Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

A hydrodynamical study of the flow in renal tubules

The hydrodynamical problem of flow in proximal renal tubule is investigated by considering axisymmetric flow of a viscous, incompressible fluid through a long narrow tube of varying cross-section with reabsorption at the wall. Two cases for reabsorption have been studied (i) when the bulk flow,Q, decays exponentially with the axial distancex, and (ii) whenQ is an arbitrary function ofx such thatQ-Q ₀ can be expressed as a Fourier integral (whereQ ₀ is the flux atx=0). The analytic expressions for flow variables have been obtained by applying perturbation method in terms of wall parameter ε. The effects of ε on pressure drop across the tube, radial velocity and wall shear have been studied in the case of exponentially decaying bulk flow and it has been found that the results are in agreement with the existing ones for the renal tubules.     

Renal slice uptake of organic acid and sugar in pouch young of the marsupial Trichosurus vulpecula

Abstract

Kidney tissue was collected from 14 pouch young of the brushtailed possum Trichosurus vulpecula. The young were aged from 5 to 143 days postpartum. Histological observation of a 6-day mesonephric kidney revealed a well-defined microvillus brushborder lining the lumen of the proximal tubule. Renal slices from all the pouch young tested, including mesonephric and metanephric tissue, accumulated p-aminohippurate and α- methyl-d-glucoside at rates comparable to adult renal cortical slices. Tissue to medium concentration ratios after 1 h of uptake averaged 3.8 for p-aminohippurate and 1.9 for α-methyl-d-glucoside in the young. It was concluded that proximal nephron morphology and the transport systems involved in organic acid secretion and sugar reabsorption by the kidney were well-developed at all stages of pouch life.     

Absence of a role of gamma-glutamyl transpeptidase in the transport of amino acids by rat renal brushborder membrane vesicles

Summary The role of the enzyme, gamma-glutamyl transpeptidase on the uptake of amino acids by the brushborder membrane of the rat proximal tubule was examined by inhibiting it with AT-125 (l-[S, 5S]--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). AT-125 inhibited 98% of the activity of gamma-glutamyl transpeptidase when incubated for 20 min at 37°C with rat brushborder membrane vesicles. AT-125 given to ratsin vivo inhibited 90% of the activity of gamma-glutamyl transpeptidase in subsequently isolated brushborder membrane vesicles from these animals. AT-125 inhibition of gamma-glutamyl transpeptidase bothin vivo andin vitro had no effect on the brushborder membrane uptake of cystine. Similarly, there was no effect of gamma-glutamyl transpeptidase inhibition by AT-125 on glutamine, proline, glycine, methionine, leucine or lysine uptake by brushborder membrane vesicles. Furthermore, the uptake of cystine by isolated rat renal cortical tubule fragments, in which the complete gamma-glutamyl cycle is present, was unaffected by AT-125 inhibition of gamma-glutamyl transpeptidase. Therefore, in the two model systems studied, gamma-glutamyl transpeptidase did not appear to play a role in the transport of amino acids by the renal brushborder membrane.     

Role of selection and gene flow in population differentiation at the edge vs. interior of the species range differing in climatic conditions

Evaluating the relative importance of neutral and adaptive processes as determinants of population differentiation across environments is a central theme of evolutionary biology. We applied the Q_ST–F_ST comparison flanked by a direct test for local adaptation to infer the role of climate‐driven selection and gene flow in population differentiation of an annual grass Avena sterilis in two distinct parts of the species range, edge and interior, which represent two globally different climates, desert and Mediterranean. In a multiyear reciprocal transplant experiment, the plants of desert and Mediterranean origin demonstrated home advantage, and population differentiation in several phenotypic traits related to reproduction exceeded neutral predictions, as determined by comparisons of Q_ST values with theoretical F_ST distributions. Thus, variation in these traits likely resulted from local adaptation to desert and Mediterranean environments. The two separate common garden experiments conducted with different experimental design revealed that two population comparisons, in contrast to multi‐population comparisons, are likely to detect population differences in virtually every trait, but many of these differences reflect effects of local rather than regional environment. We detected a general reduction in neutral (SSR) genetic variation but not in adaptive quantitative trait variation in peripheral desert as compared with Mediterranean core populations. On the other hand, the molecular data indicated intensive gene flow from the Mediterranean core towards desert periphery. Although species range position in our study (edge vs. interior) was confounded with climate (desert vs. Mediterranean), the results suggest that the gene flow from the species core does not have negative consequences for either performance of the peripheral plants or their adaptive potential.     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Influence of zero flow pressure on fractional flow reserve

Fractional flow reserve (FFR) is a commonly used index to assess the functional severity of a coronary artery stenosis. It is conventionally calculated as the ratio of the pressure distal (P_d) and proximal (P_a) to the stenosis (FFR=P_d/P_a). We hypothesize that the presence of a zero flow pressure (P _zf), requires a modification of this equation. Using a dynamic hydraulic bench model of the coronary circulation, which allows one to incorporate an adjustable P _zf, we studied the relation between pressure-derived FFR=P_d/P_a, flow-derived true FFR_Q=Q_S/Q_N (=ratio of flow through a stenosed vessel to flow through a normal vessel), and the corrected pressure-derived FFR_C=(P_d–P_zf)/(P_a–P_zf) under physiological aortic pressures (70 mmHg, 90 mmHg, and 110 mmHg). Imposed P_zf values varied between 0 mmHg and 30 mmHg. FFR_C was in good agreement with FFR_Q, whereas FFR consistently overestimated FFR_Q. This overestimation increased when P_zf increased, or when P_a decreased, and could be as high as 56% (P_zf=30 mmHg and P_a=70 mmHg). According to our experimental study, calculating the corrected FFR_C instead of FFR, if P_zf is known, provides a physiologically more accurate evaluation of the functional severity of a coronary artery stenosis.     

Cystine uptake by rat jejunal brushborder membrane vesicles

The presence of a sodium-stimulated, saturable uptake process for L-cystine is described in brushborder membrane vesicles isolated from rat jejunal mucosa. Concentration-dependence studies indicate the presence of a single transport system for cystine withK _m=0.053 mM andV _max=0.633 nmol/mg/15 s. Lysine completely inhibits the uptake of cystine.     

Continuous lipolysis in a reversed micellar membrane bioreactor

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of AOT in isooctane was carried out in a continous reversed micellar membrane bioreactor. A tubular ceramic membrane installed in an ultrafiltration module was used to retain the lipase and separate the products from the reaction media. Water filled micelles were supplemented to the reactor together with the substrate/solvent solution to compensate for the permeation of reversed micelles. The influence of substrate concentration, residence time and water content in the productivity and conversion of the system were investigated. A linear relationship between productivity and conversion degree was found for each substrate concentration tested. Operational stability of the bioreactor was tested in a long term operation confirming the high stability of this catalytic system.List of Symbols a(S ₀ ), b(S ₀ ) parameters of Eq. (3) - [AOT] mM AOT concentration - C _c mM concentration in the concentrate - C _p mM concentration in the permeate - E _t mg total amount of lipase - [lipase] mg/cm³ overall lipase concentration - [OIL] mM olive oil concentration - [OLEIC] mM oleic acid concentration in the permeate - P mol/(min · mg) oleic acid productivity - Q _in cm³/min inlet flow rate - Q _out cm³/min outlet flow rate (equal to permeate flow rate) - Q _r cm³/min recirculation flow rate - W _o ratio of water to AOT molar concentrations - X steady state conversion degree in the permeate stream - T °C temperature - rejection coefficient     

Usefulness of thermoluminescence in herbicide research

Many herbicides of different chemical structure inhibit photosynthetic electron flow by interrupting the photosyn‐thetic electron flow by interrupting the photosynthetic electron transport chain between the primary acceptor (Q_A) and the secondary acceptor (Q_B) of photosystem 2 (PS2). Thermoluminescence (TL) originates from PS2, and the bands of the glow curve can be related to the charge recombination between positively charged donors and negatively charged acceptors. The glow curve of TL is strongly influenced by addition of PS2 herbicides. The herbicide treatment shifts the peak position and activation energy of the TL band related to Q_A, suggesting that herbicide binding affects the midpoint redox potential not only of Q _B but also that of Q_A. On the basis of the band shift the herbicides of various chemical structures can be classified into different “thermodynamical” groups which relfect the differences in the binding properties of these herbicides. As a new approach TL seems to be a useful technique in studying the mechanism and site of action of herbicides that inhibit electron transport of PS2.     

Simplistic determination of operation parameters for simulated moving bed (SMB) chromatography for the separation of ketoprofen enantiomer

Since it is troublesome to estimate adequate flow rates in four sections of SMB chromatography, a systematic determination of the flow rates has been suggested by using ketoprofen as a model chiral enantiomer.S-ketoprofen, less retained species, was separated from raffinate stream and the variation in its purity was dependent on the changes of the flow rate of section 4 (Q ₄), the raffinate flow rate (Q _raf), and the feed flow rate (Q _feed) under a fixed switching timet ^*. When one parameter was changed at the given experimental condition, purities of product were changed and these phenomena has be well explained by the triangle theory.     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

全球陆地生态系统光合作用与呼吸作用的温度敏感性

基于全球647套通量数据,定量分析了全球尺度下生态系统光合作用和呼吸作用的温度敏感性(Q10)随纬度、气候和植被的分布规律。结果表明:在全球尺度下,光合作用和呼吸过程的温度敏感性(Q10,G和Q10,R)都随纬度的升高而增加,其中Q10,G和Q10,R的均值分别为3.99±0.21和2.28±0.074。除热带多树草原、常绿落叶林外,Q10,G均大于Q10,R值。不同植被类型的温度敏感性存在显著性差异,表现为:针叶林阔叶林;落叶林常绿林,其中生态系统的季节性变异是造成差异的主要原因。当植被类型和纬度区域共同影响Q10值时,植被类型对Q10值的总变异贡献更大。气候类型对Q10,G和Q10,R都有显著影响。在气候带上,干旱带的Q10,G最小,而冷温带的Q10,G最高。不同气候类型下(除温带草原气候外)的Q10,G都大于Q10,R。在极端条件下,温度可能不在是主导因素,而水分对温度敏感性的影响不可忽略,今后的研究需要更多的关注生态系统温度敏感性对水分变化的响应。     

Carbon quality and soil microbial property control the latitudinal pattern in temperature sensitivity of soil microbial respiration across Chinese forest ecosystems

Understanding the temperature sensitivity (Q₁₀) of soil organic C (SOC) decomposition is critical to quantifying the climate–carbon cycle feedback and predicting the response of ecosystems to climate change. However, the driving factors of the spatial variation in Q₁₀ at a continental scale are fully unidentified. In this study, we conducted a novel incubation experiment with periodically varying temperature based on the mean annual temperature of the soil origin sites. A total of 140 soil samples were collected from 22 sites along a 3,800 km long north–south transect of forests in China, and the Q₁₀ of soil microbial respiration and corresponding environmental variables were measured. Results showed that changes in the Q₁₀ values were nonlinear with latitude, particularly showing low Q₁₀ values in subtropical forests and high Q₁₀ values in temperate forests. The soil C:N ratio was positively related to the Q₁₀ values, and coniferous forest soils with low SOC quality had higher Q₁₀ values than broadleaved forest soils with high SOC quality, which supported the “C quality temperature” hypothesis. Out of the spatial variations in Q₁₀ across all ecosystems, gram‐negative bacteria exhibited the most importance in regulating the variation in Q₁₀ and contributed 25.1%, followed by the C:N ratio (C quality), fungi, and the fungi:bacteria ratio. However, the dominant factors that regulate the regional variations in Q₁₀ differed among the tropical, subtropical, and temperate forest ecosystems. Overall, our findings highlight the importance of C quality and microbial controls over Q₁₀ value in China's forest ecosystems. Meanwhile, C dynamics in temperate forests under a global warming scenario can be robustly predicted through the incorporation of substrate quality and microbial property into models.     

Structure and active transport in the plasma membrane of the tubules of frog kidney

The active transport of organic anions through the plasma membrane of the proximal tubules of frog kidney was studied. For this purpose a marker anion, fluorescein, was used, its flow into the tubules registered by the increase of fluorescense. The kinetics of transport was measured as function of time, concentration of substrate, concentration of a competing acid (p-aminohippuric acid) and temperature. The process is inhibited by strophantin, a specific poison for (Na⁺ + K⁺)-dependent ATPase. These data show that fluorescein transport is effected with the participation of a charged carrier, probably by the downfield mechanism postulated by Mitchell. To confirm this mechanism, a passive flow of K⁺ was created inwards across the membrane of the proximal tubules by means of valinomycin. It led to the discharge of the membrane and to the inhibition of fluorescein transport. Anions are transported downfield across the membrane, probably in a state of complexes with two Na⁺ ions.A magnetic field of 10 000–28 000 oersted inhibits the fluorescein transport strongly. This can be regarded as a proof of the liquid-crystalline structure of biological membranes and demonstrates the importance of this structure for active transport.     

Effects of floods versus low flows on invertebrates in a New Zealand gravel-bed river

1. Floods and low flows are hydrological events that influence river ecosystems, but few studies have compared their relative importance in structuring invertebrate communities. Invertebrates were sampled in riffles and runs at eight sites along 40 km of a New Zealand gravel‐bed river every 1–3 months over 2.5 years, during which time a number of large flood and low flow events occurred. Flows were high in winter and spring, and low in summer and autumn. Four flow‐related variables were calculated from hydrological data: flow on the day of sampling (Q_sample), maximum and minimum flow between successive samples (Q_max and Q_min, respectively), and the number of days since the last bed‐moving flood (N_days). 2. The invertebrate community was summarised by relative densities of the 19 most abundant taxa and four biotic metrics [total abundance, taxon richness, the number of Ephemeroptera, Plecoptera and Trichoptera taxa (i.e. EPT richness), and per cent EPT]. Invertebrate density fluctuated greatly, and was high in summer and autumn, and low during winter and spring. Stepwise multiple regression (SMR) analysis was used to investigate relationships between the invertebrate community and season, flow, habitat and water temperature. 3. Seasonal variables were included in almost 50% of the SMR models, while flow‐related variables were included in >75% of models. Densities of many taxa were negatively correlated to Q_min and Q_max, and positively correlated to N_days, suggesting that while high flows reduced invertebrate densities, densities recovered with increasing time following a flood. Although season and flow were confounded in this study, many of the taxa analysed display little seasonal variation in abundance, suggesting that flow‐related variables were more important in structuring communities than seasonal changes in density associated with life‐cycles. 4. Five discrete flood and low flow events were identified and changes to invertebrate communities before and after these events examined. Invertebrate densities decreased more commonly after floods than after low flows, and there was a significant positive relationship between the number of taxa showing reductions in density and flood magnitude. Densities of most invertebrates either remained unchanged, or increased after low flow events, except for four taxa whose densities declined after a very long period (up to 9 months) of low flow. This decline was attributed to autogenic sloughing of thick periphyton communities and subsequent loss of habitat for these taxa. 5. Invertebrate communities changed more after floods and the degree of change was proportional to flood magnitude. Community similarity increased with increasing time since the last disturbance, suggesting that the longer stable flows lasted, the less the community changed. These results suggest that invertebrate communities in the Waipara River were controlled by both floods and low flows, but that the relative effects of floods were greater than even extended periods of extreme low flow. 6. Hydraulic conditions in riffles and runs were measured throughout the study. Riffles had consistently faster velocities, but were shallower and narrower than runs at all measured flows. Invertebrate density in riffles was expressed as a percentage of total density and regressed against the flow‐related variables to see whether invertebrate locations changed according to flow. Significant negative relationships were observed between the per cent density of common taxa in riffles and Q_sample, Q_max and Q_min. This result suggests either that these animals actively drifted into areas of faster velocity during low flows, or that their densities within riffles increased as the width of these habitats declined.     

Fracture of DNA in transient extensional flow. A numerical simulation study

Using the Brownian dynamics simulation technique, we studied the fracture process of DNA chains subjected to transient extensional flow, letting the solution with DNA molecules pass through a very small orifice (radius = 0.0065 cm), thus experiencing extensional flow of the convergent (sink) type. The DNA molecules were modeled as FENE bead-spring chains with the springs obeying a modified Warner force law, as proposed by Reese and Zimm. The fracture yield was strongly dependent on flow rate and molecular weight, reaching, in our setup, a level of 100% at a flow rate of around 0.001 cm³/s for DNA with molecular weight 26 × 10⁶ (T7 DNA). There was found to exist a critical flow rate (Q_crit) below which fracture did not occur, in accordance with what was observed in studies on polystyrene in transient extensional flow. We found that for DNA, the critical flow rate depended on the molecular weight as Q_crit ∼ M⁻¹⁴ when the hydrodynamic interaction effect (HI) was not included in the simulations. When HI was accounted for, the relation was found to be Q_crit ∼ M^−1.1, close to the theoretical prediction for fracture of partly extended chains in transient extensional flow. © 1996 John Wiley & Sons, Inc.     

The function of water channels in Chara: The temperature dependence of water and solute flows provides evidence for composite membrane transport and for a slippage of small organic solutes across water channels

Using the cell pressure probe, the effects of temperature on hydraulic conductivity (Lp; osmotic water permeability), solute permeability (permeability coefficient, P_s), and reflection coefficients (σ_s) were measured on internodes of Chara corallina, Klein ex Willd., em R.D.W.. For the first time, complete sets of transport coefficients were obtained in the range between 10 and 35 °C which provided evidence about pathways of water and solutes as they move across the plasma membrane (water channel and bilayer arrays). Test solutes used to check for the selectivity of water channels were monohydric alcohols of different molecular size and shape (ethanol, n-propanol, iso-propanol, and tert-butanol) and heavy water (HDO). Within the limits of accuracy, Q₁₀ values for Lp and for the diffusive water permeability (P_d) were identical (Q₁₀ for Lp = 1.29 ± 0.17 (± SD; n = 15 cells) and Q₁₀ for P_d = 1.25 ± 0.16 (n = 5 cells)). The Q₁₀ values were equivalent to activation energies of E_a = 16.8 ± 6.4 and 16.6 ± 10.0 kJ · mol⁻¹, respectively, which is similar to that of self-diffusion or of viscous flow of water. The Q₁₀ values and activation energies for P_s of the alcohols were significantly larger (ethanol: Q₁₀ = 1.68 ± 0.16, E_a = 37.1 ± 5.9 kJ · mol⁻¹; n-propanol: Q₁₀ =  1.75 ± 0.40, E_a = 43.1 ± 15.3 kJ · mol⁻¹; iso-propanol: Q₁₀ = 2.12 ± 0.42, E_a =  52.2 ± 14.6 kJ · mol⁻¹; tert-butanol: Q₁₀ = 2.13 ± 0.56, E_a = 51.6 ± 17.1 kJ · mol⁻¹; ±SD; n = 5 to 6 cells). Effects of temperature on reflection coefficients were most pronounced. With increasing temperature, σ_s values of the alcohols decreased and those of HDO increased. The data indicate that water and solutes use different pathways when crossing the membrane. Ordinary and isotopic water use water channels and the other test solutes use the bilayer array (composite transport model of membrane). Changes in σ_s values with temperature were found to be a sensitive measure for the open/closed state of water channels. The decrease of σ_s with temperature was theoretically predicted from the temperature dependence of P_s and Lp. Differences between predicted and measured values of σ_s allowed estimation of the bypass flow (slippage) of solutes through water channels which did not completely exclude test solutes. The permeability of channels depended on the structure and size of test solutes. It is concluded that water channels are much less selective than is usually thought. Since water channels represent single-file or no-pass pores, solutes drag along considerable amounts of water as they diffuse across channels. This results in low overall values of σ_s. The σ_s of HDO was extremely low. Its response to temperature was opposite to that for the σ_s of the alcohols. This suggested a stronger effect of temperature on the hydraulic (osmotic) than on the diffusive water flow across individual water channels, i.e. a differential sensitivity of different mechanisms to temperature. Received: 10 October 1996 / Accepted: 2 December 1996     

Primary Steps in the Energy Conversion Reaction of theCytochrome bc 1 Complex QO Site

The primary energy conversion (Q_O) site of the cytochrome bc ₁ complex is flanked by bothhigh- and low-potential redox cofactors, the [2Fe–2S] cluster and cytochrome b _L, respectively.From the sensitivity of the reduced [2Fe–2S] cluster electron paramagnetic resonance (EPR)spectral g _x-band and line shape to the degree and type of Q_O site occupants, we have proposeda double-occupancy model for the Q_O site by ubiquinone in Rhodobacter capsulatus membranevesicles containing the cytochrome bc ₁ complex. Biophysical and biochemical experimentshave confirmed the double occupancy model and from a combination of these results and theavailable cytochrome bc ₁ crystal structures we suggest that the two ubiquinone molecules inthe Q_O site serve distinct catalytic roles. We propose that the strongly bound ubiquinone,termed Q_OS, is close to the [2Fe–2S] cluster, where it remains tightly associated with the Q_Osite during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Q_OW,is distal to the [2Fe–2S] cluster and can exchange with the membrane Q_pool on a time scalemuch faster than the turnover, acting as the substrate. The crystallographic data demonstratesthat the FeS subunit can adopt different positions. Our own observations show that theequilibrium position of the reduced FeS subunit is proximal to the Q_O site. On the basis of this, wealso report preliminary results modeling the electron transfer reactions that can occur in thecytochrome bc ₁ complex and show that because of the strong distance dependence of electrontransfer, significant movement of the FeS subunit must occur in order for the complex to beable to turn over at the experimental observed rates.     

Generation of easily accessible human kidney tubules on two‐dimensional surfaces in vitro

The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β₁, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.