Hypothesis. Cytochrome c oxidase as a proton pump. A transition-state mechanism

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.     

Effect of myelin basic protein on the thermotropic behavior of aqueous dispersions of neutral and anionic glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine

The thermotropic behavior of the natural glycosphingolipids galactosylceramide, asialo-Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-Cer (GM1), sulfatide, GM1, NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer (GD1a), and NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc8-2 alpha NeuAc)beta 1-4Glc beta 1-1 Cer (GT1b), and their mixtures with dipalmitoylphosphatidylcholine (DPPC) in the presence of myelin basic protein (MBP) was studied by high sensitivity differential scanning calorimetry. The transition temperature of DPPC, galactosylceramide, and asialo-GM1 is affected little by MBP while their transition enthalpy is decreased in proportion to the amount of protein in the mixture. The thermotropic behavior of anionic glycosphingolipids is considerably perturbed by MBP. The transition temperature of gangliosides increases in the presence of MBP, whereas that of sulfatide decreases. The enthalpy of the transition of anionic glycosphingolipids increases markedly in the presence of MBP. The excess heat capacity function of these systems can be resolved into two independent phase transitions. Phase separation of enriched lipid/protein domains occurs in a magnitude that depends on the amount of MBP; the rest of the lipid phase exhibits some altered thermodynamic properties. In mixtures of glycosphingolipids with DPPC, phase separation is also present but no phase transition with the characteristic of pure DPPC is found. MBP is changing the properties of the lipid mixture as a whole and does not interact exclusively with the glycosphingolipids. The proportion of MBP required to produce the maximal changes is greater the greater the complexity of the glycosphingolipids polar head group. Relatively small variations of the amount of MBP induce large shifts in the proportion of the different phases present.     

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.     

Condensing and fluidizing effects of ganglioside GM1 on phospholipid films

Mixed monolayers of the ganglioside G_M1 and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by G_M1 on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for G_M1 concentrations ranging from 5 to 70 mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of G_M1 (<25 mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher G_M1 concentrations (>25 mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that G_M1 and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/G_M1 binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of G_M1 to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the G_M1 molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the G_M1 molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the G_M1 molecule.     

Comparison of the 13C-n.m.r. spectra of gangliosides GM1 with those of GM1-oligosaccharide and asialo-GM1

The ¹³C-n.m.r. spectra of asialo-G_M1 and G_M1-oligosaccharide are completely assigned and compared to those previously found for intact G_M1 and for the series G_M4, G_M3, G_M2, G_M1, G_D1a, G_D1b, and G_T1b. Removal of the ceramide residue from G_M1 liberated a free, reducing aldehyde group, which was reflected in a doubling of the ¹³C-n.m.r. signals assignable to the d-glucose residue because of α,β equilibrium. The spectrum of asialo-G_M1 lacks the resonances from the sialic acid residue, as expected; in addition, several resonances from the neutral gangliotetraglycosyl residue shifted to different field positions after removal of sialic acid from G_M1. These resonances include that of C-4 of the inner β-d-galactosyl residue, and C-1 of the 2-acetamido-2-deoxy-d-galactosyl residue that is near the site of attachment of the sialosyl residue. The differences between the chemical shifts of the carbon resonances of oligomeric and monomeric saccharides, termed linkage shifts, provide a quantitative assignment aid. They are ～ $\text{1}\text{3}$ of those for residues linked to sialic acid than those for residues linked to the neutral hexose chain. Correlations among linkage shifts for pairs of glycosidically-linked carbon atoms for asialo-G_M1 and G_M1-oligosaccharide were compared with those for the series of gangliosides G_M4 to G_T1b, and differences are noted for resonances for carbon atoms near the sialic acid residue. The spectrum of ganglioside G_M1b, a positional isomer of G_M1 whose ¹³C-n.m.r. spectrum has not yet been observed, is predicted.     

GLYCOSPHINGOLIPIDS IN FETAL TAY-SACHS DISEASE BRAIN AND LUNG CULTURES

Abstract— A study was undertaken of the glycosphingolipids in cell cultures derived from cerebellum of Tay-Sachs disease fetal brain in order to determine the suitability of such cell strains as a model for Tay-Sachs disease. The glycosphingolipids in the Tay-Sachs disease cultured cerebellar cells were compared with those found in normal cultured cerebellar cells, normal and Tay-Sachs cultured lung cells, and normal and Tay-Sachs fetal brain. The glycolipids were separated by TLC, then analyzed by GLC of the trimethylsilyi derivatives of the methylglycosides of the sugar moieties. In the cultured cerebellar lines, the predominant gangliosides were G_M2, G_M3, and G_D3. There was a 4-fold increase of G_M2 in the Tay-Sachs as compared with the normal line. Only G_M3 and G_D3 gangliosides were found in the Tay-Sachs and the normal fetal lung cell cultures. The major neutral glycosphingolipids in all of the cultured cells which were analyzed were glucosylceramide, lactosylceramide, digalactosyl-glucosylceramide, and globoside. When the Tay-Sachs cerebellar cells were labelled with [1-¹⁴C]gluco-samine, some radioactivity was observed in the trihexosylceramide band, indicating the presence of a small amount of a galactosamine-containing trihexosylceramide which may be asialo-G_M2 (G_A2). The trihexosylceramide in Tay-Sachs fetal brain was identified as G_A2 by GLC. Both Tay-Sachs and normal fetal brain gangliosides were more complex than those found in the cultured cells. Long chain fatty acids (C_24:0 and C_24;1) predominated in all of the glycosphingolipids of the Tay-Sachs and the normal cultured cerebellar cells. In contrast, the glycosphingolipids of Tay-Sachs and normal fetal brain contained mainly the shorter chain fatty acids (C₁₆:₀, C₁₈:₀, and C_18:1). The cerebrosides in both the Tay-Sachs and normal fetal brains were mainly glucosylceramide with only small amounts of the galactosylceramide which predominates in infant brain. Cultured cells from the fetal Tay-Sachs disease     

Phase Behavior and Lipid-Membrane Structure of Phospholipid-Glycosphingolipid Liposomes and the Thermal Unfolding of Insulin

Abstract

The thermodynamic phase behavior and lipid-membrane structure of fully hydrated uni- and multilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) surface-modified by a glycosphingolipid, sulfogalactosylceramide (sulfatide), have been investigated by means of differential scanning calorimetry and fluorescence polarization spectroscopy. The calorimetric and spectroscopic scans of the two-component liposomes demonstrate a distinct influence of increasing amounts of sulfatide on the lipidmembrane thermodynamics. This is manifested as a broad gel-fluid phasecoexistence region and a low-temperature two-phase region composed of highly ordered lipids. In addition, the pre-transition is abolished for small concentrations of sulfatide. A well-defined heat capacity peak, reflecting a thermotropic chain melting transition is observed for liposomes composed of pure sulfatide. On basis of the calorimetric and spectroscopic data a phase diagram has been established in the full temperature and composition plane. Dynamic light scattering measurements of liposome sizes reveal that the incorporation of sulfatide into the DPPC membrane matrix leads to a stabilization of the characteristic size of the extruded liposomes. Furthermore, incubation with insulin does not affect the liposome size and the aggregational behavior of the two-component sulfatide-DPPC liposomes. In accordance with this, the heat capacity curves demonstrate an insignificant influence on both the lipid membrane phase behavior and the thermal unfolding characteristics of insulin.     

Effects of ganglioside GM1 on DNA synthesis in isolated nuclei and on the activity of DNA polymerase α derived from S-phase HeLa cells

Ganglioside G_M1 inhibited either DNA synthesis in isolated nuclei or the activity of DNA polymerase α fractionated from S-phase HeLa cells. The concentrations of G_M1 necessary for 50% inhibition were about 5 μM and 10 μM for nuclei and DNA polymerase α, respectively. The G_M1 inhibition of the enzyme activity was suppressed by the addition of 0.05% Triton X-100. Neither gangliotetraosylceramide (asialo-G_M1) nor free N-acetylneuraminic acid inhibited the enzyme activity. These facts suggest that G_M1, probably in the form of micelles, could influence the enzyme activity by behaving as a polyanionic macromolecule. The kinetic studies indicate that the G_M1 inhibition of the enzyme activity was not competitive with the substrate, deoxythymidine triphosphate, but rather with the template DNA. Binding of G_M1 and DNA polymerase α was suggested by the cocentrifugation of G_M1 and the enzyme fraction after their preincubation. It was also observed that other acidic glycolipids, i.e., brain sulphatide and seminolipid, also inhibited the enzyme activity, whilst neutral galactosylceramide did not. The inhibitory influences of these sulphate esters of glycolipids were, similarly to G_M1, suppressed by the addition of 0.05% Triton X-100.     

Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells

Abstract: The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain G_M3, G_M2, G_M1, G_D3, and G_D1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GaINAc(β1→4) Gal(β1→4)Glc(β1→1)Cer (GgOse₃Cer), and GaINAc(β1→3)Gal(α1→4) Gal-(β1→4)Glc(β1→1)Cer (GbOse₃Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mm -EGTA showed a two- to threefold increase in G_M3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mm -EGTA plus 0.4 mm -EDTA a similar increase in G_M3 was observed but this change was now accompanied by decreases in G_M2, G_M1 GgOse₃Cer, and GbOse₄Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca²⁺ sufficient to bind all chelator. Mn²⁺ replacement reversed the chelator effects differentially; G_M2 and G_M1 levels were the most sensitive to increases in Mn²⁺ concentration; GgOse₃Cer and GbOse₄Cer were also sensitive, whereas G_M3 was the least affected. These results suggest calcium serves an important regulatory role on G_M3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.     

Sphingosine-1-Phosphate as an Amphipathic Metabolite: Its Properties in Aqueous and Membrane Environments

Sphingosine-1-phosphate (S1P) is currently considered to be an important signaling molecule in cell metabolism. We studied a number of relevant biophysical properties of S1P, using mainly Langmuir balance, differential scanning calorimetry, ³¹P-NMR, and infrared (IR) spectroscopy. We found that, at variance with other, structurally related sphingolipids that are very hydrophobic, S1P may occur in either an aqueous dispersion or a bilayer environment. S1P behaves in aqueous media as a soluble amphiphile, with a critical micelle concentration of ≈12 μM. Micelles give rise to larger aggregates (in the micrometer size range) at and above a 1 mM concentration. The aggregates display a thermotropic transition at ∼60°C, presumably due to the formation of smaller structures at the higher temperatures. S1P can also be studied in mixtures with phospholipids. Studies with dielaidoylphosphatidylethanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (DPPC) show that S1P modifies the gel-fluid transition of the glycerophospholipids, shifting it to lower temperatures and decreasing the transition enthalpy. Low (<10 mol %) concentrations of S1P also have a clear effect on the lamellar-to-inverted hexagonal transition of DEPE, i.e., they increase the transition temperature and stabilize the lamellar versus the inverted hexagonal phase. IR spectroscopy of natural S1P mixed with deuterated DPPC allows the independent observation of transitions in each molecule, and demonstrates the existence of molecular interactions between S1P and the phospholipid at the polar headgroup level that lead to increased hydration of the carbonyl group. The combination of calorimetric, IR, and NMR data allowed the construction of a temperature-composition diagram (“partial phase diagram”) to facilitate a comparative study of the properties of S1P and other related lipids (ceramide and sphingosine) in membranes. In conclusion, two important differences between S1P and ceramide are that S1P stabilizes the lipid bilayer structure, and physiologically relevant concentrations of S1P can exist dispersed in the cytosol.     

Interaction of melittin with glycosphingolipids and phospholipids in mixed monolayers at different temperatures. Effect of the lipid physical state

The influence of the liquid-expanded or liquid-condensed state of the lipid interface induced by changes of temperature on the lipid-protein interactions and their two-dimensional miscibility was studied for mixtures of melittin with different phospholipids (DPPC, DMPC, DOPC egg PC) and gangliosides (G_M1, G_D1a) in mixed monolayers at the air/145 mM NaCl interface. The critical amount of melittin at which a phase separation takes place in the mixed film increases as the glycosphingolipid or phospholipid is more liquid-expanded. The lipid-protein interaction increases the stability of both melittin and the lipid. The interaction of melittin with gangliosides is thermodynamically more favorable as these are more liquid-expanded. The interaction of melittin with phospholipids, on the other hand, is more favorable when the lipids are in the liquid-condensed state even if these films show lateral immiscibility at a lower proportion of protein compared to lipids in the liquid-expanded state. Hydration-dehydration effects in the polar head group region are likely to participate in these lipid-protein interactions.     

Identity of the activator proteins for the enzymatic hydrolysis of sulfatide,ganglioside GM1, and globotriaosylceramide

The activator protein for the enzymatic hydrolysis of sulfatide, ganglioside G_M1, and globotriaosylceramide was purified from human kidney, brain, and urine. As far as they could be assayed, these three activities cochromatographed during all steps, indicating that they are due to the same protein. This result was corroborated by immunochemical comparison of individually purified activator preparations. In contrast, the activator for ganglioside G_M2 hydrolysis could clearly be separated from the other activities. Kinetic data were determined for the interaction of the sulfatide activator with the different glycolipids and hydrolases.     

Thermotropic phase behaviour of α-dipalmitoylphosphatidylcholine multibilayers is influenced to various extents by carotenoids containing different structural features- evidence from differential scanning calorimetry

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: β-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane- a fully saturated derivative of β-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (ΔH_p) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes- a result of their orientation in the DPPC bilayer.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Preparation of some labeled glycosphingolipids by catalytic addition of tritium

Three experiments are described in which seven tritium-labeled glycosphingolipids were prepared by catalytic addition of tritium gas to unsaturated centers. These compounds include glucosyl ceramide, galactosyl ceramide, lactosyl ceramide (cytolipin H), N-acetylgalactosaminyl (β1 → 3) galactosyl (α1 → 3) galactosyl (β1 → 4) glucosyl ceramide (cytolipin R), and three species of gangliosides: G_M1 (G₄), G_M3 (G₆), and G_D1a (G₃).     

Glycolipids are not extracted from phospholipid bilayers by binding to ferritin-lectin conjugates

A radioactively-labelled glycosphingolipid, asialo-G_M1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-G_M1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside G_M1 with human and fetal calf sera were studied, the following main results being obtained: (a) G_M1, upon incubation with both sera gave origin to two G_M1-protein complexes, which also occurred after interaction of G_M1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore G_M1 appeared to specifically bind serum albumin and to form G_M1-albumin complexes. (b) G_M1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10^?6 M), was time and concentration dependent, and resulted in a relevant degree of G_M1 complexation (up to 80% of total G_M1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 · 10^?4 M G_M1, the ratio between bound and total G_M1 increased linearly with increasing G_M1 concentration; in the second phase, occurring above 2 · 10^?4 M, the ratio remained practically constant. After these findings it should be expected that G_M1, when present in serum containing systems, forms complexes with albumin. This should be appropriately considered when studying the effects of exogeneous G_M1 in in vivo and in vitro (tissue cultures) systems.     

Effects of the endosomal lipid bis(monoacylglycero)phosphate on the thermotropic properties of DPPC: A 2H NMR and spin label EPR study

Bis(monoacylglycero)phosphate (BMP) is an endosomal lipid with a unique structure that is implicated in the formation of intraendosomal vesicular bodies. Here we have characterized the effects of dioleoyl-BMP (BMP_18:1) at concentrations of 5, 10, 15 and 20 mol% on the thermotropic behavior of dipalmitoyl phosphatidylcholine (DPPC) vesicles, and compared them to those of equimolar concentrations of dioleoyl phosphatidylglycerol (DOPG), a structural isoform of BMP_18:1. Because BMP is found in the acidic environments of the late endosome and intralysosomal vesicles, samples were prepared at pH 4.2 to mimic the pH of the lysosome. Both ²H NMR of perdeuterated DPPC and spin-labeled EPR with 16-doxyl phosphatidylcholine were utilized in these investigations. NMR and EPR results show that BMP_18:1 induces a lowering in the main phase transition temperature of DPPC similar to that of DOPG. The EPR studies reveal that BMP_18:1 induced more disorder in the L_β phase when compared to equimolar concentrations of DOPG. Analysis from dePaked ²H NMR spectra in the L_α phase reveals that BMP_18:1 induces less disorder than equal concentrations of DOPG. Additionally, the results demonstrate that BMP mixes with other phospholipids as a phospholipid and not as a detergent molecule as once speculated.     

Glycosphingolipids in feces of germ-free rats as a source for studies of developmental changes of intestinal epithelial cell surface carbohydrates

Non-acid and acid glycosphingolipids were isolated from feces of one litter of germ-free rats from day 17 to day 51. Quantitative and qualitative changes described for small intestine of conventional rats [Bouhours D, Bouhours J-F (1981) Biochem Biophys Res Commun 99:1384–89] were also found in the feces of these germ-free rats. A decrease in lactosylceramide and sialyllactosylceramide excretion and a change fromN-acetylneuraminic acid toN-glycoloylneuraminic acid, as well as an appearance of type 1 chain blood group H-active penta- and decaglycosylceramides were observed during the weaning period. Thus the dramatic changes seen in rat intestinal glycosphingolipids postnatally seem to be primarily regulated by non-microbial factors.Abbreviations G_M3 G_M3-ganglioside, II³NeuAc-LacCer or II³NeuGc-LacCer - SPG IV³NeuAc-nLcOse₄Cer - G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer