with those patients with POS < 7 days (short stay group-SS). The following variables were evaluated: age at surgery, weight, septostomy performed (BAS) and management the day prior to surgery including use of prostaglandin E1 (PGE1), inotropes, intubation status and the establishment of enteral feeds. The SS group had 25 patients and the LS group had 32 patients. Both groups (SS vs. LS) were similar in PGE1 use (48 vs. 69 %), BAS (76 vs. 59 %), age at surgery (6 vs. 7 days) and preoperative inotropes (12 vs. 38 %). The SS group had significantly higher incidence of preoperative feeding (80 vs. 31 %, p < 0.001) and less frequent intubation (12 vs. 47 %, p < 0.001). Patients who are intubated and have not yet begun to receive enteral feeds at the time of their ASO are more likely to have prolonged POS. It is unclear if prolonged stays were a result of operating on patients with worse preoperative hemodynamics or a consequence of a preoperative management strategy that did not allow for extubation and establishment of feeds prior to surgery.

Driving competences and neuropsychological factors associated to driving counseling in multiple sclerosis.

Multiple Sclerosis (MS) significantly impacts daily living activities, including car driving. To investigate driving difficulties experienced with MS, we compared 50 MS patients with minor or moderate disability and 50 healthy controls (HC) using computerized driving tests (the ASDE driver test and the Useful Field of View (UFOV) test) and neuropsychological tests. Inclusion criteria included being active drivers. We evaluated whether cognitive deterioration in MS is associated with the results of driving tests by comparing MS patients without cognitive deterioration with HC. The results indicated that the MS patients performed worse than the HCs in attention, information processing, working memory and visuomotor coordination tasks. Furthermore, MS patients with cognitive impairments experienced more difficulties in the driving tests than did the non-impaired MS patients. Motor dysfunction associated with MS also played an important role in this activity. The results of this study suggest that MS should be assessed carefully and that special emphasis should be placed on visuomotor coordination and executive functions because patients with minor motor disability and subtle cognitive impairments can pass measures predictive of driving safety.

A chloride conductance exhibiting bicarbonate conductivity in renal inner medullary collecting duct cells.

The anion conductance in primary cultures of rat inner medullary collecting duct cells was studied using perforated-patch whole-cell clamp technique. Depolarizations above 0 mv induced an outward anionic current with a time-dependent activation (Iovt) exhibiting a similar conductivity to Cl- and HCO3-. Iovt showed half-maximal activation around 32 mV with a slope factor of 23 mV, and showed a voltage-dependent activation time course that was well fitted by a sum of two exponential functions. Iovt was potentiated when external pH or external Ca2+ was increased and was blocked by external DIDS, DPC and furosemide. These characteristics of Iovt resemble that of the ClC-K1 channels mediated currents; however, anion substitution studies showed that Iovt exhibits a Br->Cl->I->NO3- conductivity sequence, different from that observed in the ClC-K1 channels-mediated conductance. We suggest that, in inner medullary collecting duct cells, ClC-K channels of an unidentified type give rise to this Cl- and HCO3- conductance. This is the first study of a channel-mediated HCO3- current in kidney tubular cells.