Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ

The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.     

Dihydroorotate dehydrogenase from Saccharomyces cerevisiae: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues

In all organisms the fourth catalytic step of the pyrimidine biosynthesis is driven by the flavoenzyme dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11). Cytosolic DHODH of the established model organism Saccharomyces cerevisiae catalyses the oxidation of dihydroorotate to orotate and the reduction of fumarate to succinate. Here, we investigate the structure and mechanism of DHODH from S. cerevisiae and show that the recombinant ScDHODH exists as a homodimeric enzyme in vitro. Inhibition of ScDHODH by the reaction product was observed and kinetic studies disclosed affinity for orotate (K(ic)=7.7 microM; K(ic) is the competitive inhibition constant). The binding constant for orotate was measured through comparison of UV-visible spectra of the bound and unbound recombinant enzyme. The midpoint reduction potential of DHODH-bound flavine mononucleotide determined from analysis of spectral changes was -242 mV (vs. NHE) under anaerobic conditions. A search for alternative electron acceptors revealed that homologues such as mesaconate can be used as electron acceptors.     

不同发育时期樟子松（Pinus sylvestris L. var. mongolica Litv.）的电阻抗参数与抗寒性的关系

采用电阻抗图谱（EIS）法和电导（EL）法对不同发育时期的樟子松（Pinus sylvestris L. var. mongolica Litv.）茎和针叶进行了抗寒性测定,试图通过比较两种方法测定抗寒性结果的相关性,找到适合冷冻处理后樟子松抗寒性测定和不经冷冻处理估测抗寒性的EIS参数,完善EIS法测定抗寒性。以8年生樟子松苗为试材,在抗寒锻炼阶段（10月份）和脱锻炼阶段（3月份）分别取样进行EIS和EL测定。结果表明,EIS法胞外电阻率（re）与EL法测定的樟子松抗寒性相关性较高（R2=0.97）,但比EL法求出的抗寒性高。针叶的细胞膜时间恒量（τm）和茎的弛豫时间（τ1）随冷冻温度变化与re表现相似的S曲线,相关分析表明,re（茎和针叶）与τ1（茎）和τm（针叶）的变化有较好的相关性（R2=0.74～0.84）。经Logistic方程拟合,EIS的τm（针叶）和τ1（茎）法与EIS（re）法、EL法测定的樟子松抗寒性相关性也较高（R2=0.88～0.91）,说明针叶τm和茎τ1也可以作为计算抗寒性的参数。另外,8年生樟子松两个发育时期（10月和3月份）未经冷冻的针叶τm与茎的τ2随抗寒性的增强而显著增加,表明不经过冷冻处理样本用τ2（茎）和τm（针叶）估计樟子松抗寒性是很有前途的方法。     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性

在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。     

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK₂) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK₂ complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V_max of 1.25 μmol P_i/min/mg and a K_m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA^Glc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.     

Protein molecular dynamics with electrostatic force entirely determined by a single Poisson-Boltzmann calculation

The electrostatic force including the intramolecular Coulombic interactions and the electrostatic contribution of solvation effect were entirely calculated by using the finite difference Poisson-Boltzmann method (FDPB), which was incorporated into the GROMOS96 force field to complete a new finite difference stochastic dynamics procedure (FDSD). Simulations were performed on an insulin dimer. Different relative dielectric constants were successively assigned to the protein interior; a value of 17 was selected as optimal for our system. The simulation data were analyzed and compared with those obtained from 500-ps molecular dynamics (MD) simulation with explicit water and a 500-ps conventional stochastic dynamics (SD) simulation without the mean solvent force. The results indicate that the FDSD method with GROMOS96 force field is suitable to study the dynamics and structure of proteins in solution if used with the optimal protein dielectric constant.     

Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease.