Intracellular ionic activities in frog skin

Summary Asymmetrical displacement currents are measured in the absence and in the presence of the lipophilic anion dipicrylamine (DPA) in the extracellular solution of nerve fibres of the frogRana esculenta. DPA (30nM-3 M) enhances the current by a component that has the properties expected for a translocation current of DPA ion across the lipid membrane. Analysis in terms of a single-barrier model yields the translocation rate constant (k), the total surface density of DPA absorbed to the membrane (N _t ), and the equidistribution voltage (). The value ofk of about 10⁴ s^–1 is similar to that for a solvent-free artificial bilayer formed by the Montal-Mueller method. The surface densityN _t varies with the DPA concentration as it does in the artificial bilayer, but is about tenfold smaller at all concentrations. The DPA ions sense an intrinsic electric field that is offset by a transmembrane voltage between 0 and 30 mV (inside positive). The part of the axolemma probed by the DPA ion appears as a thin (<2.5 nm), fluid bilayer of lipids. DPA ions seem, however, to be excluded from the major part of the axolemma as if this area is occupied by integral proteins or negative charges.     

Extrinsic charge movement in the squid axon membrane

The absorption of the lipophilic anions dipycrilamine (DPA^-) and tetraphenylborate (TPhB^-) by the lipid matrix of the squid axon membrane, and the kinetics of their translocation, were studied by the charge pulse relaxation technique. The axons were treated with tetrodotoxin (TTX) and 4-aminopyridine to block the ionic currents responsible for nerve excitation. At high enough concentrations of absorbed ions ( 10^-12 mol/cm²) the membrane voltage relaxation following a brief current pulse consisted mainly of two exponential components, whose time constants and relative amplitudes were used for estimating the translocation rate constant, K, and the density of absorbed ions, N. These measurements were performed at different hydrostatic pressures in the range 1–100 MPa ( 1,000 atm), and at different temperatures in the range 5° C–20° C. Both K and N were found to be little affected by pressure. The pressure dependence of K indicated that the translocation of lipophilic ions across the nerve membrane involves activation volumes of the order of 5 cm³/mol. In all experiments the passive membrane resistance was little affected by pressures up to 80 MPa. However, above 100 MPa it fell dramatically to low values, presumably because of phase separation phenomena between the membrane components. The temperature dependence of K, both for DPa^- and TPhB^-, implied an activation energy for ion translocation of the order of 60 kJ/mol, close to that measured in artificial lipid bilayers.It is concluded that the lipid bilayer structure of the nerve membrane is not modified by pressures below 80 MPa and that the intramembrane movements of relatively small charged groups cannot account for the large activation volumes involved in the gating of ionic channels.     

Overexpression of ornithine-δ-aminotransferase increases proline biosynthesis and confers osmotolerance in transgenic plants

Accumulationof proline is a way to increase tolerance to water stress in plants. Therefore,considerable attention has been devoted to optimise proline biosynthesis intransgenic plants. Glutamate and ornithine are both precursors of proline butwhile genes of the glutamate pathway were overexpressed in transgenic plants,no gene encoding an enzyme of the ornithine pathway was considered until now. Thepresent study aims to establish if the overexpression ofornithine--aminotransferase (-OAT) represents an additional wayto increase proline content. To achieve this goal, anArabidopsis -OAT cDNA was fused to the CaMV35Spromoter and introduced via Agrobacterium transformationinto Nicotiana plumbaginifolia. Overexpression of the-OAT cDNA in the analysed transgenic lines was linked to an increase in-OAT enzyme activity. The transgenic lines presenting high enzymaticactivity synthesized more proline than the control plants and showed a higherbiomass and a higher germination rate under osmotic stress conditions. Thesestudies reveal a new and efficient way to increase proline content in plantsand to enhance crop tolerance.     

Kinetics of ion transport in lipid membranes induced by lysine-valinomycin and derivatives

Summary Lysine-valinomycin and two N-acyl derivatives are compared with respect to their potency to transport Rb⁺ ions across thin lipid membranes. Lysine-valinomycin acts as a neutral ion carrier only above a pH of about 7 of the aqueous solutions, while at lower pH the molecules seem to be positively charged due to a protonation of the -NH₂ group of the lysine residue.A kinetic analysis based on voltage jump relaxation experiments and on the nonlinearity of the current-voltage characteristics showed that the conductance increment per carrier molecule for uncharged lysine-valinomycin is similar to that of natural valinomycin. The attachment of a rather bulky side group such as the dansyl or para-nitrobenzyloxycarbonyl group reduced by approximately one order of magnitude.Some of the relaxation data of the valinomycin analogues were influenced by an unspedfic relaxation of the pure lipid membrane. This structural relaxation represents a limitation to the possibility of analyzing specific transport systems in thin lipid membranes by the voltage jump or charge pulse techniques. It is shown that the time dependence of this structural relaxation — which was first published by Sargent (1975) — is at variance with a three capacitor equivalent circuit of the membrane, which was suggested by Coster and Smith (1974) on the basis of a.c. measurements. A modified equivalent circuit has been found to represent a satisfactory analogue for the current relaxation in the presence of valinomycin. It turned out, however, that such an equivalent circuit provides little insight into the molecular mechanism of transport.     

Electron microscopic studies on the polytene chromosomes of Chironomus thummi salivary glands

The method of ultrathin sections of unsquashed salivary gland polytene chromosomes of Ch. thummi was applied to their ultrastructural mapping. There was a good agreement between electron micrographs and Hägele's light microscopic map (1970) with respect to the pattern and number of bands. 94% of bands were identified in larval and prepupal chromosomes. In Ch. thummi, band thickness varied from 0.05–0.5 m. Most characteristic were 0.2–0.3 m bands. Morphologically, bands were classified as: continuous (frequently with holes and gaps), discrete, dotted and continuous-discrete, discrete-dotted.Band morphology is related to band size, such that smaller bands, as a rule, were also dotted. Bands beginning to puff likewise became dotted. Interbands in unsquashed chromosome sections were from 0.05–0.15 m. The smallest interbands contained only fibrils, in the larger interbands few granules could be observed. This makes interbands distinguishable from a typical puff with many such granules.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Rapid micropropagation of <Emphasis Type="Italic">Curcuma longa</Emphasis> using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Multiple shoots of Curcuma longa were induced by culture of bud explants for 1week in Murashige and Skoog (MS) liquid medium supplemented with 72.64M thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8weeks. The regeneration rate was up to 11.4±1.7shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.     

Measurements of electrical potential differences across yeast plasma membranes with microelectrodes are consistent with values from steady-state distribution of tetraphenylphosphonium inPichia humboldtii

Summary Electrical potential differences across the plasma membrane () of the yeastPichia humboldtii were measured with microelectrodes (filled with 0.1m KCl) inserted into cells immobilized in microfunnels. The registered signals were reproducible and stable for several minutes. On attainment of stable reading for the specific membrane resistanceR _sp was determined by applying square-current pulses to the preparation. Both andR _sp were pH dependent and displayed equal but opposite deflection, reaching its maximal value of –88±9 mV (n=13) andR _sp its minimal value of 10 k·cm² (maximal conductance) at pH 6.5. Uncouplers and the polyene antibiotic nystatin depolarized the cells, decreasing to –21±15 mV (n=10) with concomitant decrease ofR _sp. Comparison of values from microelectrode measurements with those calculated from the steady-state distribution of tetraphenylphosphonium ions agreed within 10 mV under all physiological conditions tested, except at pH values above 7.0. During microelectrode insertion transient voltage signals (a few msec long) were detected by means of an oscilloscope. These voltage signals were superimposed on the stable recordings described above. These short voltage signals disappeared in uncoupled cells. The closely related values obtained by two independent methods (direct measurements with microelectrodes and calculation from steady-state distribution of a lipophilic cation) provide evidence that these values reffect the true membrane potential of intact cells.     

Adsorption to dipalmitoylphosphatidylcholine membranes in gel and fluid state: pentachlorophenolate, dipicrylamine, and tetraphenylborate.

We measured the dependence of electrophoretic mobility of dipalmitoylphosphatidylcholine (DPPC) vesicles on the aqueous concentration of negatively charged ions of pentachlorophenol (PCP), dipicrylamine (DPA), and tetraphenylborate (TPhB). The objective was to determine how the physical state of hydrocarbon chains of lipids affects adsorption of lipophilic ions. The studies were done at 25 and 42 degrees C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of zeta-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, Ps, and the linear partition coefficient, beta. Results: K, (x 10(4)M-1): K(gel): PCP (0.49 +/- 0.28), DPA (25 +/- 10), TPhB (31 +/- 10); K(fluid): PCP (4.5 +/- 0.9), DPA (74 +/- 21), TPhB (59 +/- 14); Ps, (nm2): Ps(gel): PCP (5.4 +/- 2.3), DPA (5.9 +/- 2), TPhB (5.0 +/- 1.7); Ps(fluid): PCP (4.5 +/- 0.4), DPA (5.2 +/- 0.4), TPhB (4.1 +/- 0.2); beta, (x 10(-5) m): beta(gel): PCP (0.15 +/- 0.09), DPA (7.1 +/- 0.3), TPhB (10 +/- 7); beta(fluid): PCP (1.7 +/- 0.3), DPA (24 +/- 7), TPhB (24 +/- 6). It was interesting to find that the adsorption site area for PCP, DPA, and TPhB were very similar for both the gel and fluid membranes; also, the areas were independent of the size and molecular structure of the adsorbing species. Using a simple discrete charge model the adsorption site areas for all species were consistent with a dielectric constant of 8-10 and with an ion adsorption depth of 0.4-0.6 nm below the water/dielectric interface. The delta delta G0 = delta G0(gel) - delta G0(fluid) was found to be about twice as large for PCP than for DPA and TPhB. This indicates that PCP will be significantly more adsorbed in the fluid and disordered regions of biomembranes, whereas the distribution of DPA and TPhB is expected to be relatively more even.     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

Novel glutathione analogues containing the dithiol and disulfide form of the Cys-Cys dyad

Summary The glutathione analogue -(H-Glu-OH)--OH (5), containing the 8-membered disulfide ring- replacing the native -Cys-Gly fragment, has been synthesized and characterized together with its reduced dithiol form -(H-Glu-OH)-Cys-Cys-OH (6).Abbreviations DBU 1,8-diazabicyclo [5.4.0] undec-7-ene; - DCCI N,N-dicyclohexylcarbodiimide - NMM N-methylmorpholine - THF tetrahydrofuran; (n-Bu)₃P, tri-n-butylphosphine     

Monitoring of the mitochondrial and plasma membrane potentials in human fibroblasts by tetraphenylphosphonium ion distribution

The lipophilic cation tetraphenylphosphonium (TPP⁺) is accumulated by human skin fibroblasts across both the plasma and mitochondrial membranes. We show here that TPP⁺ uptake is indeed greatly decreased under conditions leading to de-energization of mitochondria. The TPP⁺ accumulation in the presence of the proton ionophore FCCP has been used for determination of the plasma membrane potential across the plasma membrane, after correction for potential-independent binding of TPP⁺ to cellular components. Following this procedure, a value of 75 mV has been obtained. Through the amount of TPP⁺ released by FCCP treatment, an estimate of thein situ mitochondrial membrane potential has been made. Furthermore, we report that the mitochondrial component of TPP⁺ accumulation decreases with aging of fibroblast cultures.Abbreviations _m membrane potential across thein situ mitochondria - _p membrane potential across the plasma membrane - TPP⁺ tetraphenylphosphonium - HEPES N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

Dependence of the electrical breakdown voltage on the charging time inValonia utricularis

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. For a charging time of 420 sec the breakdown voltage is about 750 mV (18°C), a value that is in close agreement with earlier results obtained with current pulses (Coster & Zimmermann, 1975;J. Membrane Biol. 22:73). If the membrane is charged to the breakdown voltage in a shorter time, the breakdown voltage is found to be a function of the duration of the charge pulses. Whereas towards smaller pulse lengths down to 10 sec only a small, but significant, increase in the breakdown voltage is observed (1.1 V at 10 sec pulse length and 18°C), a strong increase in the breakdown voltage is found for even shorter charging times. For a pulse length of 800 nsec the breakdown voltage has a value of about 2.4 V (18°C) and a plateau seems to be reached for a pulse duration of 500 nsec. The influence of temperature on the breakdown voltage as observed for short charging times is very similar to that reported earlier for current pulses of 500 sec duration. For charge pulses of 1 to 2 sec duration the breakdown voltage decreases from 3.6 V at 3°C to 1.6 V at 25°C by more than a factor of two.Voltage relaxation studies in the low-field range suggest that the time constants of the two membranes arranged in series, tonoplast and plasmalemma, are similar. From this, it is suggested that both membranes show electrical breakdown, whereby the breakdown voltage of a single membrane is probably half the value of the total breakdown voltage. Its dependence on pulse length is therefore considered to be an intrinsic property of one single membrane. The strong dependence of the breakdown voltage on the charging time of the membrane further supports the interpretation of the breakdown phenomenon on the basis of the electro-mechanical model proposed earlier. In this model it is assumed that the electrical and mechanical compressive forces are counter balanced by elastic restoring forces within the membrane. However, towards very short pulses (less than 800 nsec), where a plateau seems to be reached, other processes may be generated by the application of the electric field. We discuss whether one of these processes is the ion movement through the membranes induced by a high electric field (Born energy).     

Purification of bovine colostrumβ-galactosideα(2–6)sialyltransferase to near homogeneity by affinity chromatography

Cytidine-5-monophospho-N-acetylneuraminic acid:-galactoside 2-6sialyltransferase was purified from bovine colostrum by two sequential affinity chromatography steps on CDP-ethanolamine-Sepharose and CDP-ethanolamine-(N-caproylamino-)-Sepharose, respectively. While the conditions for elution were those of Paulsonet al. [J Biol Chem (1977) 252:3256–62], the ligand of the second affinity column was coupled to Sepharose by using 6-aminocaproic acid as linker. The ease of this procedure allows rapid synthesis of bulk quantities of ligand.Highly purified preparations of sialyltransferase were obtained which moved on gradient gel electrophoresis as a single band of 76 kDa and on dodecylsulphate electrophoresis as a single band of 54 kDa. The product of the reaction between lactose and CMP-N-acetylneuraminic acid catalyzed by the purified sialyltransferase was identified by high-resolution 500 MHz¹H-NMR spectroscopy as Neu5Ac2-6Gal1-4Glc.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Effects of Livagen and Epitalon, New Peptide Bioregulators, on Enkephalin-Degrading Enzymes from Human Serum

The effects of new peptide bioregulators—Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)—on the endogenous opioid system was studied. In particular, attention was focused on their ability to change the activity of enkephalin-degrading enzymes of blood serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of ³H-Leu-enkephalin hydrolysis in the presence of Livagen and Epitalon. These peptides inhibited enkephalin-degrading enzymes of human serum. Livagen proved to be more efficient than some well-known peptidase inhibitors, such as puromycin, leupeptin, and D-PAM. The dose–inhibitory effect curves for Livagen and Epitalon were plotted; their IC₅₀ equaled 20 and 500 M, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [³H][D-Ala², D-Leu⁵]-enkephalin. No interaction was observed between the peptides and - or -opioid receptors of the membrane fraction from the rat brain.     

A comparative assessment of TLC overlay technique and microwell adsorption assay in the examination of influenza A and Sendai virus specificities towards oligosaccharides and sialic acid linkages of gangliosides

Influenza A and Sendai viruses bind toneolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C_24:1 and C_16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac2-3Gal1-4Glc-R of G_M3, and Neu5Ac2-3Gal1-4GlcNAc-R as well as Neu5Ac2-6Gal1-4GlcNAc-R ofneolacto-series gangliosides with nLcOse₄Cer and nLcOse₆Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficientr _s was calculated according to Spearman from each virus binding towards G_M3, IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data. Based on these results, analyses of virus binding on thin-layer chromatograms as well as in microwells were found equivalent tools for ganglioside receptor studies. Abbreviations: BSA, bovine serum albumin; GSL(s), glycosphingolipids; HPTLC, high performance thin-layer chromatography; PBS, phosphate buffered saline; Neu5Ac,N-acetylneuraminic acid [35];r _s = rank correlation coefficient according to Spearman. The designation of the glycosphingolipids follows the IUPAC-IUB recommendations [36]. LacCer or lactosylceramide, Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; G_M3 (according to Svennerholm [37]) or II³Neu5AcLacCer.