Factors affecting sulfisoxazole transport through excised rat skin during iontophoresis

Permeation of sulfisoxazole (SIX) across the excised rat skin were studied using two chamber cell with four electrodes, under three successive experimental conditions: without current for 3 h (treatment I), with current for 4 h (treatment II) and without current for 3 h (treatment III). Transport of SIX was significantly increased by iontophoresis. The enhancement ratio of SIX flux were reasonably predicted by Goldman's equation. There was no significant difference (p less than 0.05) between the flux in treatment I and treatment III. On the basis of the flux reversibility, it was concluded that skin alteration did not occur when the applied electric potential was below 5.025 V. Although a prominent current-induced volume flow (from the anodal side to the cathodal side) was observed during current exposure, SIX flux was not influenced by the volume flow. The flux enhancement of SIX was mainly dependent on transdermal potential difference.     

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.     

Electron transport through a self-assembled monolayer of thiol-end-functionalized tetraphenylporphines and metal tetraphenylporphines

The monolayers of several thiol-end-functionalized tetraphenylphophines (SH-TPP) and metal tetraphenylporphines (SH-MTPP) were self-assembled on gold surfaces and identified by cyclic voltammetry (CV), electrochemical impedance spectroscopy, scanning electrochemical microscopy, and the contact angle. The CV peaks of the [Fe(CN)6]3-/ [Fe(CN)6]4- couple were used to identify the efficiency of electrons transferring through the self-assembled monolayer (SAM). The results suggested that SH-TPP and SH-MTPP could form high-quality SAMs on gold surfaces. The SAMs blocked electron transport from the gold electrode to solution. When the length of the thiol-end-link spacer (alkyl group) increased, the electron transport ability of the SAM decreased because of the increased insulator properties of the alkyl chain. With the insertion of metallic ions, the electron transport ability of the SAM of SH-MTPP increased compared to that of the SAM of SH-TPP, which was probably due to the fact that (i) the insertion of metallic ions changed the molecular structure and the molecular structure of SH-MTPP played an important role in electron transport through the SAM and (ii) the insertion of metallic ions increased the electron tunneling probability through the monolayer.     

Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS)

Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity.     

Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography

High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein’s non-polar core. This partitioning was described by overall affinity constants of 1.2 (±0.3)?×?10⁶ M^?1 for R-propranolol and 2.4 (±0.6)?×?10⁶ M^?1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (±2.3)?×?10⁴ M^?1 for R-propranolol and 9.6 (±2.2)?×?10⁴ M^?1 for S-propranolol. Comparable results were obtained at 20 and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes.     

Modulation of rat Kupffer cells on high density lipoprotein receptors on hepatocytes

The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned media transfer. This indicated that the transferable factors produced by Kupffer cells modulate HDL receptors on hepatocytes.     

Importance of monolayer quality for interpreting current transport through organic molecules: alkyls on oxide-free Si

We study the effect of monolayer quality on the electrical transport through n-Si/C(n)H(2n+1)/Hg junctions (n = 12, 14, and 18) and find that truly high quality layers and only they, yield the type of data, reported by us in Phys. Rev. Lett. 2005, 95, 266807, data that are consistent with the theoretically predicted behavior of a Schottky barrier coupled to a tunnel barrier. By using that agreement as our starting point, we can assess the effects of changing the quality of the alkyl monolayers, as judged from ellipsometer, contact angle, XPS, and ATR-FTIR measurements, on the electrical transport. Although low monolayer quality layers are easily identified by one or more of those characterization tools, as well as from the current-voltage measurements, even a combination of characterization techniques may not suffice to distinguish between monolayers with minor differences in quality, which, nevertheless, are evident in the transport measurement. The thermionic emission mechanism, which in these systems dominates at low forward bias, is the one that is most sensitive to monolayer quality. It serves thus as the best quality control. This is important because, even where tunneling characteristics appear rather insensitive to slightly diminished quality, their correct analysis will be affected, especially if layers of different lengths are also of different quality.     

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor ��

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor γ (PPARγ) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPARγ activity or knockdown of PPARγ expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPARγ through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPARγ siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress-induced gene expression by suppressing translation via activation of PPARγ in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPARγ.     

Mutations affecting the binding, internalization, and lysosomal hydrolysis of low density lipoprotein in cultured human fibroblasts, lymphocytes, and aortic smooth muscle cells.

Studies comparing the metabolism of low density lipoprotein (LDL) in normal cells and in cells cultured from patients with homozygous familial hypercholesterolemia have disclosed the existence of a receptor for plasma LDL. This receptor has been identified on the surface of human fibroblasts, lymphocytes, and aortic smooth muscle cells. An extension of these studies to cell strains derived from patients with other single gene defects in cholesterol metabolism has provided additional insight into the normal mechanisms by which cells regulate their cholesterol content and how alterations in these genetic control mechanisms may predispose to atherosclerosis in man.     

Comparative analysis of foetal calf and human low density lipoprotein: relevance for pharmacodynamics of photosensitizers

The effects of differences in lipoprotein content on the distribution of the novel hydrophobic photosensitizer n-butyl-3-[18-(2-butylcarbamoyl-ethyl)-3,7,12,17-tetramethyl-18,13-divinyl-22,24-dihydro-porphin-2-yl]propionamide (PP-N-3) and haematoporphyrin ester (HpE), a relatively hydrophilic photosensitizer, in human (HS) and foetal calf sera (FCS), were investigated. The binding characteristics of human and foetal calf low-density lipoprotein (LDL) were characterised using a human fibroblast line (Vag 12). The uptake into cells of HpE and PP-N-3 was also examined. A comparison of the lipoprotein content, composition and receptor-binding characteristics of foetal calf and human serum was also carried out. LDL content was measured directly using sequential ultracentrifugation to isolate LDL. In our study, we found haematoporphyrin ester to bind to human very low-density lipoprotein (VLDL), LDL and high-density lipoprotein (HDL) in the ratio 2:31:65. In the case of PP-N-3 this ratio was 56:10:33. As VLDL was not detected in foetal calf serum, only binding to LDL and HDL was observed. Using the sequential ultracentrifugation technique, foetal calf serum was found to contain LDL which in turn did bind to human LDL receptors. The uptake of PP-N-3 and HpE in the presence of low density lipoprotein from foetal calf serum (FC-LDL) was not significantly different to values observed in the presence of human serum low density lipoprotein (HS-LDL).     

Current density analysis of electron transport through molecular wires in open quantum systems

The current density in molecular wires connected to contacts is investigated within the nonequilibrium Green's function formalism combined with the Landauer approach. Energy-dependent and total current density through a series of molecular junctions are calculated in real space representation. A rich variety of current patterns including pronounced ring currents (“vortices”) are found even in the defect-free minimal building blocks of molecular devices. The influences of contact positions, functional groups as well as atomic defects on the transport properties are examined systematically for prototypical ortho-, meta-, and para-substituted benzenes as well as heteroaromatic systems. It is found that substitutional functional groups mainly shift the molecular levels and retain characteristic transport channels, while a significant change of electronic pathways and conductance is induced by hetero-aromaticity. The current distribution is used to calculate the static magnetic field distribution in the carbon-based conductors. © 2017 Wiley Periodicals, Inc.     

非对称场流分离技术在蛋黄浆质低密度脂蛋白粒径表征中的应用

通过自组装的非对称场流分离系统(AF4)与紫外可见光检测器联用分离表征了笼养鸡蛋、柴鸡蛋、鹌鹑蛋和鸭蛋蛋黄浆质中的低密度脂蛋白(LDL)。在近似蛋黄浆质生理条件下,研究了进样量、交叉流流速、膜的类型对AF4蛋黄浆质中LDL分离表征的影响;考察了该方法的精密度。在优化的AF4分析条件下,检测出了笼养鸡蛋、柴鸡蛋、鹌鹑蛋和鸭蛋蛋黄浆质中LDL的水力学粒径分布。LDL的AF4洗脱峰高和峰面积的日内精密度分别为1.3%和1.9%(n=7),日间精密度分别为2.4%和2.3%(n=7)。研究结果表明,该方法可用于分离禽类蛋黄浆质中的LDL,同时能够得到LDL水力学粒径分布。     

Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells.

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3 beta-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37 degrees C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

An in vitro method for studying the iontophoretic enhancement of drug transport through skin

An electrodiffusion cell and measurement system has been developed to study the factors governing the transport of ionized drugs through skin under the influence of an electric field. The system allows the determination of transport rates in vitro under conditions of either constant current or constant voltage across the skin. Both accelerating and retarding voltages may be applied. To test the method, the iontophoretic transport of a negatively charged bone resorption agent, etidronate disodium (ethanehydroxydiphosphonate, EHDP) was measured across excised human skin. An enhancement factor of 50-70 over passive diffusion was obtained using a constant current density of 140 μA-cm⁻², and a factor of approximately 100 was obtained with a constant applied voltage of -0.5 V. At the lowest power levels tested (14 μA-cm⁻² or -0.25 V) the enhancements were in reasonable agreement with the predictions of a constant field model for electrodiffusion; however, at higher power levels a phenomenological parameter, the drug transference number in the membrane, appeared to be more useful. Neither the constant current nor the constant voltage mode was found to be a completely satisfactory way of controlling drug delivery in this model. A current-voltage regimen in which both parameters are varied may be required in order to achieve consistent results.     

The uptake behaviors of kaempferol and quercetin through rat primary cultured cortical neurons

The objective of this study was to investigate whether kaempferol and quercetin could be transported into primary cultured cerebral neurons, to establish a practical HPLC method with UV detection for the two flavonols in the neurons, and to study the uptake and transport behaviors of them through the neurons. The present results showed that the level of kaempferol in the neurons increased linearly and then reached a plateau with incubation time at the high concentration of 10 microg/mL, but not at the other two concentrations of 1 and 0.1 microg/mL. However, the levels of quercetin in the neurons were not detected at the three incubating concentrations, and there was a new peak detected in the cell whose retention time was shorter (3.42 min) than that of quercetin (4.65 min). This phenomenon suggested that quercetin might be transported into the neurons and then metabolized quickly to some derivative. Kaempferol could be transported into the neurons in a concentration- and time-dependent manner when the neurons were incubated with the culture medium containing kaempferol at the high dose. There was an apparent correlation between the concentrations of kaempferol in the medium and in the cell, indicating that the uptake of kaempferol in the cell increased along with its dose (10 microg/mL). However, there was a negative correlation between the concentrations of quercetin in the medium and in the cell. The results suggested that kaempferol and quercetin were disposed by the neurons at different way, and this might be an important factor for their different effects on primary cultured cortical cells.     

Enantiomeric difference in percutaneous penetration of propranolol through rat excised skin.

The percutaneous penetration of R-(+)- and S-(-)-propranolol (PL) through rat excised skin was investigated in vitro. The flux of S-(-)-PL after application to normal skin was high compared with that of R-(+)-PL. On the other hand, in damaged rat skin, the flux of R-(+)-PL was almost equivalent to that of S-(-)-PL. It is suggested that there is an enantiomeric difference between S-(-)- and R-(+)-PL in terms of penetration through rat stratum corneum.     

Nonequilibrium molecular dynamics simulation of water transport through carbon nanotube membranes at low pressure

Nonequilibrium molecular dynamics (NEMD) simulations are used to investigate pressure-driven water flow passing through carbon nanotube (CNT) membranes at low pressures (5.0 MPa) typical of real nanofiltration (NF) systems. The CNT membrane is modeled as a simplified NF membrane with smooth surfaces, and uniform straight pores of typical NF pore sizes. A NEMD simulation system is constructed to study the effects of the membrane structure (pores size and membrane thickness) on the pure water transport properties. All simulations are run under operating conditions (temperature and pressure difference) similar to a real NF processes. Simulation results are analyzed to obtain water flux, density, and velocity distributions along both the flow and radial directions. Results show that water flow through a CNT membrane under a pressure difference has the unique transport properties of very fast flow and a non-parabolic radial distribution of velocities which cannot be represented by the Hagen-Poiseuille or Navier-Stokes equations. Density distributions along radial and flow directions show that water molecules in the CNT form layers with an oscillatory density profile, and have a lower average density than in the bulk flow. The NEMD simulations provide direct access to dynamic aspects of water flow through a CNT membrane and give a view of the pressure-driven transport phenomena on a molecular scale.     

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins

The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N-terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH 8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation.