Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein

Hyperfibrinogenemia is a common feature of the nephrotic syndrome, and contributes to increased tendency for thrombosis and atherosclerosis. Its genesis is not certain, but the increase in liver fibrinogen mRNA in nephrotic rats indicates increased synthesis. Data in humans are scarce. We presently compared synthesis rates of fibrinogen and albumin in nephrotic adults (N = 7; plasma albumin 22.3 +/- 0.7 g/liter, proteinuria 12 g/day) and healthy control subjects (N = 8) using a primed/continuous infusion of the stable isotope L-[1-13C]valine for six hours. Absolute synthesis rate (ASR) of fibrinogen was 31 +/- 3 mg/kg/day in nephrotic subjects and 21 +/- 1 mg/kg/day in control subjects (P < 0.05), and positively correlated with plasma fibrinogen (P = 0.0317). The plasma fibrinogen pool was disproportionately increased in the nephrotic patients (271 +/- 30 mg/kg) compared to the controls (126 +/- 8 mg/kg), suggesting decreased fractional catabolic rate as well. The ASR of albumin was increased from 71 +/- 4 mg/kg/day in the controls to 160 +/- 19 mg/kg/day in the patients (P < 0.0001), and strongly correlated with the ASR of fibrinogen (P = 0.0046). Plasma alpha 2-macroglobulin was also elevated and correlated with the albumin synthesis rate, whereas plasma serum amyloid A and C-reactive protein were not elevated. These data suggest that in nephrotic patients the increased albumin synthesis is associated with an increase in synthesis of a specific and coordinated group of proteins, among which is fibrinogen.     

Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P-glycoprotein

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports. Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167-16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977-5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CHRC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.     

Separation of paraproteins from human plasma by membrane chromatography

The purpose of this study was to assess the effects of acute pharmacological interventions on the ischemia-reperfusion damage in a canine model of hypothermic global myocardial ischemia. Three experimental groups each consisting of seven animals were subjected to 2 h of global ischemia followed by 1 h of reperfusion. Group A (control) used Tyers' iso-osmolar potassium cardioplegia solution; group B received allopurinol (40 mg/kg), 95% intravenously (IV) systemically with 5% added to the final infusion of Tyers' solution. In group C, superoxide dismutase (6.5 mg/kg) was used, one third of the total dose in the final delivery of the Tyers' cardioplegia solution and two thirds IV during the initial 5 min of reperfusion. In all three groups, myocardial temperature was maintained between 15 and 19 degrees C. Methods of evaluation included hemodynamic and echocardiographic parameters of ventricular function. Assessment was performed at three time periods: pre-cardiopulmonary bypass (control), 60 min postreperfusion and immediately post-volume loading (at 2 h after cardiopulmonary bypass). No significant deterioration of myocardial function was observed in either of the experimental groups after the use of these preservation techniques. Comparison of regression slopes based on analysis of covariance for myocardial performance, systolic function, and diastolic compliance did not demonstrate any significant differences between the groups. Two hours of global ischemia was not sufficient to cause measurable damage to the myocardium on the basis of which the pharmacological intervention with allopurinol and superoxide dismutase could be evaluated. The controversy surrounding the use of allopurinol and superoxide dismutase is discussed with the findings of this experimental protocol and is brought up for scientific dialogue.     

Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins

BACKGROUND: Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. METHODS: The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. RESULTS: Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. CONCLUSION: Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.     

Lipid domain structure of the plasma membrane revealed by patching of membrane components

Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone

We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K+ and olfactory channels. Expression of a subclone of amino acids 482-702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K+ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K+ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.     

超声波辅助提取花生衣中的白藜芦醇

[目的]以乙醇为溶剂,采用超声波辅助提取的方法提取花生衣中白藜芦醇.[方法]以白藜芦醇的提取率为考察指标,研究花生衣中白藜芦醇提取的优化工艺.[结果]正交试验表明,花生衣中白藜芦醇的适宜提取条件:常温条件下料液比为15:1、乙醇浓度为60%、超声处理时间为60 min、超声功率为150 W,白藜芦醇的平均提取率是0.299%.料液比和乙醇浓度是影响白藜芦醇提取率的显著因素.[结论]该工艺简单快捷,可用于花生衣中白藜芦醇的提取.     

Isolation of human platelet membrane microparticles from plasma and serum

Methods have been developed to isolate human platelet membrane fragments from plasma and serum. Rabbit antibody produced against the human platelet membrane glycoprotein complex, IIb/IIIa, was utilized in an immunoelectrophoretic assay to evaluate the amount of this antigen in various microparticle preparations. The serum concentration of platelet microparticles was more than tenfold greater than that observed for plasma (65 micrograms/ml versus 4.4 micrograms/ml, respectively). Ultrastructural evaluation of either plasma or serum-derived microparticles disclosed a variety of membrane fragments and membrane-bound vesicles with occasional fragments of red blood cells, white blood cells, and platelets. In contrast, microparticle preparations derived from isolated washed platelets after thrombin stimulation contained a heterogeneous array of membrane fragments, vesicles, and granules but no identifiable red cell, white cell, or platelet fragments. Thus, these studies demonstrate that normal human plasma and serum contain platelet membrane fragments that are produced during cell activation. If a similar loss of platelet membranes occurs in vivo following reversible platelet activation, it is possible that the resulting membrane modifications may be of importance in both the structural and functional changes that develop during platelet senescence.     

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.     

Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells

We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.     

Influenza viruses select ordered lipid domains during budding from the plasma membrane

During the budding of enveloped viruses from the plasma membrane, the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the host membrane. Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus family, vesicular stomatitis virus (VSV), and Semliki Forest virus (SFV). Analysis of detergent extractability of proteins, cholesterol, phosphoglycerolipids, and sphingomyelin in virions showed that FPV contains high amounts of detergent-insoluble complexes, whereas such complexes are largely absent from VSV or SFV. Cholesterol depletion from the viral envelope by methyl-beta-cyclodextrin results in increased solubility of sphingomyelin and of the glycoproteins in the FPV envelope. This biochemical behavior suggests that so-called raft-lipid domains are selectively incorporated into the influenza virus envelope. The "fluidity" of the FPV envelope, as measured by the fluorescence polarization of diphenylhexatriene, was significantly lower than compared with VSV or SFV. Furthermore, influenza virus hemagglutinin incorporated into the envelope of recombinant VSV was largely detergent-soluble, indicating the depletion of raft-lipid assemblies from this membrane. The results provide a model for lipid selectivity during virus budding and support the view of lipid rafts as cholesterol-dependent, ordered domains in biological membranes.     

Modulation of synaptosomal plasma membrane-bound enzyme activity through the perturbation of plasma membrane lipid structure by bupivacaine

To determine whether young capuchin monkeys, Cebus apellaselectively interacted with others concerning novel foods, 11 infants (4.5-12 months) living in two groups were observed following presentation of familiar or novel foods. Foods were presented either to the whole group or to infants in a section of the home cage to which only they had access. Infants showed more frequent interest in others' food and picked up foods more frequently when foods were novel, and they tended to eat novel foods more frequently than familiar foods. The pattern was the same whether the foods were presented to the group or only to infants. Infants expressed interest in others' novel foods equally often before and after sampling these foods themselves. The frequency of interest in others' food correlated positively with age. It is concluded that acceptance of novel foods in these monkeys occurs readily regardless of socially provided information about edibility. Social interactions do not appear to make important contributions to acceptance of novel foods by infant capuchin monkeys.     

Internalization of the lymphocytic surface protein CD22 is controlled by a novel membrane proximal cytoplasmic motif

CD22 is a key receptor on B-lymphocytes that modulates signaling during antigenic stimulation. We have defined a novel cytoplasmic motif in human CD22 that controls its unusually rapid turnover at the plasma membrane. Chimeric and mutated CD22alpha cDNA vectors were constructed and stably transfected in CD22-negative Jurkat T-lymphocytic cells. Two assays were employed to measure CD22alpha internalization: first, cytoplasmic uptake of radioiodinated anti-CD22 monoclonal antibody; and second, lethal targeting of a toxin, saporin, into cells via CD22 using bispecific F(ab')2 ([anti-CD22 x anti-saporin]) antibody. Results showed that CD22alpha lacking a cytoplasmic tail was not internalized and that replacement of the cytoplasmic tail of CD19 with that of CD22alpha resulted in a chimeric molecule that behaved like CD22alpha and internalized rapidly. Step-wise deletion of the cytoplasmic tail of CD22alpha located the internalization motif to a polar region of 11 residues (QRRWKRTQSQQ) proximal to the plasma membrane, a part of the molecule predicted to form a coil or turn structure. Interestingly, additional CD22 mutants showed that the two glutamine residues sandwiching the serine are critical to internalization but that the serine itself is not.     

微波辅助法提取山楂黄酮类化合物的工艺条件研究

[目的]探讨微波辅助法提取山楂中黄酮类化合物的工艺.[方法]采用单因素试验和正交试验,研究微波处理时间、微波功率、乙醇浓度和液固比4个因素对提取山楂黄酮类化合物的影响.[结果]最佳提取工艺条件为:液固比60:1、乙醇浓度80%、微波处理时间60 s、微波功率420 W.在此工艺条件下,提取率达到3.47%.[结论]该工艺条件适合于提取山楂中黄酮类化合物.     

Repair of commissural prolapse by extended leaflet sliding

Three patients had chronic mitral regurgitation due to commissural chorda rupture with commissural prolapse. Two of them had myxomatous valve disease and 1 had fibroelastic deficiency. Characteristic intraoperative epicardial and transesophageal echocardiographic findings are demonstrated. A technique is proposed that involves creating a new commissure and avoiding annular plication by extended sliding plasty and rotating the posterior mitral leaflet.     

Isolation of plasma membrane from eukaryotic cells on polylysine-coated polyacrylamide beads

The conditions for covalently binding polylysine to polyacrylamide beads used for membrane isolation have been analyzed. Larger amounts of bead bound polylysine were required for maximimizing plasma membrane purification from HeLa cells than from Dictyostelium discoideum. The least was needed for erythrocytes. The amount of polylysine bound to the bead was dependent on the carboxyl content of the bead and on the concentration of the polylysine used during the bead-polylysine coupling reaction.     

Regulation of the dynamic properties of platelet plasma membrane by intracellular sodium ions

Our previous experiments in human and rat platelets demonstrated that the absence of extracellular Na+ increased the fluorescence anisotropy of TMA-DPH (trimethylamino-diphenylhexatriene, probe preferentially incorporated into the outer leaflet of the plasma membrane). Here we investigated further the in vitro effects of Na+ ions on membrane dynamic properties. Na(+)-dependent changes were reversible and they required about 10-20 min to be induced. They were specifically located in the TMA-DPH environment because they were not observed with diphenylhexatriene (probe non-selectively incorporated into all hydrophobic domains of the cell). To evaluate the possible influence of the intracellular Na+, the effects of sodium replenishment, monensin, ouabain and thrombin on TMA-DPH anisotropy were measured. A rise in intracellular Na+ above the physiological level was associated with unchanged or slightly decreased TMA-DPH anisotropy whereas its decrease was accompanied by a pronounced rise in TMA-DPH anisotropy. Our data indicate that the changes in intracellular Na+ concentration, rather than those in extracellular Na+ concentration, are responsible for the alterations in platelet membrane fluidity probed by TMA-DPH.     

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.