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Biomedical subjects

P Pohl

Publications and source records attributed to P Pohl.

At least 19 recordsLinked to original sources
PubMed2001

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Aquaporins↗
PubMed2001

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

Amino Acid Sequence↗
PubMed2001

[Imaging of coronary vascular abnormality with aneurysm and arteriovenous fistula using MRI bolus tracking].

Coronary arteriovenous fistulas are very rare findings and predominantly drain into the right atrium or the right ventricle. Echocardiography and x-ray coronary angiography are established methods in the detection of this anomalies. It is reported on a patient, in whom an anomaly with dilatation of the circumflex coronary artery, aneurysma and drainage over an arteriovenous fistula in the right ventricle was observed. By means of magnetic resonance tomography with bolustracking technique the vessel course and the dynamics of perfusion are noninvasive shown beside the imaging using TSE-sequence. The method of multiple injections of 2 ml Gd-DTPA makes the bolustracking technique to an ideal tool for the determination of the individual vessel course, the haemodynamics and draining direction of an coronary anomaly with fistula.

Adult↗
PubMed2001

[Effect of ultrasound on membrane surface potentials].

The electrostatic equilibrium on the surface of an ion-impermeable membrane was not influenced by ultrasound fields. Only after incorporation of an ion transporter did the ultrasound induce changes of the membrane surface potential. Because the ultrasound effect was completely reversible, measurements of the surface potential of a flat lipid bilayer membrane containing the calcium transporter calcimycin were performed, simultaneously to the ultrasound exposure. The ultrasound-induced volume flow, also called quartz wind, favored the mass transfer through the diffusion boundary close to the membrane, thereby leading to increased calcium concentrations in the immediate vicinity of the membrane. This, in turn, became manifest as a reduction of the negative surface charge density.

Calcimycin↗
PubMed2001

Methods for the computational localization of atrio-ventricular pre-excitation syndromes.

BACKGROUND: The site of atrioventricular pre-excitation can roughly be estimated with the help of schemes basing on a few number of electrocardiogram (ECG) leads. Computer algorithms have been developed which utilize the body surface mapping of the pre-excitation signal for the localization purpose. We tested several new algorithms. METHOD: A patient suffering from Wolff-Parkinson-White syndrome was investigated prior the catheter ablation. The body surface mapping was performed with a 62-lead magnetocardiograph. The site of pre-excitation was calculated by using different methods: the dipole method with fixed and moving dipoles, the dipole scan on the endocardium, and different current density methods (L1 norm method, L2 norm method, low resolution electromagnetic tomography (LORETA) method, and maximum entropy method). Three-dimensional (3D) magnetic resonance imagings (MRIs) of the heart were used to visualize the results. The source positions were compared to the site of catheter ablation. RESULTS: The accessory pathway was successfully ablated left laterally. This site was correctly identified by the conventional dipole method. By scanning the entire endocardial surface of the heart with the dipole method we found a circumscribed source area. This area too, was located at the lateral segment of the atrio-ventricular grove. The current density methods performed differently. Whereas the L1 norm identified the site of pre-excitation, the L2 norm, the LORETA method and the maximum entropy method resulted in extended source areas and therefore were not suited for the localization purpose. CONCLUSION: The dipole scan and the L1 norm current density method seem to be useful additions in the computational localization of pre-excitation syndromes. In our single case study they confirmed the localization results obtained with the dipole method, and they estimated the size of the suspected source region.

Adult↗
PubMed2001

Localization of late potential sources in myocardial infarction.

INTRODUCTION: Late potentials (LP) are markers of arrhythmogenic events after myocardial infarction (MI). The localization of LP sources would help to identify arrhythmogenic myocardium. The purpose of this study was to localize these LP sources from non-invasive body surface mapping data. METHODS AND RESULTS: Six patients were investigated with cardiac MRI and signal averaged 62-lead magnetocardiography after MI. Three of them were suffering from sustained ventricular tachycardia (VT). Sophisticated computer algorithms were used in order to compute the current density on the surface of the left ventricle. We compared these current density distributions for the entire QRS complex and the high frequency LP signals. In the three patients which had premature ventricular complexes (PVCs) we localized the exit sites of these arrhythmias. We found a close matching of the low current density areas based on the QRS complexes and the high current density areas based on the LP signals. These areas predominantly corresponded to sites of the infarctions. Exit sites of PVCs were located close to these areas. CONCLUSIONS: By means of sophisticated computer algorithms we were able to localize LP sources. This would be useful in steering catheter ablation and coronary revascularization therapies. However, the method has to be proven with the help of invasive mapping in a larger number of patients.

Adult↗
PubMed2001

Investigation of arsenic speciation in oyster test reference material by multidimensional HPLC-ICP-MS and electrospray tandem mass spectrometry (ES-MS-MS).

Multidimensional (size-exclusion-anion-exchange-cation-exchange) liquid chromatography with ICP-MS detection was developed to produce a map of water-soluble species in an oyster test reference material. The presence of arsenobetaine, trimethyl(2-carboxyethyl)arsonium inner salt, arsenocholine, dimethylarsonic acid, tetramethylammonium ion, As(v) and two arsenosugars was demonstrated by ES-MS-MS. A previously unreported compound was isolated and identified by ES-MS-MS as 5-dimethylarsinoyl-beta-ribofuranose. Anion-exchange chromatography was optimized to produce a chromatographically pure peak of arsenobetaine (accounting for ca. 64% of all water-soluble As present) that was used to quantify this compound.

Animals↗
PubMed2001

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation.

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.

Animals↗
PubMed2000

Changes of intrinsic membrane potentials induced by flip-flop of long-chain fatty acids.

The passive transbilayer movement-flip-flop-was investigated on planar bilayer lipid membranes (BLMs), containing myristic, stearic, or linoleic long-chain fatty acids (FA). In response to a transbilayer pH gradient, a difference in the surface charges between inner and outer leaflets appeared. Because the BLM was formed from FA and neutral lipid, a surface potential difference was originated solely by a concentration difference of the initially equally distributed ionized FA. As revealed by zeta-potential measurements, the corresponding surface potential difference DeltaPhi(s) was at least twice the value expected from a titration of the FA alone. The additional surface charge was attributed to FA flip-flop induced by the transbilayer pH gradient. DeltaPhi(s) was derived from capacitive current measurements carried out with a direct current (dc) bias and was corrected for changes of membrane dipole potential Phi(d). Dual-wavelength ratiometric fluorescence measurements have shown that Phi(d) values of the pure DPhPC bilayers and BLMs containing 40 mol % FA differ by less than 6%. It is concluded that fast FA flip-flop is not restricted to membranes with high curvature. The role of pH gradient as an effective driving force for the regulation of FA uptake is discussed.

Buffers↗
PubMed2000

Membrane photopotential generation by interfacial differences in the turnover of a photodynamic reaction.

The adsorption of a membrane-impermeable photosensitizer to only one membrane leaflet is found to trigger a localized photodynamic reaction; i.e., the amount of carbonyl cyanide m-chlorophenylhydrazone (CCCP) molecules damaged in the leaflet facing the photosensitizer is roughly identical to the total amount of CCCP inactivated. Whereas the latter quantity is assessed from the drop in membrane conductivity G, the former is evaluated from the photopotential phi that is proportional to the interfacial concentration difference of the uncoupler. Localized photodestruction is encountered by CCCP diffusion to the site of photodamage. A simple model that accounts for both photoinhibition and diffusion predicts the dependence of the photopotential on light intensity, buffer capacity, and pH of the medium. It is concluded that only a limited amount of the reactive oxygen species responsible for CCCP photodamage diffuses across the membrane. If the concentration of reactive oxygen species is decreased by addition of NaN(3) or by substituting aqueous oxygen for argon, phi is inhibited. If, in contrast, their life time is increased by substitution of H(2)O for D(2)O, phi increases.

Biophysical Phenomena↗
PubMed2000

Desformylgramicidin: a model channel with an extremely high water permeability.

The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1.10(-12) cm(3) s(-1), desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double barreled microelectrodes in the immediate vicinity of a planar bilayer. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17 pS (pH 2.5), 7 and 3 pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A.

Aquaporins↗
PubMed2000

Solvent drag across gramicidin channels demonstrated by microelectrodes.

The competition of ion and water fluxes across gramicidin channels was assessed from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double-barreled microelectrodes in the immediate vicinity of a planar bilayer. Because water movement across the membrane led to accumulation of solutes on one side of the membrane and depletion on the other, the permeable cation was not only pushed by water across the channel (true solvent drag); it also flowed along its concentration gradient (pseudo-solvent drag). For the demonstration of true solvent drag, a difference between the bulk concentrations on the hypertonic and the hypotonic sides of the membrane was established. It was adjusted to get equal cation concentrations at both membrane/water interfaces. From the sodium and potassium fluxes measured along with membrane conductivity under these conditions, approximately five water molecules were found to be transported simultaneously with one ion through the channel. In diphytanoyl phosphatidylcholine membranes, a single-channel hydraulic permeability coefficient of 1.6 x 10(-14) cm(3) s(-1) was obtained.

Biophysical Phenomena↗
PubMed2000

Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels.

The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 </= n </= 4) was found to correlate with their affinity for membrane lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS(2) was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AlPcS(1), AlPcS(2), AlPcS(3), and AlPcS(4), respectively. At high concentrations alpha was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcS(n) (2 </= n </= 4) binding to LUVs and sensitized photoinactivation of gramicidin channels. It is concluded that photosensitizer binding to membrane lipids is a prerequisite for the photodynamic inactivation of gramicidin channels.

Binding Sites↗
PubMed2000

Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects.

Cationic liposomes (CL) are widely used vectors for gene transfer. Recently, ultrasound (US) was reported to enhance liposome-mediated gene transfer to eucaryotic cells in culture. The present study was aimed at studying the effects of 2-MHz pulsed Doppler US on malignant brain tumor cells transfection by cationic liposome/plasmid-DNA complexes (lipoplexes). Cationic liposomes consisting of DOSPA/DOPE were complexed with a plasmid carrying the cDNA encoding green autofluorescent protein (EGFP). Rodent (9L) and canine (J3T) glioma cells were exposed to pulsed US in the presence of EGFP-lipoplexes. A diagnostic transcranial Doppler device (MultiDop L) was used for insonation for 30, 60, and 90 s at 2 MHz/0.5 W/cm(2). To eliminate US reflection and cavitation, a custom-made absorption chamber was designed, where US is applied through a water tank before interacting with the cells and is fully absorbed after passing through the cell layer. Expression of the marker gene EGFP was quantified by FACS analysis and intravital fluorescent microscopy. Cell viability was accessed by Trypan Blue staining. US treatment of tumor cells on microplates for 60 s yielded a significant increase in transfection rates without damaging the cells, but 90-s treatment killed most of the cells. In the absorption chamber, no significant effects of US on transfection were noted. Additional experiments employed US contrast agent (Levovist, Schering) which was able to significantly increase tumor cell transfection rate by enhancing cavitation effects, and also severely damaged most cells when applied at a concentration of 200 mg/mL. In conclusion, our results support the assumption that US effects on lipoplex transfection rates in brain tumor cells in culture are mediated by cavitation effects.

Animals↗
PubMed2000

[Inpatient management of children and adolescents with psychiatric diseases in Germany].

Employing official statistics from the year 1997 the present article analyses in-patient treatment of children and adolescents with psychological disorders in all 16 German Bundeslaender. Sixty per cent of the children and adolescents who are referred to and treated in a German clinic for psychological disorders on the basis of a psychiatric diagnosis are not given professionally adequate treatment. Furthermore, evidence is presented documenting inpatient treatment of children only 0 to 12 months of age in clinics throughout Germany on the basis of psychiatric diagnoses. A new measure is introduced for future planning of inpatient health care for children and adolescents with psychological disorders in Germany.

Adolescent↗
PubMed2000

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Actins↗
PubMed1999

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans.

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.

Animals↗