Streptococcus mutans membrane lipid composition: Virulence factors and structural parameters.

OBJECTIVE: The aim of the present work was to analyze whether the location of the dental biofilm was associated with shifts in the membrane fatty acid profile, and whether such shifts could affect certain virulence factors of strains of Streptococcus mutans. DESIGN: An experimental study was conducted to assess the behavior of S. mutans strains isolated from dental biofilm collected from sound and carious smooth and occlusal surfaces of the oral cavity of children. The lipid composition of the bacterial membrane, structural membrane parameters, acid survival, and ATPase activity were tested at pH 7 and 5. RESULTS: At pH 5, an increase in both the unsaturated and the long chain fatty acids as well as in proton ATPase hydrolytic activity was observed in strains isolated from carious smooth surface biofilm but not in carious occlusal surface strains. The observed changes correlated with the studied structural parameters, and were found to be associated with membrane organization. The changes correlated with a decreased in ΔC (sn-1 and sn-2 acyl chain inequivalence), indicative of increased acyl chain interaction. CONCLUSIONS: The obtained results suggest that the acidic environment (pH 5) of smooth surface caries affects membrane organization, inducing a shift in membrane lipid profile, which would likely induce better protein/lipid hydrophobic matching, resulting in increased ATPase activity and higher acid survival.

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways.

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.

Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition.

The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane lipid composition on sorting of the endocytosed material. This suggests that changes in the membrane organization can be one of the underlying mechanisms for regulating the endocytosis/exocytosis processes and membrane intracellular trafficking.

Differential response of the urothelial V-ATPase activity to the lipid environment.

The vesicle population beneath the apical plasma membrane of the most superficial urothelial cells is heterogeneous and their traffic and activity seems to be dependent on their membrane composition and inversely related to their development stage. Although the uroplakins, the major proteins of the highly differentiated urinary bladder umbrella cells, can maintain the bladder permeability barrier, the role of the membrane lipid composition still remains elusive. We have recently reported the lipid induced leakage of the vesicular content as a path of diversion in the degradative pathway. To extend the knowledge on how the lipid environment can affect vesicular acidification and membrane traffic through the regulation of the V-ATPase (vacuolar ATPase), we studied the proton translocation and ATP hydrolytic capacity of endocytic vesicles having different lipid composition obtained from rats fed with 18:1n-9 and 18:2n-6 fatty acid enriched diets. The proton translocation rate decreases while the enzymatic activity increases in oleic acid-rich vesicles (OAV), revealing an uncoupled state of V-ATPase complex which was further demonstrated by Western Blotting. A decrease of the very long fatty acyl chains length (C20-C24) and increase of the C16-C18 chains length in OAV membranes was observed, concomitant with increased hydrolytic activity of the V-ATPase. This response of the urothelial V-ATPase was similar to that of the Na-K ATPase when the activity of the latter was probed in reconstituted systems with lipids bearing different lengths of fatty acid chains. The studies describe for the first time a lipid composition-dependent activity of the urothelial V-ATPase, identified by immunofluorescence microscopy which is related to an effective coupling between the channel proton flux and ATP hydrolysis.

Pre-cancerous changes in urothelial endocytic vesicle leakage, fatty acid composition, and As and associated element concentrations after arsenic exposure.

The urothelium covering the luminal surface of the urinary bladder has developed an efficient permeability barrier that protects it against the back-flow of toxins eliminated in the urine. The subapical endocytic vesicles containing the urinary bladder fluid phase are formed during the micturition cycle by endocytosis processes of the superficial cells. In normal conditions, the permeability barrier of the endocytic vesicles blocks the passage of the fluid phase to the cellular cytoplasm and the fluid is recycled to the bladder lumen. The aim of this work was to investigate the alteration of the endocytic vesicle membrane permeability barrier to toxins such as iAs (inorganic arsenic) administered in drinking water. By using an induced endocytosis model and the fluorescence requenching technique, it is shown that the exposure of rats to ingestion of water containing iAs not only induced pre-cancerous morphological changes, but allowed the differential leakage of an endocytosed fluorescent marker, HPTS, and its quencher, DPX, (hydroxypyrene-1,3,6-trisulfonic acid and p-xylene-bis-pyridinium bromide, respectively) out of the vesicular lumen. The leakage of the cationic DPX was almost complete, while the release of the anionic HPTS molecule was partial and higher in arsenic-treated-rats than in controls. Such membrane alteration would allow the toxins to elude the permeability barrier and to leak out of the endocytic vesicles, thus establishing a "bypass" to the permeability barrier. The retention of As in the urinary bladder, assessed by synchrotron radiation X-ray fluorescence spectrometry (SR-µXRF), was lower than the kidney accumulation of arsenic previously observed by our group and was accompanied by altered concentrations of K, Ca, Fe, Cu and Zn, all ions related to cellular metabolism. The results support the hypothesis that low amounts of endocytosed As can accumulate in the interior of the urothelial superficial cells and initiate the cytotoxic effects reflected in the morphological alterations observed.

Dietary lipids modulate eicosanoid release and apoptosis of cells of a murine lung alveolar carcinoma.

Dietary arachidonic acid (AA) and eicosanoids influence neoplastic cell (NC) growth, differentiation and apoptosis. Plasma membrane fatty acid and cyclooxygenase (COX) and lipoxygenase (LOX) products were investigated in lung alveolar carcinoma cells from mice fed on different diets. Two groups were fed on a basic diet plus 6% of: corn oil (rich in 18:2n-6; CO) and on olein oil (rich in 18:1n-9; O), respectively. Control group (C) received commercial diet. NC fatty acids were analyzed by GLC, and apoptosis by flow cytometry and microscopy. In NC from CO group AA levels and LOX metabolites were increased, whereas COX metabolites decreased. NC from CO compared to O group diet showed a higher count of apoptosis and increased LOX:COX ratio. High levels of AA and decreased COX eicosanoids has been involved in anti-tumoral mechanisms by increasing tumor cell apoptosis. Present data emphasizes the implications of the dietary fatty acids on the neoplastic process in this tumoral model.

Fatty acids specifically related to the anisotropic properties of plasma membrane from rat urothelium.

Four different luminal surfaces of rat urothelium differing in their fatty acid composition were prepared by dietary induction. In order to induce lipid changes, each of four groups of rat received a basal diet rich in one of the unsaturated n-3, n-6 or n-9 fatty acid families and a commercial (control) diet. The effects of the dietary regime on the fatty acid composition of luminal urothelial membranes and their relation to the mobility of fluorescent probes were studied. In comparison with the control diet membrane, all three fatty acid-rich diets induced a decrease of the percentage amount of saturated fatty acid while that of the unsaturated fatty acids was increased. Accordingly, all three diets increased the unsaturation index in comparison with the control diet. The anisotropy across each membrane fraction was assessed using the n-(9-anthroyloxy) fatty acid fluorescent probes 3-AS, 7-AS and 12-AS, which locate at different depths in the membrane. Two different anisotropy profiles were observed. One profile showed the highest anisotropy at the C7 depth, whereas the other exhibited a continuous decrease of the anisotropy from the surface to the center of the bilayer. The molecular properties (isomerization) of 18:2n-9 fatty acid may account, at least in part, for the observed V-shaped profile (the ascending trend) of the membrane anisotropy values as a function of the respective 18:2n-9 fatty acid contents. Nevertheless, the minimum value of the profile did not correspond to the minimum 18:2n-9 fatty acid content, but rather to the higher amount of docosahexaenoic (22:6n-3) fatty acid. Thus, a modulating role of the 22:6n-3 fatty acid on the rigidifying effect of 18:2n-9 fatty acid is suggested, possibly mediated by relationships between fatty acid composition, saturated and unsaturated chain lengths, and freedom of motion of the phospholipid acyl chains.

Lipid and fatty acid composition of different fractions from rat urinary transitional epithelium.

The phospholipid composition of rat urinary transitional epithelium (TE) and the fatty acid composition of microsomal, mitochondrial, cytosolic, and plasma membrane (PM) subcellular fractions were investigated. PM marker enzymes and electron microscopy analysis were used to characterize the PM fraction, which showed a distinctive lipid composition compared to the general profile of PM from different sources. The levels of cholesterol and sphingomyelin were not enriched in the PM fraction; on the other hand, the increased amounts of glycosphingolipids and phosphatidylserine, and the decreased level of phosphatidylcholine followed the general features of a PM profile. This differential PM lipid composition may reflect the unique morphology of this mammal TE, consisting of concave plaques with an asymmetrical membrane unit. The distribution of the double bond across the PM indicated a higher unsaturation of the inner relative to the outer part of the PM hemileaflet. In addition, the presence of 20:3n-9 nonessential fatty acid in a normal TE may represent a characteristic fatty acid metabolism of this epithelium.

Surface behavior of myelin monolayers.

Myelin can be spread as a stable monomolecular layer, with reproducible properties, at the air-water interface. The major lipids and proteins of myelin are represented in this monolayer in molar ratios similar to those in the original membrane. A well-defined collapse point of the myelin monolayer occurs at ca. 46 mN/m. At a surface pressure of ca. 20 mN/m, the surface pressure-molecular area isotherm of the myelin monolayer shows a change in its compressibility, exhibited as a diffuse but reproducible inflection with a clearly marked change of the surface compressional modulus; the surface potential-area curve shows a change of slope at the same surface pressure. The myelin monolayer shows considerable hysteresis during the first compression-decompression cycle; no detectable protein unfolding under expansion; and decreased hysteresis after the first cycle. The average molecular areas, the inflection at 20 mN/m, the variation of the surface potential per unit of molecular surface density, and the hysteresis properties of the myelin monolayer indicate that this membrane undergoes changes of intermolecular organization mostly ascribed to the protein fraction, above a lateral surface pressure of ca. 20 mN/m. The behavior is consistent with a surface pressure-dependent relocation of protein components in the film. This has marked effects on the stability, molecular packing, and dipolar organization of the myelin interface.

Lipid composition and phospholipid asymmetry of membranes from a Schwann cell line.

We report the total lipid composition and phospholipid asymmetry of a plasma membrane preparation isolated from a Schwann cell line (NF1T) derived from a human neurofibroma. The specific activities of three plasma membrane markers (5'-nucleotidase, Na-K-ATPase, and CNPase) were 8-fold, 12-fold, and 16-fold higher, respectively, in the plasma membrane fraction compared to the specific activities found in the total homogenate. The specific activities of the marker enzymes of intracellular membranes in the isolated plasma membrane fraction indicated little contamination with intracellular organelles. The enrichment of cholesterol (3-fold), sphingomyelin (3-fold), and glycolipids (cerebrosides 8-fold, sulfatides 5-fold) also indicated a high degree of purity of the plasma membrane fraction. The high content of phosphatidylinositol and phosphatidylcholine (10% and 44% of total phospholipid) and the low phosphatidylserine and phosphatidylethanolamine content (3% and 14% of the total phospholipid) were also characteristic of the plasma membrane fraction derived from this cell line. The transbilayer phospholipid distribution of the plasma membrane in intact cells and in the isolated plasma membrane fraction was investigated by using phospholipase A2 (bee venom) and sphingomyelinase (S. aureus). The phospholipid asymmetry of NF1T plasma membrane followed the general features of phospholipid asymmetry in eukaryotic cells: sphingomyelin and phosphatidylcholine were preferentially located in the outer leaflet (90% and 89%, respectively) while the aminophospholipids phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were in the inner half of the membrane (85%, 96%, and 69%, respectively). A high percentage of the total plasma membrane phosphatidylinositol (31%) was found in the outer side of the membrane indicating a decreased asymmetric distribution for this negatively charged phospholipid. The phospholipid asymmetry found in the plasma membrane vesicle fraction corroborated the phospholipid asymmetry of the intact cells, thus confirming that the plasma membrane vesicles maintained the original orientation and lipid asymmetry after homogenization and/or sonication.

Ganglioside GD3 and GD3-lactone mediated regulation of the intermolecular organization in mixed monolayers with dipalmitoylphosphatidylcholine.

The interactions of dpPC with ganglioside GD3 and two lactones. GD3LacI or GD3LacII, in lipid monolayers occur with reduced, unaltered, or increased molecular area and surface potential/molecule, respectively. dpPC is fully miscible with GD3 and GD3LacI but films with GD3LacII show immiscibility above 75 mol% lactone. At low proportions of GD3 in mixtures with dpPC, GD3 undergoes condensation and depolarization; dpPC is depolarized and its molecular area is reduced above 50 mol% GD3. GD3LacI forms ideally mixed films with dpPC. Mixtures of dpPC with GD3LacII at mole fractions below 0.3 show increased mean molecular area and surface potential/molecule mostly due to lactone alterations. Between mole fractions of 0.3 and 0.75 the surface parameters of dpPC are altered, and above these proportions both lipids are immiscible. Defined variations of molecular properties induced by ganglioside lactonization are selectively transduced to changes of the intermolecular organization and surface electrostatics in mixed interfaces with dpPC. Thus, changes in the relative proportions of a ganglioside and its lactone forms may act as sensitive biotransducers for membrane-mediated cellular functions, without the need for metabolically altering the concentration of gangliosides.

Modulation of Schwann cell Po glycoprotein and galactocerebroside by the surface organization of axolemma.

The nature of the axon signal for the induction of proliferation and differentiation of peripheral glial cells is still unknown. Besides the existence of interactions among surface molecules the cellular responses can also be regulated by physicochemical parameters of the membrane. We have previously reported that planar axolemma monolayers coated on glass cover-slips at different defined surface molecular packing affected the Schwann cell (SC) morphology and their proliferative response (Calderon et al.: J Neurosci Res 34:206-218, 1993). In this paper we report that relative to SC cultured on uncoated coverslips, the level of expression of both glycoprotein Po and galactocerebroside (GC) (as revealed by immunofluorescence) was increased 2-4 times in SC cultured on axolemma monolayers with either high or low molecular packing. However, the cellular distribution of these antigens was profoundly influenced by the molecular packing density of the axolemma monolayer. SC cultured on an axolemma monolayer at high molecular packing showed preferential expression of Po at the SC surface whereas GC was concentrated intracellularly. On the other hand, SC grown on an axolemma monolayer at low molecular density GC showed preferential expression at the cell surface whereas Po was concentrated intracellularly.

Lipid composition of neuronal cell bodies and neurites from cultured dorsal root ganglia.

The lipid composition of neuronal somata and neuritic processes of cultured root ganglia has been determined. Neuronal soma contained 37% of dry weight as lipid (15.4% cholesterol, 4.8% galactolipid, and 57.1% phospholipid). The major phospholipids were phosphatidylcholine and phosphatidyl ethanolamine. Galactolipids consisted of cerebroside and sulfatide in molar ratio 2:1. The neuronal soma contained tetrasialo-, disialo-, and monosialoganglioside. In contrast, neurites contained 15% of the dry weight as lipid (22.1% cholesterol, 7.7% galactolipid with cerebroside and sulfatide in molar ratio 2:1, and 56.4% total phospholipid). The neuritic galactolipid content was higher, as was the percentage of sphingomyelin, and phosphatidyl serine. The higher cholesterol content in neuritic lipid reflected the higher percentage of plasma membrane in this compartment. The ganglioside pattern of neurites was distinct from that of the neuronal soma and consisted entirely of gangliosides GQ1b, GT1b, GD1b, GD1a, and GD3, with no monosialogangliosides. The results indicate a preferential phospholipid and glycolipid sorting to the neuritic plasma membrane that may be related to the distinctive functions of this neuronal compartment.

Mitochondrial schwannopathy and peripheral myelinopathy in a rabbit model of dideoxycytidine neurotoxicity.

BACKGROUND: The reverse transcriptase inhibitor, 2',3'-dideoxycytidine (ddC), causes a dose-limiting peripheral neuropathy in humans, the mechanism of which is unknown. Rabbits given ddC develop peripheral myelinopathy and axonopathy, but it has not been determined if either the myelin or axonal changes are primary or if they occur concurrently. EXPERIMENTAL DESIGN: To characterize sequential development of the ddC-induced neuropathy, 40 rabbits were given either vehicle or ddC by oral intubation at a dose of 35 mg/kg per day for 24 weeks. Electrophysiologic studies, pathologic examination of peripheral and central nervous system and skeletal muscle, and biochemical analysis of the sciatic nerve were performed at baseline (electrophysiology only) and after 8, 12, 16, 20, and 24 weeks of treatment. RESULTS: Neuropathologic changes in peripheral nerves were first evident at 16 weeks and were more pronounced at 20 and 24 weeks; onset of paresis occurred at week 20, whereas clear electrophysiologic deficits were seen only at week 24. Electrophysiologic changes were prolonged F-waves (measure of proximal motor conduction) and minor changes in distal conduction measurements. Pathologic changes included myelin splitting, intramyelinic edema, demyelination, and remyelination of the largest diameter nerve fibers in the ventral root and sciatic nerve. Axonal degeneration and reduction in axonal diameter were seen. Enlarged mitochondria with abnormal ultrastructure were present in Schwann cells of those animals with a myelinopathy. Mitochondrial abnormalities or other signs of degeneration were not seen in neurons of the dorsal root ganglia or in skeletal muscle. Significant changes were not present in myelin protein composition, myelin lipid composition, or activity of the myelin-specific enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase. Major reductions in levels of protein zero (P0, the homophilic adhesion protein of myelin) were not seen; however, the turnover rate of P0 was reduced as P0 messenger RNA expression in ddC-treated sciatic nerves decreased to 30 to 50% of control values. CONCLUSIONS: The peripheral neuropathy caused by ddC in rabbits is characterized as a myelinopathy of the proximal portion of the nerve fibers and as an axonopathy involving both proximal and distal fibers. The myelinopathy was associated with enlarged and abnormally shaped mitochondria in Schwann cells and is consistent with an effect of ddC on structure and function of Schwann cell mitochondria. Altered Schwann cell metabolism was evident by reduced levels of P0 messenger RNA, loss of homophilic myelin adhesion at the intraperiod line, and subsequent intramyelinic edema. Because axonal degeneration occurred concurrently with the myelin changes, it could not be determined if axonal changes were secondary to serve myelinic edema or if they represented a primary effect of ddC on neurons.

Surface behavior of axolemma monolayers: physico-chemical characterization and use as supported planar membranes for cultured Schwann cells.

The axolemma membrane forms a stable and reproducible monomolecular layer at the air-aqueous interface. The major lipids and proteins are present in this monolayer in molar ratios similar to the original membrane. Acetylcholinesterase and Na-K-ATPase activities are preserved in the monolayer to levels of 64% and 25%, respectively. The total lipid fraction forms a homogeneously mixed phase. The presence of proteins in the monolayer introduces surface inhomogeneties. Among other features, this is revealed by the presence of two values of lateral pressure at which the monolayer shows partial or total collapse: a broad partial collapse at surface pressures between 13 to 30 mN/m and a sharp collapse point at 46 mN/m. The average molecular areas, the broad collapse point, and the variation of the surface potential per molecule suggest the relocation of protein components at surface pressures between 13 to 30 mN/m. The behavior is consistent with the extrusion and exposure of proteins toward the aqueous medium that depends on the lateral pressure. Schwann cells grown on coverslips coated with axolemma monolayers at 13 mN/m (beginning of the broad collapse) and 34 mN/m (above the broad collapse) recognize the difference in the surface organization of axolemma caused by the lateral pressure which affects their proliferation, morphology, and spatial pattern of organization. Our results show for the first time that response of Schwann cells depends on the intermolecular organization of the axolemma surface with which they interact. These results suggest that the local expression of putative surface molecules of axolemma that may mediate membrane recognition and the signalling of morphological and proliferative changes can be modulated by long range supramolecular properties.

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential.

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.

Trypanosoma cruzi: involvement of proteolytic activity during cell fusion induced by epimastigote form.

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by 4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca2+-dependent while lysis is delayed in the absence of exogenous Ca2+. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.

Thermal denaturation of staphylococcal nuclease.

The fully reversible thermal denaturation of staphylococcal nuclease in the absence and presence of Ca2+ and/or thymidine 3',5'-diphosphate (pdTp) from pH 4 to 8 has been studied by high-sensitivity differential scanning calorimetry. In the absence of ligands, the denaturation is accompanied by an enthalpy change of 4.25 cal g-1 and an increase in specific heat of 0.134 cal K-1 g-1, both of which are usual values for small globular proteins. The temperature (tm) of maximal excess specific heat is 53.4 degrees C. Each of the ligands, Ca2+ and pdTp, by itself has important effects on the unfolding of the protein which are enhanced when both ligands are present. Addition of saturating concentrations of these ligands raises the denaturational enthalpy to 5.74 cal g-1 in the case of Ca2+ and to 6.72 cal g-1 in the case of pdTp. The ligands raise the tm by as much as 11 degrees C depending on ligand concentration. From the variation of the denaturational enthalpies with ligand concentrations, binding constants at 53 degrees C equal to 950 M-1 and 1.4 X 10(4) M-1 are estimated for Ca2+ and pdTp, respectively, and from the enthalpies at ligand saturation, binding enthalpies at 53 degrees C of -15.0 and -19.3 kcal mol-1.

Trypanosoma cruzi: fusogenic ability of membranes from cultured epimastigotes in interaction with human syncytiotrophoblast.

Trypanosoma cruzi epimastigotes cultured in vitro were disrupted by successive freezing and thawing and subsequent sonication. The total homogenate was fractionated by differential centrifugation to obtain an enriched plasma membrane fraction. The proteins of subcellular parasite fractions were labeled with 131I and their binding to membrane fractions from human placenta syncytiotrophoblast was studied. Syncytiotrophoblast fractions enriched in plasma showed higher specific activity for binding an enriched T. cruzi plasma membrane fraction compared with other fractions of syncytiotrophoblast. The properties of this interaction were studied with digestive enzymes (trypsin and phospholipase A2). The results showed that both proteins and lipids could be involved in this interaction. The Ca2+ requirements for the membrane-membrane interaction are different for the two membranes studied. Also the enriched plasma membrane T. cruzi fraction had a higher capacity to induce fusion processes than the other subcellular fractions. The above results indicate that a preferential syncytiotrophoblast-T. cruzi interaction may occur between the two cell surfaces as compared to intracellular membranes and that the parasite surface is able to induce an instability process leading to membrane fusion. These results may have implications in regard to the mechanism of entry of the parasite into cells.