Cerebroside Sulfate Research Articles

Croatian Population Data for Arylsulfatase A Pseudodeficiency-Associated Mutations in Healthy Subjects, and in Patients with Alzheimer-Type Dementia and Down Syndrome

Background Arylsulfatase A (ASA) is a lysosomal enzyme involved in catabolism of cerebroside sulfate, whose deficiency causes metachromatic leukodystrophy, a rare autosomal recessive disorder characterized by storage of cerebroside sulfate, mainly in the nervous system. Low ASA activities have also been reported in healthy individuals and several neuropsychiatric disorders due to the condition termed ASA pseudodeficiency. The aim of this study was to establish frequency of two mutations associated with ASA pseudodeficiency in healthy individuals in the Croatian population as well as in persons with Alzheimer-type dementia and Down syndrome. Methods Presence of N350S and 1524+95 A→G pseudodeficiency mutations was detected in genomic DNA extracted from leukocytes of healthy subjects ( n = 125) and of patients with Alzheimer-type dementia ( n = 18) and Down syndrome ( n = 21). Arylsulfatase A activity was measured in leukocyte homogenates by spectrophotometry (λ = 515 nm) using p-nitrocatechol sulfate as chromogenic substrate. Results Frequency of N350S mutation and mutation 1524+95 A→G was estimated at 6.8 and 2.8% for healthy controls, 11 and 5.5% for Alzheimer-type dementia, and 12 and 9.5% for Down syndrome, respectively. Arylsulfatase A activity was slightly but not significantly decreased in leukocytes derived from subjects with dementia and Down syndrome in comparison with age-matched control samples. Conclusions Frequency of two mutations associated with ASA pseudodeficiency in the Croatian population is slightly below the range reported for other populations. Additionally, despite the proposed role of arylsulfatase A pseudodeficiency as one of the predisposing factors for neuropsychiatric disorders, our preliminary results did not show significantly higher frequencies of either mutation in Alzheimer-type dementia or Down syndrome.

Isolated Peripheral Neuropathy in Atypical Metachromatic Leukodystrophy: A Recurrent Mutation

Metachromatic leukodystrophy (MLD) is a genetic neurodegenerative disorder resulting from a deficiency of arylsulfatase A. Late onset forms are relatively rare. Central nervous system (CNS) involvement is characteristic at all ages. A patient in her late 40s with peripheral neuropathy was assessed by EEG, evoked potentials, CT and nerve conduction studies. Nerve and muscle biopsy samples were investigated by electron microscopy. Arylsulfatase A activity in leukocytes and excreted cerebroside sulfate were determined. The arylsulfatase A gene was investigated for mutations using polymerase chain reaction (PCR) and DNA sequencing. The identified mutation was expressed transiently in African green monkey kidney (COS) cells to determine the effect of the mutation on arylsulfatase A activity. Central nervous system functions were normal. Nerve conduction velocities were decreased. Sural nerve biopsy showed inclusions typical of MLD. Arylsulfatase A was less than 5% of normal. A homozygous mutation thr286pro was identified in the arylsulfatase A gene and demonstrated to be deleterious through transient expression studies. Our patient has a progressive peripheral neuropathy but has apparently intact CNS function at her present age of 57 years. Biochemical, physiological and pathological findings are consistent with a diagnosis of MLD. A homozygous mutation, thr286pro, found in her arylsulfatase A gene, decreased enzyme activity to a level consistent with a late onset form of MLD.

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.

Neurophysiological Studies In Metachromatic Leukodistrophy

Metachromatic Leukodystrophy (MLD) is a lysosomal storage disease caused by deficiency of Arylsulfatase A (ASA) with accumulation of cerebroside sulfate in the white matter of central and peripheral nervous system. Late infantile, juvenile and adult forms have been described according to the age of onset and severity. Several studies demonstrated the diagnostic usefulness of neurophysiological tests in leukodystrophies, but their role in the progression of the disease and/or on the therapeutic monitoring is still to be established. Four patients with late juvenile MLD were studied by means of visual, auditory, somatosensory and motor evoked potentials to monitor central nervous system involvement and by means of motor and sensory conduction velocity to monitor peripheral nerve function. All neurophysiological tests demonstrated their ability in detecting abnormalities of the related neurological system. Abnormal findings of multimodal Evoked Potentials and sensory nerve conduction study consisted of disappearance of responses while motor nerve conduction study showed quantitative data which consisted of slowing of conduction velocity. As motor nerve fibers are relatively spared in MLD patients, motor nerve conduction studies would provide a simple method for evaluating the course of the disease and therapeutical efficacy. Unfortunately no neurophysiological tests are presently available for long‐term follow‐up of CNS damage in MLD.

Comparative lipid binding study on the cerebroside sulfate activator (saposin B).

Cerebroside sulfate activator (saposin B) is a small protein involved in glycosphingolipid metabolism. It binds certain membrane lipids, making them available to water-soluble enzymes. Defects in this protein are responsible for a form of metachromatic leukodystropy, a progressive neurodegenerative condition. The protein participates in the catabolism of a number of lipids but does show lipid binding selectivity. However, the basis of this selectivity is unclear. Here we assess the relative binding of a number of lipids compared to cerebroside sulfate (sulfatide). We utilize a competitive binding paradigm, in which the lipids compete for protein under favorable conditions and are then switched to a condition in which the complex is stable. This study is unique in that a single molecular species of the activator is employed, and an expanded selection of natural and semisynthetic membrane lipids is surveyed. No simple "binding rule" can be ascertained from these data, but ligands with longer and/or more complex lipoidal and polar adducts appear to be favored.

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein.

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.

Disulfide Connectivity in Cerebroside Sulfate Activator Is Not Necessary for Biological Activity or α-Helical Content but Is Necessary for Trypsin Resistance and Strong Ligand Binding

Cerebroside sulfate activator (CSAct) protein is exceptionally resistant to heat denaturation and proteolytic digestion. Although water soluble the protein binds membrane-associated lipids. Its biological role is thought to be to transfer certain lipids between membranes and to facilitate their catabolism in the lysosomes. An example of the latter is the removal of the sulfate group from cerebroside sulfate by arylsulfatase A. The mechanism of lipid sequestration from membranes and presentation of the lipid–protein complex to catabolic enzymes is a crucial aspect of the function of this protein. The widespread occurrence of the protein class of which CSAct is one of the best known members underscores the significance of this protein. The preparation, purification and chemical and biological properties of a stable disulfide blocked derivative of CSAct is described. The pyridoethylated protein was susceptible to tryptic attack and devoid of a significant population of solvent-protected exchange resistant protons. It apparantly formed a CS complex. However, unlike the complex with the native protein, this was not sufficiently stable to remain intact during size exclusion chromatography. The disulfide-blocked protein had a similar CD spectrum as native protein, indicating similar α-helical content. Unexpectedly, the activities of disulfide-blocked protein in the arylsulfatse A catalyzed sulfate hydrolysis from cerebroside sulfate were substantial. Hitherto, it had been assumed that the disulfide connectivities were essential for the protein to maintain a correctly folded configuration to bind lipid ligands and potentiate their hydrolysis. Some revision of our thoughts on the importance of the disulfide connectivities in the structure and function of the protein are necessary.

Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein.

Hydrogen-deuterium exchange can be a sensitive indicator of protein structural integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydrochloride plus dithiothreitol. Native protein has three internal disulfide bonds and treated protein has no internal disulfide bonds. The comparisons were made using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry, percentage alpha-helical content measured by circular dichroism and biological activity measured by the ability to support arylsulfatase A-catalyzed sulfate hydrolysis from cerebroside sulfate. In acidic solvent native protein has 59 exchange refractory protons and treated protein has 20 exchange refractory protons (44 and 14% of the exchangeable proton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature and the presence of a ligand. It is uninfluenced by the presence or absence of glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduced but not obliterated in treated protein. The hydrogen-deuterium exchange profile appears to be a sensitive signature of the correctly folded protein, and reflects a dimension of the protein structure that is not apparent in circular dichroic spectra or in the ability of the protein to support arylsulfatase A-catalyzed sulfate hydrolysis from sulfatide. The hydrogen-deuterium exchange profile will be a valuable criterion for characterizing mutant forms of CSAct produced by recombinant and synthetic paradigms and also the native and mutant forms of related proteins.

Trans Interactions between Galactosylceramide and Cerebroside Sulfate across Apposed Bilayers

The two glycosphingolipids galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS), are present at high concentrations in the multilayered myelin sheath and are involved in carbohydrate-carbohydrate interactions between the lipid headgroups. In order to study the structure of the complex of these two glycolipids by Fourier transform infrared (FTIR) spectroscopy, GalC dispersions were combined with CBS dispersions in the presence and absence of Ca 2+. The FTIR spectra indicated that a strong interaction occurred between these glycolipids even in the absence of Ca 2+. The interaction resulted in dehydration of the sulfate, changes in the intermolecular hydrogen bonding interactions of the sugar and other oxygens, decreased intermolecular hydrogen bonding of the amide C O of GalC and dehydration of the amide region of one or both of the lipids in the mixture, and disordering of the hydrocarbon chains of both lipids. The spectra also show that Ca 2+ interacts with the sulfate of CBS. Although they do not reveal which other groups of CBS and GalC interact with Ca 2+ or which groups participate in the interaction between the two lipids, they do show that the sulfate is not directly involved in interaction with GalC, since it can still bind to Ca 2+ in the mixture. The interaction between these two lipids could be either a lateral cis interaction in the same bilayer or a trans interaction between apposed bilayers. The type of interaction between the lipids, cis or trans, was investigated using fluorescent and spin-label probes and anti-glycolipid antibodies. The results confirmed a strong interaction between the GalC and the CBS microstructures. They suggested further that this interaction caused the CBS microstructures to be disrupted so that CBS formed a single bilayer around the GalC multilayered microstructures, thus sequestering GalC from the external aqueous phase. Thus the CBS and GalC interacted via a trans interaction across apposed bilayers, which resulted in dehydration of the headgroup and interface region of both lipid bilayers. The strong interaction between these lipids may be involved in stabilization of the myelin sheath.

The inhibition of complement-dependent hemolysis by liposomes containing cerebroside sulfate

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20-30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92-1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.

Preparation of the Cerebroside Sulfate Activator (CSAct or Saposin B) from Human Urine

The purification of cerebroside sulfate activator (CSAct) or saposin B from pooled human urine is described. Urinary proteins are concentrated by ammonium sulfate precipitation. A suspension of the precipitate is heat-treated and the heat-stable proteins are fractionated through a series of chromatographic steps. An initial concanavalin A column retains little of the CSAct activity, but is important for subsequent purification. Passing the Con A effluent directly onto an octyl Sepharose column removes the protein of interest which is recovered by affinity elution with octyl glucoside. Subsequent ion-exchange and gel filtration chromatographies yield a protein of 80–90% purity, although it is sometimes necessary to repeat one or more steps. A small amount of CSAct can sometimes be recovered from the initial Con A Sepharose column by methyl mannoside elution and purified by a parallel chromatographic protocol. Mass spectral analysis suggests that the final material is a mixture of two major and several minor glycoforms of a 79 amino acid protein with the structure predicted from the human prosaposin cDNA by truncation of both N- and C-terminal regions. Sugar analysis revealed the presence of glucosamine, mannose, and fucose, consistent with the major isoforms bearing a five-sugar Man2GluNac2Fuc or a single GluNac substituent. The human urinary material is similar to the previously characterized pig kidney protein in most respects, but varies in some details.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

Divalent Cation-Mediated Interaction Between Cerebroside Sulfate and Cerebrosides: An Investigation of the Effect of Structural Variations of Lipids by Electrospray Ionization Mass Spectrometry

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the α-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.

Binding of human cytomegalovirus to sulfated glucuronyl glycosphingolipids and their inhibitory effects on the infection.

Interactions between human cytomegalovirus (HCMV) and various carbohydrate structures were analysed using sulfated glucuronyl glycosphingolipids (SGGLs) and the structurally related glycosphingolipids (GLs). A thin-layer chromatography-overlay assay and a solid-phase binding assay revealed that HCMV strongly bound to sulfated glucuronyl lactosaminylparagloboside, one of the SGGLs having the repeating lactosamine structure (3Gal beta1-4GlcNAc1-)2 in addition to the 3-O-sulfated glucuronyl moiety. The virus bound less strongly to other 3-O-sulfated GLs, which included sulfated glucuronyl paragloboside and cerebroside sulfate ester, and also to (3Gal beta1-4GlcNAc1-)2-containing GLs that included nLc6Cer. Thus, a (3Gal beta1-4GlcNAc1-)2 and a 3-O-sulfated saccharide seem to be important structures for the binding by HCMV. When virus particles were preincubated with these GLs, inhibitory effects were observed both on expression of the viral immediate-early gene and on plaque formation by HCMV. These effects were very well correlated with the abilities of the GLs to bind to the virus. Pretreatment of host cells with HNK-1 monoclonal antibody, which specifically recognizes SGGLs, resulted in partial inhibition of plaque formation by HCMV. These results clearly show that HCMV recognizes and binds to the sulfated carbohydrate structure in SGGL and also suggest that binding of HCMV to the specific sugar structure may play an important role in HCMV infection.

Metachromatic leukodystrophy: molecular genetics and an animal model.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA; EC 3.1.6.8). Deficiency of this enzyme causes intralysosomal storage of the sphingolipid cerebroside sulphate. This lipid is abundant in myelin and it may thus not be surprising that storage mainly affects oligodendrocytes. Patients suffer from a progressive demyelination causing various neurological symptoms. The disease is fatal and treatment is not available. The human ASA gene has been cloned and more than 40 mutations have been analysed that cause metachromatic leukodystrophy. Few of these alleles are frequent among patients, whereas most mutant alleles have only been found in single families. Since MLD has only been described in humans and no naturally occurring animal model has been described, ASA-deficient mice have been generated by homologous recombination. The ASA knockout mice are unable to degrade sulphatide and store the lipid intralysosomally. The pattern of lipid storage in neuronal and non-neuronal tissues resembles that described for patients. In the nervous system, lipid storage is found in oligodendrocytes, astrocytes and some neurons. Animals display an astrogliosis and a decreased average axonal diameter. Purkinje cells and Bergmann glia of the cerebellum are morphologically aberrant. Demyelination is seen in the acoustic ganglion and occurs between the ages of 6 and 12 months. The animals are deaf at this age and display various neuromotor abnormalities. However, compared to humans the mice have a surprisingly mild phenotype, since they have a normal life span and do not develop widespread demyelination. ASA-deficient mice have been transplanted with bone marrow, which was transduced with a retroviral vector expressing arylsulphatase A. The majority of transplanted animals display sustained expression of arylsulphatase A from the retroviral construct up to 5 months after transplantation. However, preliminary data suggest that this therapeutic approach does not reduce storage material.

Porcine Cerebroside Sulfate Activator (Saposin B) Secondary Structure: CD, FTIR, and NMR Studies

Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical–chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of α-helical conformation and little if any β-sheet. Circular dichroism (CD) studies indicate 45–50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85°C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.

Depressed autoantibody synthesis in Trypanosoma cruzi-infected rats born to mothers undergoing this infection during pregnancy

Earlier work indicated that Trypanosoma cruzi infection in pregnant rats decreased the amount of myocardial damage that developed in their chronically infected offspring. Given the suspected role of autoimmune mechanisms in the generation of chronic myocarditis, we evaluated whether this maternal intervention was likely to affect the synthesis of autoantibodies in infected young. Autoantibodies were investigated against molecules exhibiting cross-reactivity with T. cruzi antigens or not, that is cerebroside sulphate (sulphatide) and actin, respectively. Female `l' rats (75 days old) that had been mated with syngeneic sires were separated into two groups, one challenged with living trypomastigotes at 7, 14 and 21 days following mating, and the other one given physiologic saline at the same intervals. At the time of weaning, offspring were injected with 10 6/ T. cruzi to constitute two infected groups: young born to infected mothers (InMoTc) and young delivered by uninfected mothers (CoMoTc). Serum antibodies were investigated by ELISA at 30 and 60 days post-infection, which represents acute and chronic infection, respectively. T. cruzi infection was associated with the production of anti-sulphatide antibodies, but the phenomenon was significantly less evident in InMoTc young and virtually unnoticeable during their chronic infection. Unlike the anti-sulphatide results, levels of anti-actin antibodies showed no differences between CoMoTc and InMoTc rats when compared during acute or chronic infection. The decreased production of anti-sulphatide autoantibodies of InMoTc offspring may be due to a modification of the immune repertoire of offspring because of the contact with parasite antigens during ontogeny.

Characterization of the interaction of Ca2+ with hydroxy and non-hydroxy fatty acid species of cerebroside sulfate by Fourier transform infrared spectroscopy and molecular modeling.

Ca2+-mediated interactions between the carbohydrate groups of glycolipids, including that of cerebroside sulfate (galactosylceramide I3-sulfate), have recently been implicated as a basis of cell recognition and adhesion. Hydroxylation of the fatty acid of this lipid has an effect on these interactions. Therefore, FT-IR spectroscopy was used to study the interaction of Ca2+ with semisynthetic hydroxy (HFA) and non-hydroxy fatty acid (NFA) species of cerebroside sulfate (CBS). Ca2+ caused partial dehydration of the sulfate group and reduced hydrogen bonding of the sugar hydroxyls of both species. The amide I and II bands of the lipids in the absence of Ca2+ (NH4+ salt forms) suggested that the N-H of the HFA species is involved in a bent intramolecular hydrogen bond, probably with the fatty acid hydroxyl group and the glycosidic oxygen, while that of the NFA species is involved in a linear intermolecular hydrogen bond with the C=O and/or other oxygens. Ca2+ caused a rearrangement of the hydrogen-bonding network in the interfacial region of the HFA species involving the amide group. The results suggested increased hydrogen bonding of the C=O and a shift in hydrogen bonding of the N-H of the Ca2+ salt form of the HFA species from a bent intramolecular hydrogen bond to a linear intermolecular hydrogen bond, probably with the C=O of neighboring molecules, similar to the NFA species. The involvement of the fatty acid alpha-hydroxyl group in the rearranged network was indicated by a reduction in mobility of the alpha-CH group of the HFA species, in contrast to that of the NFA species. Participation of the alpha-OH group in hydrogen-bonding networks in the interfacial region of both the NH4+ and Ca2+ salt forms caused a significant increase in the interchain packing, as evident from correlation field splitting of the HFA-CBS methylene scissoring mode, while this did not occur for the NFA species. The absence of intramolecular hydrogen bonding of the N-H with the glycosidic oxygen for both salt forms of the NFA species and for the Ca2+ salt form of the HFA species may destabilize the "bent shovel", bilayer planar conformation of the sugar and cause it to be in the extended, bilayer perpendicular conformation. Calculations of the three-dimensional interaction energy of Ca2+ with CBS showed strong binding around the sulfate and the surface of galactose facing the bilayer in the bent shovel conformation. Ca2+ binding at this surface would disrupt intra- and intermolecular hydrogen-bonding interactions of the head group, thus accounting for its effect in inducing a transition to the extended conformation.

Opioid receptor affinity of multivalent ligand system consisting of polymerized liposome.

A multivalent ligand system, in which enkephalin/phospholipid conjugates were immobilized on a polymerized liposome, has been prepared. A hydrophilic spacer chain, -(Sar-Sar-Pro)2-, was placed between Tyr-D-Ala-Gly-Trp-Leu (an enkephalin part) and a phospholipid part to diminish steric hindrance of the liposome against receptor binding of the enkephalin unit. The affinity of the immobilized enkephalin conjugate for opioid receptor was dependent on the density of the enkephalin unit on the surface of polymerized liposome, indicating that more than two enkephalin units on a liposome simultaneously bind to receptors in the membrane. IC50 values for the delta- and mu-receptors were 15 and 26 nM, respectively, when the conjugate was immobilized at the molar ratio of [enkephalin]/[phospholipid] of 340. The latter affinity is better than that of Tyr-D-Ala-Gly-Trp-Leu (50 nM). The enkephalin part was located at the membrane surface, as indicated by fluorescence spectroscopy. When 12% cerebroside sulfate was mixed in the polymerized liposome, delta-receptor affinity of the immobilized enkephalin conjugate was improved to 7.4 nM. The environment around the enkephalin part became hydrophilic upon incorporation of cerebroside sulfate into the polymerized liposome, resulting in changes in the receptor affinities.