Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.     

糖尿病肾病腹膜透析血管内皮功能变化及内皮功能不全的相关因素探讨

目的：探讨分析糖尿病肾病腹膜透析患者血管内皮功能的变化。方法：将来我院行腹膜透析的糖尿病肾病患者56例作为观察组,非糖尿病肾病患者64例作为对照组,测量血压及血容量,采用血流介导的肱动脉扩张法测定血流介导的血管扩张。结果：观察组患者的收缩压、脉压、细胞外液均明显高于对照组,血管扩张则显著低于对照组（P〈0．05）;血管扩张与身高、体重、收缩压、细胞外液均呈负相关（P〈0．05）;细胞外液、有无糖尿病肾病是血管扩张的独立预测因素。结论：糖尿病肾病腹膜透析患者具有严重的内皮功能不全,其中细胞外液、有无糖尿病肾病是血管扩张的独立预测因素。     

Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut

In this work, we describe the expression, purification, and disulfide mapping of the named 'peanut seed cDNA 33' (PSC33) peanut allergen. A variant of PSC33 (with N(63), E(64), Q(69) instead of D(63), Q(64), E(69)) has been identified in peanut by proteomic analysis of a highly IgE immunoreactive purification fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and amplified it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally amplified. Since expression of the natural PSC33 (DQE) gene was very low in Escherichia coli even with supplementation of rare codon tRNAs, a synthetic gene optimized for expression in E. coli of PSC33 (DQE) was introduced into a pET9-c vector. A high production of protein occurred in the inclusion bodies that was submitted to refolding using an additive-introduced stepwise dialysis protocol which consists in the gradual removal of the denaturing agent guanidine-HCl with controlled introduction of oxidized and reduced glutathione and l-arginine as a chemical chaperone. After reverse phase HPLC purification, 1mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100ml of bacterial culture. Trypsin and CNBr hydrolysis of the protein combined with MALDI-TOF mass spectrometry allowed us to assign disulfide bridges and show that the native and refolded proteins were identical. The four disulfides of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.     

Characterization of In Vivo Dopamine Release as Determined by Brain Microdialysis After Acute and Subchronic Implantations: Methodological Aspects

Infusion of tetrodotoxin (TTX) through the dialysis membrane and perfusion with calcium-free Ringer solution (calcium depletion) were used to evaluate the dopamine release determined by in vivo brain dialysis. Several hours after implantation, the dopamine release recorded by the U-shaped cannula did not respond to calcium depletion and was only partly (approximately 50%) TTX dependent. The half-life of the TTX-independent dopamine overflow was determined to be 2 h. In contrast, when a transstriatal cannula was used, the dopamine output displayed calcium and TTX dependency. Differences in the dimensions of the two types of probes are a likely explanation for the observed effects. Twenty-four hours after implantation, both types of cannula fulfilled the criteria of calcium and TTX dependency. The results indicate that infusion of TTX-containing or calcium-free Ringer solution can be used to estimate the functional damage caused by the implantation of the cannula.     

Does fatty acid-binding protein play a role in fatty acid transport?

Summary The possible property of fatty acid-binding proteins (FABPs) to transport fatty acid was investigated in various model systems with FABP preparations from liver and heart. An effect of FABP, however, was not detectable with a combination of oleic acid-loaded mitochondria and vesicles or liposomes due to the rapid spontaneous transfer. Therefore, the mitochondria were separated from the vesicles in an equilibrium dialysis cell. The spontaneous fatty acid transfer was much lower and addition of FABP resulted in an increase of fatty acid transport. Oleic acid was withdrawn from different types of monolayers by FABP with rates up to 10%/min. When two separate monolayers were used, FABP increased fatty acid transfer between these monolayers and an equilibrium was reached.Abbreviations FABP(s) fatty acid-binding protein(s) - PC phosphatidylcholine - PS phosphatidylserine - PE phosphatidylethanolamine     

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.     

A dynamic dialysis method for studying protein-ligand binding using chromatographic theory

A new dynamic dialysis method has been developed for studying protein-ligand binding phenomena. The method depends on analysis of the elution pattern of ligand in a single dialyzing process where the ligand concentration in the sample compartment changes greatly with time. The dialyzer is composed of a long, narrow chamber (the sample compartment) between two sheets of semipermeable membrane and two outside chambers (the sink compartment) connected as a single path. Eluting buffer flows in the sink compartment to exchange the ligand with the solution in the sample compartment. Therefore, the ligand concentration gradient in the sink compartment is in the longitudinal direction. The mathematical expressions to analyze the experimental data were derived from a modified theory of chromatography. Examination of the binding of sulfanilamide to bovine serum albumin using this method shows that these equations are valid for use in studying protein-ligand binding.     

An automated continuous-flow dynamic dialysis technique for investigating protein-ligand binding

An automated, continuous-flow dynamic dialysis technique has been developed to investigate protein-ligand binding. The method depends on a comparison of the diffusion of the low molecular mass ligand, in the presence and absence of protein, through a semipermeable membrane. The ligand passes from the sample compartment of a dialysis cell into the sink compartment through which a constant flow of eluting buffer is maintained. Digitized spectrophotometric determinations of the ligand concentration in the eluting buffer at successive, equally spaced time intervals, punched onto paper tape, provide the primary data (normally about 1000 data points). A mathematical treatment of the data based on a model of the diffusion system, whereby the protein-ligand binding isotherm may be evaluated, is discussed. The validity of the method is demonstrated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15, 20, and 25°C. The method yields a large number of points on the binding isotherm (usually several hundred) which, in terms of a Scatchard model, provide values for the number of binding sites on the BSA molecule and binding constants for the phenol red-BSA interaction. The results obtained are consistent with values reported in the chemical literature but which are based on much scantier data.     

曲美他嗪人血浆蛋白结合率的测定

目的:建立测定曲美他嗪与人血浆蛋白结合率的高效液相色谱法(HPLC),测定曲美他嗪在人血浆中的蛋白结合率。方法:采用平衡透析法,结合HPLC测定曲美他嗪在人血浆中的蛋白结合率。结果:低、中、高3个浓度下,曲美他嗪的血浆蛋白结合率分别为14.7%,16.2%和15.5%。结论:本方法灵敏度高,重现性好,操作简单,能满足生物样品分析的要求。曲美他嗪与血浆蛋白结合率较低。