High-resolution computer simulation of the dynamics of isoelectric focusing using carrier ampholytes: focusing with concurrent electrophoretic mobilization is an isotachophoretic process

Focusing of four hemoglobins with concurrent electrophoretic mobilization was studied by computer simulation. A dynamic electrophoresis simulator was first used to provide a detailed description of focusing in a 100-carrier component, pH 6-8 gradient using phosphoric acid as anolyte and NaOH as catholyte. These results are compared to an identical simulation except that the catholyte contained both NaOH and NaCl. A stationary, steady-state distribution of carrier components and hemoglobins is produced in the first configuration. In the second, the chloride ion migrates into and through the separation space. It is shown that even under these conditions of chloride ion flux a pH gradient forms. All amphoteric species acquire a slight positive charge upon focusing and the whole pattern is mobilized towards the cathode. The cathodic gradient end is stable whereas the anodic end is gradually degrading due to the continuous accumulation of chloride. The data illustrate that the mobilization is a cationic isotachophoretic process with the sodium ion being the leading cation. The peak height of the hemoglobin zones decreases somewhat upon mobilization, but the zones retain a relatively sharp profile, thus facilitating detection. The electropherograms that would be produced by whole column imaging and by a single detector placed at different locations along the focusing column are presented and show that focusing can be commenced with NaCl present in the catholyte at the beginning of the experiment. However, this may require detector placement on the cathodic side of the catholyte/sample mixture interface.

Surfactant mediated adsorption of negatively charged latex particles to a cellulose surface

The adsorption of anionic, carboxyl functionalized latex particles, recharged by a cationic surfactant acting as fabric softener/conditioner, to a cellulose surface was investigated with evanescent wave video microscopy. This technique allows to monitor the deposition and release of individual particles in real-time with an excellent selectivity and sensitivity. Since the recharged particles and the conditioner compete for the free surface, the initial deposition rate and final surface coverage are found to be strongly dependent on the ratio of particle and conditioner concentrations.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

High-resolution computer simulation of electrophoretic mobilization in isoelectric focusing

Cationic and anionic electrophoretic mobilization for focusing of hemoglobins (Hb's) in the presence of 100 carrier ampholytes covering a pI range of 6.00-7.98 was studied by computer simulation at a constant current density of 300 A/m(2). Electropherograms that would be produced by whole column imaging and by single detectors placed at different locations along the focusing column are presented. Upon mobilization, peak heights of the Hb zones decrease, but the zones retain a relatively sharp constant profile and are migrating at a constant velocity. A further peak decrease occurs during readjustment at the locations of the original buffer/column interfaces, indicating that detection sensitivity is the lowest at these locations. An anionic carrier ampholyte mobility smaller than that of its cationic species produces a cathodic drift which is smaller than the transport rate used for electrophoretic mobilization. Compared to the case with equal mobilities of carrier ampholyte species, a small increase (decrease) is predicted for the cationic (anionic) mobilization rate within the focusing column. Simulation data suggest that electrophoretic mobilization after focusing and focusing with concurrent electrophoretic mobilization are comparable isotachophoretic processes that occur when there is an uninterrupted flux of an ion through the focusing column. Cathodic drift caused by unequal mobilities of the species of carrier ampholytes, electrophoretic mobilization, and decomposition occurring at the pH gradient edges are related electrophoretic processes.

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Effect of natural seasonal pollen exposure and repeated nasal allergen provocations on elevation of exhaled nitric oxide

BACKGROUND: Exhaled nitric oxide (FENO) is a marker for allergic airway inflammation. We wondered whether in patients with intermittent allergic rhinitis only (i) natural pollen exposure and (ii) artificial pollen exposure by repeated nasal allergen provocations may lead to an elevation of FENO. METHODS: In two prospective studies, we compared the FENO of nonatopic controls with the FENO of nonasthmatic individuals with mild intermittent rhinitis to tree and/or grass pollen. Study I: 13 atopic individuals and seven controls had measurements of FENO, blood eosinophils and eosinophilic cationic protein (ECP) before, during and after pollen season. Study II: 16 atopic individuals and 12 controls had nasal allergen provocations on four following days out of pollen season, with daily measurements of FENO before, 2 and 6 h after provocation, and determination of blood eosinophils, ECP and FEV1 at baseline, on days 5 and 10-12. RESULTS: Natural pollen exposure (study I) caused a significant elevation of FENO in allergic individuals. Nasal allergen provocations (study II) did not elicit a statistically significant rise neither of FENO nor of blood eosinophils between baseline and day 5. However, a subgroup of four individuals with a rise of blood eosinophils during nasal allergen provocations showed also a rise of FENO. CONCLUSIONS: We suppose that in allergic rhinitis a concomitant reaction of the bronchial system is dependent on a strong local inflammation leading to a generalized immune stimulation.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

The SLC3 and SLC7 families of amino acid transporters

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

Convergent synthesis and cellular uptake of multivalent cell penetrating peptides derived from Tat, Antp, pVEC, TP10 and SAP

Cell penetrating peptides (CPP) are peptides of 10 to 30 residues derived from natural translocating proteins. Multivalency is known to enhance cellular uptake for the Tat peptide and closely related polycationic sequences. To test whether multivalency effects on cellular uptake might also occur with other CPP types, we prepared multivalent versions of the strongly cationic Tat, the amphipathic sequences Antp, pVEC and TP10, and the polyproline helix SAP by convergent thioether ligation of the linear CPP onto multivalent scaffolds, and evaluated their uptake in HeLa and CHO cells, intracellular localization, cytotoxicity and hemolysis. While multivalency did not increase the cellular uptake of pVEC or SAP, multivalency effects on uptake comparable to Tat were observed with TP10 and Antp, which are attributable to their polycationic nature. The efficient synthetic protocol for these divalent CPP and their localization in the cytoplasm suggest that CPP might be useful for application in cargo delivery into cells.

N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide

Cupiennins are small cationic a-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.