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A simple procedure is described for preparing lyso-GM1, a GM1 derivative that lacks the fatty acid moiety, starting from GM1 ganglioside using a one-pot reaction. Ganglioside deacylation was carried out in KOH/propan-1-ol in the absence of oxygen. The yield of lyso-GM1 under optimal conditions (6 h, 90 degrees C, 0.2 N KOH, 1 mM GM1) was 54%. The chemical structure of lyso-GM1 was determined by 1H-NMR and FAB-MS analyses, thus proving that the acetamide groups of galactosamine and sialic acid units were not affected during the deacylation reaction.

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.

GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively.

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition. In particular, long chain bases were submitted to gas-liquid chromatographic-mass spectrometric analyses as their trimethylsilyl (TMS) or N-acetyl-TMS derivatives, and chain length, presence or absence of C4-C5 double bond, and C-3 steric configuration were ascertained. The final preparations of individual molecular species of GM1 and GD1a gangliosides were more than 99% homogeneous in their saccharide moiety, contained a single long chain base (homogeneity higher than 99%), and had a fatty acid composition primarily of stearic acid (92 to 97%). All the individual molecular species of GM1 and GD1a gangliosides were also prepared in radioactive form by selective tritiation at C-3 of the long chain base. Their specific radioactivity ranged from 1.3 to 1.45 Ci/mmol. The availability of these molecular species of gangliosides is expected to facilitate studies aimed at ascertaining the role played by the hydrophobic portion in the functional behavior of gangliosides.

Headache is a common condition with a substantial burden of disease worldwide. Concerns have been raised over the potential impact of long-term mobile phone use on headache due to radiofrequency electromagnetic fields (RF-EMFs). We explored prospectively the association between mobile phone use at baseline (2009-2012) and headache at follow-up (2015-2018) by analysing pooled data consisting of the Dutch and UK cohorts of the Cohort Study of Mobile Phone Use and Health (COSMOS) (N = 78,437). Frequency of headache, migraine, and information on mobile phone use, including use of hands-free devices and frequency of texting, were self-reported. We collected objective operator data to obtain regression calibrated estimates of voice call duration. In the model mutually adjusted for call-time and text messaging, participants in the high category of call-time showed an adjusted odds ratio (OR) of 1.04 (95 % CI: 0.94-1.15), with no clear trend of reporting headache with increasing call-time. However, we found an increased risk of weekly headache (OR = 1.40, 95 % CI: 1.25-1.56) in the high category of text messaging, with a clear increase in reporting headache with increasing texting. Due to the negligible exposure to RF-EMFs from texting, our results suggest that mechanisms other than RF-EMFs are responsible for the increased risk of headache that we found among mobile phone users., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Maternal and paternal occupational exposure to pesticides was linked to leukemia in the offspring in some previous studies. Risks for other cancers, particularly from maternal exposure, are largely unknown. We examined the association between maternal and paternal exposure to pesticides and childhood cancer in a Swedish register-based case-control study (1960-2015). Cancer cases <20 years old were identified from the Cancer Register (n = 17313) and matched to controls (1:25) on birth year and sex. Employment history of each biological parent around the child's birth was retrieved from six censuses and a nationwide register, and exposure to any of herbicides, insecticides, and fungicides was evaluated using the Swedish job-exposure matrix (SWEJEM) in 9653/172194 mothers and 12521/274434 fathers of cases/controls. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated from conditional logistic regression models for any cancer, leukemia, lymphoma, central nervous system [CNS], and other solid tumors. We found an OR of 1.42 (95% CI 0.78, 2.57; 12 exposed cases) for lymphoma and 1.30 (95% CI 0.88, 1.93; 27 exposed cases) for other solid tumors associated with maternal occupational exposure to pesticides. No associations were observed between maternal exposure and leukemia or CNS tumors, or paternal exposure and any of the cancers examined, except for a potential association between pesticides exposure and myeloid leukemia (OR 1.15 [95% CI 0.73, 1.79; 22 exposed cases]). Although these findings merit further investigation, they indicate that parental exposure to pesticides may lead to higher risks of childhood cancer even in settings of low exposure., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The innate immune response of insects provides a robust line of defense against pathogenic microbes and eukaryotic parasites. It consists of two types of overlapping immune responses, named humoral and cellular, which share protective molecules and regulatory mechanisms that closely coordinate to prevent the spread and replication of pathogens within the compromised insect hemocoel. The major feature of the humoral part of the insect immune system involves the production and secretion of antimicrobial peptides from the fat body, which is considered analogous to adipose tissue and liver in vertebrates. Previous research has identified and characterized the nature of antimicrobial peptides that are directed against various targets during the different stages of infection. Here we review this information focusing mostly on the diversity and mode of action of these host defense components, and their critical contribution to maintaining host homeostasis. Extending this knowledge is paramount for understanding the evolution of innate immune function and the physiological balance required to provide sufficient protection to the host against external enemies while avoiding overactivation signaling events that would severely undermine physiological stability., (Copyright © 2018. Published by Elsevier B.V.)

Background: Hypoxia is a common feature of many congenital heart defects (CHDs) and significantly contributes to their pathophysiology. Thus, understanding the mechanism underlying cell response to hypoxia is vital for the development of novel therapeutic strategies. Certainly, the hypoxia inducible factor (HIF) has been extensively investigated and it is now recognized as the master regulator of cell defense machinery counteracting hypoxic stress. Along this line, we recently discovered and reported a novel mechanism of HIF activation, which is mediated by sialidase NEU3. Thus, aim of this study was to test whether NEU3 played any role in the cardiac cell response to chronic hypoxia in congenital cyanotic patients., Methods: Right atrial appendage biopsies were obtained from pediatric patients with cyanotic/non-cyanotic CHDs and processed to obtain mRNA and proteins. Real-Time PCR and Western Blot were performed to analyze HIF-1α and its downstream targets expression, NEU3 expression, and the NEU3 mediated effects on the EGFR signaling cascade., Results: Cyanotic patients showed increased levels of HIF-1α, NEU3, EGFR and their downstream targets, as compared to acyanotic controls. The same patients were also characterized by increased phosphorylation of the EGFR signaling cascade proteins. Moreover, we found that HIF-1α expression levels positively correlated with those recorded for NEU3 in both cyanotic and control patients., Conclusions: Sialidase NEU3 plays a central role in activating cell response to chronic hypoxia inducing the up-regulation of HIF-1α, and this represent a possible novel tool to treat several CHD pathologies., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Tobacco metabolites and carcinogens can be found in placental and umbilical cord tissues of fetuses exposed to maternal smoking. However, studies regarding maternal smoking during pregnancy and childhood brain tumor (CBT) have shown inconsistent results., Methods: All children born in Sweden between 1983 and 2010 and with information about maternal smoking during pregnancy, obtained from the Swedish Medical Birth Register, were included in this population based cohort study (n=2,577,305). CBT cases were identified from the National Cancer Register. Cox regression models were used to estimate the effect of maternal smoking during pregnancy on the risk of CBTs., Results: We identified 1039 cases of CBT in the cohort. Overall, there was little or no effect of maternal smoking during pregnancy on the risk of CBTs. However, in analyses stratified by age at diagnosis and child's sex, positive associations were found among 5-9 years old children. In this age interval, maternal smoking during pregnancy was associated with an increased risk of all CBTs combined only among male children (RR=1.50, 95% CI 0.96-2.34), while for astrocytoma there was a positive association in both male (RR=2.00, 95% CI 1.02-3.91) and female children (RR=1.80, 95% CI 0.85-3.82)., Conclusion: Results from this large Swedish cohort study suggest that even though maternal smoking during pregnancy has a limited overall effect on CBTs, it may increase the risk of astrocytomas., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Little is known about the aetiology of childhood brain tumours. We investigated anthropometric factors (birth weight, length, maternal age), birth characteristics (e.g. vacuum extraction, preterm delivery, birth order) and exposures during pregnancy (e.g. maternal: smoking, working, dietary supplement intake) in relation to risk of brain tumour diagnosis among 7-19 year olds. The multinational case-control study in Denmark, Sweden, Norway and Switzerland (CEFALO) included interviews with 352 (participation rate=83.2%) eligible cases and 646 (71.1%) population-based controls. Interview data were complemented with data from birth registries and validated by assessing agreement (Cohen's Kappa). We used conditional logistic regression models matched on age, sex and geographical region (adjusted for maternal age and parental education) to explore associations between birth factors and childhood brain tumour risk. Agreement between interview and birth registry data ranged from moderate (Kappa=0.54; worked during pregnancy) to almost perfect (Kappa=0.98; birth weight). Neither anthropogenic factors nor birth characteristics were associated with childhood brain tumour risk. Maternal vitamin intake during pregnancy was indicative of a protective effect (OR 0.75, 95%-CI: 0.56-1.01). No association was seen for maternal smoking during pregnancy or working during pregnancy. We found little evidence that the considered birth factors were related to brain tumour risk among children and adolescents., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.

Pax3/7 paired homeodomain transcription factors are important markers of muscle stem cells. Pax3 is required upstream of myod for lateral dermomyotomal cells in the amniote somite to form particular muscle cells. Later Pax3/7-dependent cells generate satellite cells and most body muscle. Here we analyse early myogenesis from, and regulation of, a population of Pax3-expressing dermomyotome-like cells in the zebrafish. Zebrafish pax3 is widely expressed in the lateral somite and, along with pax7, becomes restricted anteriorly and then to the external cells on the lateral somite surface. Midline-derived Hedgehog signals appear to act directly on lateral somite cells to repress Pax3/7. Both Hedgehog and Fgf8, signals that induce muscle formation within the somite, suppress Pax3/7 and promote expression of myogenic regulatory factors (MRFs) myf5 and myod in specific muscle precursor cell populations. Loss of MRF function leads to loss of myogenesis by specific populations of muscle fibres, with parallel up-regulation of Pax3/7. Myod is required for lateral fast muscle differentiation from pax3-expressing cells. In contrast, either Myf5 or Myod is sufficient to promote slow muscle formation from adaxial cells. Thus, myogenic signals act to drive somite cells to a myogenic fate through up-regulation of distinct combinations of MRFs. Our data show that the relationship between Pax3/7 genes and myogenesis is evolutionarily ancient, but that changes in the MRF targets for particular signals contribute to myogenic differences between species.

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.

The behavior of the 2 sialidase forms present in the erythrocyte membrane was investigated in 117 subjects with type 2 diabetes mellitus versus 95 healthy controls. A significant increase of the acidic form of sialidase, which is anchored to the membrane by a glycosylphosphatidylinositol bridge, was observed in erythrocyte resealed membranes. On the contrary, the neutral form of the enzyme, the only one capable of removing lipid- and protein-bound sialic acid from endogenous and exogenous sialoderivatives, was significantly reduced with a consequent increase of erythrocyte membrane total sialic acid content. Disease duration, therapy, glycemia, parameters of metabolic control, and presence of complications, except nephropathies, had no influence on the tested enzyme activities. Diabetic subjects showed a different erythrocyte age distribution, with an almost double proportion of young red cells and only one quarter of senescent ones compared with controls. In young erythrocytes, diabetic and control subjects had the same distribution of the 2 enzymes, while in senescent cells the acidic enzyme was increased 3.5-fold and the neutral form was reduced by half in the diabetic subjects. The increase of both acidic sialidase and total membrane-bound sialic acid, together with an overpresence of young red cells in diabetics, suggests that in this pathological condition there might be an altered aging process with a diminished expression of the neutral form of the enzyme and an increase of bound sialic acid. It has been suggested that the expression of the neutral enzyme requires some activation mechanism that is impaired in diabetes.

We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.

The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37 degrees C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.

The metabolic processing of sialoglycolipids (gangliosides) was investigated in cultures of skin fibroblasts obtained from two patients affected with Salla disease. Cultured fibroblasts were fed with GM1 ganglioside [3H]-radiolabelled at the sialic acid ([NeuAc-3H]GM1) or sphingosine ([Sph-3H]GM1) moiety. Formation of metabolites was followed in pulse-chase experiments. It was observed that: (a) Salla fibroblasts, fed with [NeuAc-3H]GM1 accumulate radioactive free sialic acid in the lysosomal compartment and show a much lower sialic acid re-cycling for biosynthetic purposes than control fibroblasts, as demonstrated by decreased incorporation of the label into glycolipids and glycoproteins; (b) Salla fibroblasts, fed with [NeuAc-3H]GM1 or [Sph-3H]GM1, tend to accumulate gangliosides GM2 and GM3, and to reduce the breakdown products following the desialosylation step, presumably as a consequence of the inhibition of sialidase by free sialic acid; (c) owing to (b) the basal production of the bioregulators of sphingoid nature, ceramide and sphingosine, is reduced, as well as re-cycling of these substances for biosynthetic purposes, with further reduction of the turnover rate of sphingolipids. The decreased turnover rate of sialoglycoconjugates and sphingolipids, together with the diminished formation of bioregulators of sphingoid nature, may play a relevant role in the pathogenesis of the disease.

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

An in vitro test was set up to assess the release of lysosomal enzymes from cells and the effect on this process of the commonly used preservatives, parabens. Human peripheral lymphocytes, cultivated in vitro for 24 h in the presence or absence of phytohaemagglutinin (PHA; 5 mg/l), were used. After 1 day of incubation, PHA treatment caused an increased release (from 220 to 500%) of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, alpha-L-fucosidase and alpha-D-galactosidase. This enhancement was analytically reliable, and detectable with 1-5 micrograms of cell protein. Leakage of lactate dehydrogenase (LDH) underwent only a 20% increase on PHA treatment, indicating that the increased release of lysosomal enzymes was presumably due to secretion, not to cell damage. In PHA-stimulated lymphocytes, methyl-, ethyl-, propyl- and butyl-parabens caused a concentration-dependent diminution of the secretion of lysosomal enzymes. Butyl-paraben appeared to be the most potent inhibitor, causing a 45-50% inhibition at 0.06 mmol/l. With the other parabens, the inhibitory effect became statistically significant at about 0.25 mmol/l for alpha-L-fucosidase and alpha-D-galactosidase, and at 0.5 mmol/l for N-acetyl-beta-D-glucosaminidase and beta-D-glucuronidase. At 1 mmol/l inhibition was greater than 50% for all the enzymes and was more marked with the propyl derivative. Parabens did not influence the release of LDH, suggesting that they affected particularly the secretion of lysosomal enzymes. This supports the hypothesis that parabens are capable of affecting cellular function at concentrations which are likely to be reached in blood or tissues under conditions of common use.

We have adapted for routine analysis a pre-existing method for separating the three major N-acetyl-beta-D-glucosaminidase (NAG) isoenzyme forms--A, B+I1 and I2--by chromatofocusing followed by fluorimetric assay of the enzyme activity. This method combines good resolution, accurate quantification of the different isoenzymes and high reproducibility with an acceptable degree of analytical precision. We have applied it to studying the isoenzyme levels in the plasma of a general population of 417 subjects and have analysed these enzyme activities as functions of age, sex, body mass and declared alcohol consumption. Unlike the levels of unfractionated enzyme, levels of all the isoenzymes were higher in men than in women at all ages except in the 20-29 year group. Isoenzyme I2 showed the greatest sex difference. On the whole, with increasing age, both sexes showed more or less regular increases in plasma levels of all the isoenzymes. We also found significant correlations for the population as a whole with age and with body mass index. The only significant correlation with alcohol consumption was for B+I1 in men.

The levels of some enzymes of lysosomal origin were assayed during days 2 and 5 of life in plasma from 11 sets of twin neonates and from 25 neonates from single pregnancies (13 of weight appropriate for gestational age and 12 small for their gestational age) as controls. The plasma enzyme levels were also determined in the correspondent twin and control mothers 2 days after delivery. N-Acetyl-beta-D-glucosaminidase isoenzymes were assayed after chromatofocusing separation. All the plasma enzyme levels were higher in the group of twin neonates and of their mothers than in the respective control groups with differences highly statistically significant for two enzymes, beta-D-galactosidase and alpha-D-glucosidase. In neonate plasma lysosomal enzymes are increased at the fifth day of life with respect to the second day. Full term control neonates showed the same enzyme trend. For the N-acetyl-beta-D-glucosaminidase the more significant differences concerned the isoenzyme I2-P (pregnancy). The pattern of the lysosomal enzymes in the twins resembled that of neonates of diabetic mothers who had had no insulin therapy. Since lysosomal enzymes are considered to be particularly sensitive indicators of carbohydrate metabolism abnormalities, we conclude that twin pregnancies are more at risk for these abnormalities than single ones.

A simple procedure is described for preparing lyso-GM1, a GM1 derivative that lacks the fatty acid moiety, starting from GM1 ganglioside using a one-pot reaction. Ganglioside deacylation was carried out in KOH/propan-1-ol in the absence of oxygen. The yield of lyso-GM1 under optimal conditions (6 h, 90 degrees C, 0.2 N KOH, 1 mM GM1) was 54%. The chemical structure of lyso-GM1 was determined by 1H-NMR and FAB-MS analyses, thus proving that the acetamide groups of galactosamine and sialic acid units were not affected during the deacylation reaction.

The antigen designated as Chol-1 beta, detected by an antiserum specific for cholinergic neurons, has been purified to homogeneity from ganglioside mixtures extracted from Torpedo electric organ and pig brain. The final products from the two sources behaved identically in a wide range of tests and gave coincident immunopositive and Ehrlich-positive spots after thin layer chromatography in seven different solvent systems; they were thus considered to be identical and to constitute a single, pure chemical species. Gas-chromatographic analysis revealed the presence of long-chain bases, glucose, galactose, N-acetylgalactosamine, and sialic acid in integral molar ratios of 1:1:2:1:3; the compound's reactivity to cholera toxin after Vibrio cholerae sialidase treatment on thin layer chromatography and the recovery of GM1 as sole product of exhaustive sialidase treatment identified it as a member of the gangliotetrahexosyl series. From the products of partial enzymatic desialylation and treatment with beta-galactosidase and a comparison of the compound's immunoreactivity to anti-Chol-1 antisera with that of other trisialogangliosides of defined molecular structure, we were able to assign a disialosyl residue alpha-Neu5Ac-(2----8)-alpha-Neu5Ac-(2----3)- to the inner galactose, and we suggest GalNAc as a possible site of linkage of the third sialic acid.

beta-D-N-Acetylglucosaminidase isozymes were separated and assayed in the plasma of control healthy individuals and pregnant women by an automated method consisting in chromatofocusing on polybuffer exchanger PBE-94 column, flow-through fluorimetric determination of activity and computer assisted quantification. Under the established optimal conditions the method fractionated beta-D-N-acetylglucosaminidase into four isozymes. A, I2, I1 and B, with the analytical coefficients of variation of 1.8, 2.2, 6.4 and 4.1%, respectively. Duration of a single analysis was 25 min including washing, and 10-15 successive runs could be performed on the same column with good reproducibility. A linear activity response was observed from 1-5 microliters of plasma (depending on the individual isozyme) to 50 microliters, and the detection limit was 0.016 mUnits. Isozyme A was heat labile. Upon sialidase treatment, isozymes A, I2 and I1 released sialic acid and were eluted from the column at less acidic pHs. In healthy individuals isozymes A, I2, I1 and B covered about 62.8, 6.9, 15.0 and 15.1% of the total beta-D-N-acetyl-glucosaminidase activity, respectively. During pregnancy the plasma concentration of all isozymes increased. Isozyme I2 showed the highest enhancement (30-fold), followed by I1 (8-fold), B (5.6-fold) and A (3-fold). Interruption of pregnancy by either physiological delivery or ambulatory abortion was followed by a sharp fall of the concentration of all isozymes reaching, in a few days, the control levels.

The optimal assay conditions and the stability of the following enzymes of lysosomal origin in human cerebrospinal fluid (CSF) were studied: acid phosphatase, beta-D-N-acetylglucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase, alpha-L-fucosidase, alpha-D-mannosidase, beta-D-glucuronidase. The microsomal alpha-D-mannosidase, pH 5.7, was used as a reference non-lysosomal glycohydrolase. All the examined enzymes, with the only exception of beta-D-glucuronidase, underwent a more or less rapid loss of activity upon CSF storage in the temperature range from 37 degrees C to -80 degrees C. Storage in liquid nitrogen (-196 degrees C) was the only condition in which full activity for all tested enzymes was maintained for at least 15 days. Addition of human serum albumin to CSF, immediately after withdrawal, had a double effect in favouring enzyme stabilization and causing enzyme activation in some cases, and enzyme inhibition in others. Using conditions warranting enzyme stability the fluorimetric methods for lysosomal enzymes determination in cerebrospinal fluid appear to be highly reproducible (CV less than 5%) and simple enough for routine use.

A simple method for recognition and quantification of alkali-labile gangliosides is described. The method was worked out using authentic alkali-labile gangliosides in pure form (9-O-Ac-GT1b; 9-O-Ac-GQ 1b; lactone form of GD 1b) and applied to ganglioside mixtures from the brain of mouse, rat, rabbit, pig, and pigeon. The method consists of two-dimensional thin-layer chromatography on silica gel high-performance thin-layer chromatography plates employing the same solvent, chloroform/methanol/0.2% aqueous CaCl2, 50/40/10, for both runs. Prior to the second run the plate is exposed at room temperature for 5 h to ammonia vapors in order to split alkali-labile linkages. At the end of chromatography alkali-stable gangliosides appear lined along a diagonal starting from the origin; the spots corresponding to alkali-labile gangliosides lie out of the diagonal and can be individually detected and quantified on the basis of their sialic acid content. Up to 15 different spots, corresponding to as many alkali-labile gangliosides, can be recognized by this procedure.

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition. In particular, long chain bases were submitted to gas-liquid chromatographic-mass spectrometric analyses as their trimethylsilyl (TMS) or N-acetyl-TMS derivatives, and chain length, presence or absence of C4-C5 double bond, and C-3 steric configuration were ascertained. The final preparations of individual molecular species of GM1 and GD1a gangliosides were more than 99% homogeneous in their saccharide moiety, contained a single long chain base (homogeneity higher than 99%), and had a fatty acid composition primarily of stearic acid (92 to 97%). All the individual molecular species of GM1 and GD1a gangliosides were also prepared in radioactive form by selective tritiation at C-3 of the long chain base. Their specific radioactivity ranged from 1.3 to 1.45 Ci/mmol. The availability of these molecular species of gangliosides is expected to facilitate studies aimed at ascertaining the role played by the hydrophobic portion in the functional behavior of gangliosides.

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-fucosidase-in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible. Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10-14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

The condition for maximal activity (pH, buffer, saturating substrate concentration, range of linear relationships between enzyme activity versus incubation time, and versus enzyme concentration) in the fluorimetric assay of several glycohydrolases of lysosomal origin in human plasma and serum have been established. The following enzymes were studied: alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-glucuronidase, alpha-mannosidase, alpha-fucosidase. All examined enzymes turned out to be more or less unstable upon storage at 37 degrees C, 4 degrees c, and -20 degrees C in both serum and plasma. The only exceptions were beta-glucuronidase, which was stable in plasma and serum, and alpha-fucosidase which was stable only in plasma. Generally the degree of instability was greater in serum than in plasma. The levels of some enzymes (alpha-galactosidase, beta-galactosidase, beta-N-acetyl glucosaminidase, beta=glucuronidase) were markedly higher in serum than in plasma; conversely the levels of the same enzymes in "platelet free" serum equalled those in plasma. This stresses the necessity to use freshly prepared plasma for lysosomal glycohydrolase assay. Under the procedural conditions recommended for the assay the methods for the determination of lysosomal glycohydrolases in plasma appeared to be simple, sensitive and reproducible.

Gangliosides that contain the disialosyl residue alpha-Neu5Ac-(2--8)-alpha-Neu5Ac-(2--3)- can lactonize in the presence of traces of acid and this reaction has been studied in detail on GD1b [beta-Gal-(1--3)-beta-GalNAc-(1 --4)-[alpha-Neu5Ac-(2--8)-alpha-Neu5Ac-(2 --3)]-beta-Gal-(1--4)-beta-Glc-1--1)-Cer]. Lactonization occurs rapidly at a proton-ganglioside molar ratio of less than 1. At equilibrium, the ratio of GD1b to its lactone is 3:7. The data suggest the possibility that a proton-driven lactonization of gangliosides may occur in vivo.

The optimal assay conditions and the levels of seven lysosomal glycohydrolases (alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-N-acetyl-D-glucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase), alpha-D-mannosidase, alpha-L-fucosidase) were determined in human peripheral unseparated lymphocytes, T and non-T lymphocyte subpopulations. From fifteen adult volunteers the enzymes were assayed by fluorimetric procedures using the corresponding 4-methylumbelliferyl glycosides as substrates. The enzyme assay procedures displayed good precision and reproducibility. All the tested enzymes had higher activities in non-T than T lymphocytes. This difference was statistically highly significant, especially when the enzyme contents were expressed on a DNA, rather than mg protein, basis. Unseparated lymphocytes displayed levels of lysosomal enzymes which corresponded to the proportion of T and non-T lymphocytes in the unseparated preparation, indicating that the process of lymphocyte fractionation caused neither loss nor activation of lysosomal enzymes. It is concluded that the observed difference in lysosomal enzyme levels is an authentic imprint of the two lymphocyte subpopulations, implying a differential role played by the lysosomal apparatus in the same cells.

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.

The following enzymes of lysosomal origin were fluorimetrically determined in maternal plasma from the second to the ninth month of pregnancy at 1-mth intervals: beta-D-N-acetylglucosaminidase (EC 3.2.1.30), beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1.51) and alpha-D-mannosidase (EC 3.2.1.24) (pH 4.0). As reference microsomal alpha-D-mannosidase (pH 5.7) was also studied. Thirty-eight healthy women, aged 18-37 yr, who had a normal pregnancy followed by normal parturition, were studied. All enzymes, with the only exception of beta-D-galactosidase, showed a progressive and statistically significant increase of activity throughout pregnancy. At the end of pregnancy, the increase ranged from a maximum of 5.6-fold for beta-D-N-acetylglucosaminidase to a minimum of 0.55-fold for alpha-D-mannosidase, pH 5.7. In the case of beta-D-N-acetylglucosaminidase, the level at the fifth month of pregnancy was significantly higher than that at the third month, and from the sixth to the ninth month each level significantly differed from that of the month immediately preceding.

The circadian and circannual group rhythms in the plasma concentrations of the following lysosomal enzymes were studied in women and men: beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, alpha-D-galactosidase, alpha-L-fucosidase and alpha-D-mannosidase. The circadian rhythm was detected in all the tested enzymes of women, and only in alpha-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase and beta-D-N-acetylglucosaminidase of men. A statistically significant difference between genders in the circadian rhythm was exhibited by beta-D-galactosidase, beta-D-glucosidase, beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase and alpha-L-fucosidase. A circannual rhythm was detected in all the tested enzymes, with the exception of beta-D-glucuronidase and beta-D-N-acetylglucosaminidase, without any statistically significant difference between genders. The group rhythms of some of the enzymes (alpha-D-galactosidase, beta-D-glucosidase, beta-D-galactosidase) showed similar values of both circadian and circannual acrophases, suggesting that they may be subjected as a group to the same chronobiological coordination. The chronobiological rhythms of lysosomal enzymes were different from those of lactate dehydrogenase and alpha 1-antitrypsin, indicating that these rhythms are not merely reflecting fluctuations of the water content of plasma. No in-phase relationship was observed between the circadian and circannual rhythms of plasma cortisol and those of the tested lysosomal enzymes, excluding a direct chronobiological relationship between this hormone and lysosomal enzymes.

GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively.