Primary structure of a low-molecular-mass N-linked oligosaccharide from hemocyanin of Lymnaea stagnalis. 3-O-methyl-D-mannose as a constituent of the xylose-containing core structure in an animal glycoprotein

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high-molecular-mass copper-containing oxygen-transport protein, which occurs freely dissolved in the hemolymph. It is a glycoprotein containing fucose, xylose, 3-O-methylmannose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues as sugar constituents. The N-glycosidic carbohydrate chains of this glycoprotein were released by hydrazinolysis of a pronase digest and subsequently fractionated as oligosaccharide-alditols on Bio-Gel P-4 followed by Lichrosorb-NH2. Investigation with 500-MHz 1H-NMR spectroscopy, in conjunction with sugar and methylation analysis revealed the lowest-molecular-mass glycan chain to have the structure: (Formula: see text).     

Apiose and mono-O-methyl sugars as minor constituents of the leaves of deciduous trees and various other species

1. Leaves of a number of species were hydrolysed with aqueous sulphuric acid and the resulting mixtures of sugars were fractionated by chromatography on activated charcoal. Paper chromatography of the fractions showed the presence in all the hydrolysates of minor constituents with R(F) values similar to or greater than those of the common hexoses and pentoses. 2. Two of these were identified as 2-O-methylxylose and 2-O-methylfucose. Estimates of the amounts present in whole leaves, and in fractions prepared from them, showed that they were associated with the hemicelluloses. 3. A third constituent was identified, by the formation of its di-isopropylidene derivative, as apiose. It also was associated chiefly with the hemicellulose fraction; none could be found in aqueous extracts from leaves of Tilia vulgaris, nor in aqueous extracts of Zostera marina, in which apiose is a major constituent of the water-insoluble polysaccharide. 4. A further constituent, after further purification by preparative paper chromatography, was tentatively identified, by gas-liquid chromatography of derivatives, as 3-O-methylgalactose, and was probably accompanied by small amounts of 4-O-methylgalactose. 5. These observations confirm the widespread occurrence of 2-O-methylxylose, 2-O-methylfucose and apiose, but 3-O-methylgalactose was hitherto known only in slippery-elm mucilage, and 4-O-methylgalactose in soil polysaccharides. Some experiments on the digestion of leaf hemicellulose fractions by snail crop-juice suggested that the mono-O-methyl sugars might confer resistance to enzymic degradation.     

Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus ebracteatus: their chemical and physical features

Crude water-soluble polysaccharides were isolated from Acanthus ebracteatus by hot water extraction followed by ethanol precipitation after pre-treatment with 80% ethanol. The crude polysaccharides were separated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (A1001) was rich in galactose, 3-O-methylgalactose and arabinose, whereas the acidic polysaccharide (A1002) consisted mainly of galacturonic acid along with rhamnose, arabinose and galactose as minor components indicating a pectin-type polysaccharide with rhamnogalacturonan type I (RG-1) backbone. 3-O-Methylgalactose is also present in the acidic fraction. Both neutral and acidic fractions showed potent effects on the complement system using pectic polysaccharide PM II from Plantago major as a positive control. A small amount of 3-O-methylgalactose present in the pectin seemed to be of importance for activity enhancement in addition to the amount of neutral sugar side chains attached to RG-1. The relationship between chemical structure and effect on the complement system of the isolated polysaccharides is considered in the light of these data. The presence of the rare monosaccharide 3-O-methylgalactose may indicate that this can be used as a chemotaxonomic marker. The traditional way of using this plant as a medical remedy appears to have a scientific basis.     

3-O-methyl-D-galactose residues in lycophyte primary cell walls

Acid hydrolysis of cell wall-rich material from young leaves of the lycophyte Selaginella apoda (L.) Spring yielded substantial amounts of 3-O-methyl-D-galactose (1) in addition to the usual major monosaccharides (glucose, galactose, arabinose, xylose and galacturonic acid). The yield of 1 approximately equalled that of galacturonic acid. Compound 1 was identified as 3-O-methylgalactose by its 1H and 13C NMR spectra, and shown to be the D-enantiomer by its susceptibility to D-galactose oxidase. Compound 1 was detected in acid hydrolysates of the alcohol-insoluble residues from young leaves of all lycophytes tested, both homosporous (Lycopodium, Huperzia and Diphasiastrum) and heterosporous (Selaginella). It was not detectable in the charophyte green algae Coleochaete scutata, Chara coralina or Klebsormidium flaccidum, any bryophytes [a hornwort (Anthoceros), four liverworts and three mosses], or any euphyllophytes [a psilopsid (Psilotum), a horsetail (Equisetum), eusporangiate and leptosporangiate ferns, the gymnosperm Gnetum, and diverse angiosperms]. A high content of 1 is thus an autapomorphy of the lycophytes.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

Teichoic acid from the cell wall of Actinomadura carminata--a producer of the antibiotic carminomycin

The cell walls of Actinomadura carminata, producing the antibiotic carminomycin, contain a poly(glycerol phosphate) teichoic acid. The polymer belongs to 1,3-type and consists of about 8 glycerol phosphate units, two of them have 2-acetamido-2-deoxy-alpha-D-galactopyranosyl substituent and one--3-O-methyl-beta-D-galactopyranosyl-(1----3)-2- acetamido-2-deoxy-alpha-D-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was established by chemical analysis and 13C-NMR spectroscopy. The teichoic acid accounted for about 10% of the cell wall dry weight. 3-O-methylgalactose in the structure of the teichoic acid was found for the first time.     

Studies on resistance in snails. 3. Tissue reactions to Echinostoma lindoense sporocysts in sensitized and resensitized Biomphalaria glabrata.

The resistance of Biomphalaria glabrata snails that have been sensitized by various levels of irradiated or nonirradiated Echinostoma lindoense miracidia increased after a second challenge infection with nonirradiated miracidia of the same species. This was demonstrated by increased suppression of migrating capacity of invading sporocysts, an accelerated host tissue reaction, and a greater tendency of snail amebocytes to flatten while attacking the parasite. Three methods of elimination of invading sporocysts were observed: (1) encapsulation by amebocytes followed by destruction of the sporocysts; (2) expulsion of the sporocyst through the host epithelium after its encapsulation in the subepithelial tissues; (3) blockade of the parasite's entry into subepithelial tissues by a localized amebocyte aggregation. The basic mechanism of host snail response to a single or a repeated challenge infection appears to be similar, though an anamnestic reaction is evident in the accelerated response following a second challenge exposure.     

Molluscicidal activity of 2-hydroxy-3-alkyl-1,4-naphthoquinones and derivatives

In the search for new molluscicidal agents we tested the activity of lapachol and other 2-hydroxy-3-alkylnaphthoquinones possessing nitrogenated alkyl chains, against the snail Biomphalaria glabrata. Lapachol, isolapachol and nor-lapachol showed strong molluscicidal activity against the adult snail (LD(90)<10 ppm) and significant toxicity against snail egg masses (LD(90)<0.2 ppm). As lapachol is easily extracted, and the derivatives can be synthesised without any difficulty, large-scale synthesis and field tests can be conducted, with a view to large-scale molluscicidal programs.     

Snail mediates PDGF‐BB‐induced invasion of rat bone marrow mesenchymal stem cells in 3D collagen and chick chorioallantoic membrane

We previously reported that membrane type‐1 matrix metalloproteinase (MT1‐MMP) enables mesenchymal stem cells (MSCs) to move through both three‐dimensional (3D) type I collagen and basement membrane barriers; however, its upstream regulating factors were unidentified. Here, we report that PDGF‐BB upregulates both mRNA and protein expression of snail in rat bone marrow MSCs (rBMMSCs). PDGF‐BB enhances rBMMSC invasion in 3D collagen, which is blocked by snail specific siRNA transfection. Snail overexpression induced by plasmid transfection results in increased rBMMSC invasion in 3D collagen. Snail expression induced by PDGF‐BB in MSCs is inhibited by LY294002 and PD98059, which are inhibitors of the PI3K/AKT and MAPK1/2/ERK1/2 signaling pathways, respectively. MT1‐MMP expression in rBMMSCs, both as mRNA and protein, is decreased by snail siRNA transfection, but increased by snail overexpression, indicating that they are controlled by snail. Finally, snail controls MSC transmigration through chorioallantoic membrane of 11‐day‐old chick embryos. Taken together, these in vitro and in vivo data identify snail as a critical mediator for rBMMSC invasion induced by PDGF‐BB. J. Cell. Physiol. 228: 1827–1833, 2013. © 2013 Wiley Periodicals, Inc.     

Protoplast formation and yeast cell-wall structure. The action of the enzymes of the snail, Helix pomatia

1. The digestive juice of the snail Helix pomatia was used in the study of the degradation of isolated cell-wall preparations from a strain of Saccharomyces carlsbergensis. 2. The crude enzyme system was fractionated by gel filtration and the activities of the more specific fractions thus obtained were examined. 3. Results are discussed with respect to (a) the nature of various factors that are essential for protoplast formation and cell-wall dissolution and (b) structures envisaged in yeast cell walls that are responsible for the observed variations in susceptibility to attack by snail juice.     

A somatically diversified defense factor, FREP3, is a determinant of snail resistance to schistosome infection

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished.     

鲶—草—螺人工生态群落的效益研究

近年来,科学工作者为充分发挥土地最佳经济效益,正在致力于把各种养殖业与种植业进行综合培植,并获得较好的试验效果。为探索发挥池塘水体的生产力,自1987年开始我们以革胡子鲶(Clarias leather)、凤眼莲(Eichhornia crassipes)、福寿螺(Ampullarum crossean)为试验材料。连续两年进行了鲶、草、螺人工生态群落的效益研究。     

The helix C3-motoneurone contains ACh and fmrfamide

1. The identifiable C3 neurone of the snail (Helix aspersa) is a motoneurone for the tentacle retractor muscle (TRM) and is immunoreactive for the molluscan peptide FMRFamide.2. Using bioassay, we show that this cell, as well as its target muscle, contains acetylcholine (ACh) and can synthesize ACh from its usual precursors.3. Acetylcholinesterase was also detected in the TRM.     

Primary structure of the low-molecular-weight carbohydrate chains of Helix pomatia alpha-hemocyanin. Xylose as a constituent of N-linked oligosaccharides in an animal glycoprotein

alpha-Hemocyanin of Helix pomatia is a copper-containing glycoprotein which serves as an oxygen carrier in the hemolymph. Its carbohydrate moiety has as constituents fucose, xylose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine, and N-acetyl-glucosamine residues. Alkaline borhydride did not split off any carbohydrate material, suggesting the absence of O-glycosidic chains. The N-glycosidic carbohydrate chains of this glycoprotein were liberated by hydrazinolysis of a Pronase digest then fractionated as alditols on Bio-Gel P-4. The fractions containing the low-molecular-weight glycans were investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar and methylation analysis. The largest, and most abundant, compound was established to be: (Formula: see text). Another compound was characterized as the afuco analogue of this structure. H. pomatia alpha-hemocyanin is the first example of an animal glycoprotein having xylose as a constituent of N-glycosidic carbohydrate chains.     

Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1)

Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.     

Coenological study of snails (Mollusca: Gastropoda) in forest phytocoenoses of Medvednica mountain (NW Croatia,Yugoslavia)

The coenological research of land snails has produced 42 taxa gathered on 68 sites in 11 forest phytocoenoses of the Medvednica mountain area (NW Croatia, Yugoslavia). Relative abundance, constancy and density have been established for every taxon of a snail community in the specific phytocoenosis. The total number of snail taxa, the average number of snail taxa per site, the Shannon-Wiener index (the qualitative characteristics) and the community density (the quantitative characteristic) have been established for every snail community. The snail communities from thermophilic basophilous phytocoenoses, especially the community in the association Querco-Ostryetum have been proved to be the richest in regard to quality and quantity. Markedly acidophilic phytocoenoses have had the poorest snail community: a snail community in the association Querco-Castanetum has proved the poorest in quality and the snail community in the association Luzulo-Fagetum has been the poorest in quantity. The Shannon-Wiener index has got its highest values in the snail communities belonging to climatogenous phytocoenoses. The attempts at establishing the relation of snail communities using objective methods have been made by calculating the similarity of snail communities according to Sørensen (1943) and Kulczyński (1927) and also by clustering according to weight variable-group method (Sokal & Sneath 1963: 310, 311). The following common features have been established among the results obtained by applying the stated methods:

- the grouping of snail communities belonging to thermophilic phytocoenoses on the calcareous soil

- the grouping of snail communities from the predominantly acidophilic woodlands

- the grouping of snail communities belonging to the acid soils of the highest region of Medvednica.

     

Extracellular glycoconjugates produced by cyanobacterium Wollea saccata

In order to survive in a highly competitive environment, freshwater or marine phototrophic microorganisms have to develop defense strategies that result in a tremendous diversity of compounds from different metabolic pathways. Recent trends in drug research from natural sources have shown that algae and cyanobacteria are promising organisms to furnish novel biochemically active compounds. In this study, we have analysed the extracellular mucilaginous proteoglycan produced by fresh-water heterocytous filamentous cyanobacterium Wollea saccata, strain Hindák 2000/18. This mucilaginous material is an acidic proteoglycan containing 30% protein and 52% carbohydrates on the basis of fraction dry weight. The constituent sugars of the carbohydrate component include glucose, fucose, 3-O-methylfucose, xylose, galactose, 3-O-methylgalactose, mannose, rhamnose, arabinose and glucuronic acid. The extracellular proteoglycan has been separated into five fractions (WF1-WF5) by anion exchange chromatography. Individual polymeric fractions varied in protein (16-57%) and carbohydrate (31-66%) contents, and in the composition of constituent monosaccharides.     

Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin

Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2-e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92-143 of FU RtH2-e with a glycosylation site at Asn-127. Besides the core structure Man(3)GlcNAc(2) for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(alpha1-3) or the Man(alpha1-6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed, indicating the existence of microheterogeneity at the glycan level. The tetrasaccharide unit contains a central fucose moiety substituted with 3-O-methylgalactose and N-acetylgalactosamine, and linked to GlcNAc at the reducing end. This structure represents a novel N-glycan motif and is likely to be immunogenic. A second potential site for N-glycosylation in FU RtH2-e at Asn-17 was shown to be not glycosylated.     

Identification of cholinoreceptors on the soma of snail neurons RPa3 and LPa3

Identification of cholinoreceptors (CR) of the soma of neurons RPa3 and LPa3 of the snail is performed using selective cholinomimetics and cholinolytics during the recording of transmembrane ionic currents. Agonists of the nicotinic (NCR) and muscarinic (MCR) types of cholinolytics evoked a brief activation of the receptors, with the exception of carbamylcholine, followed by an irreversible blocking. All selective cholinomimetics bonded with the same membrane centers which acetylcholine (AC) activated. The nicotinic and muscarinic cholinolytics decreased the amplitude of the input current elicited by AC; however, the use of scopolamine and platyphylline was without effect. It is speculated that the soma of neurons RPa3 and LPa3 exhibits NCR and MCR which have a number of pharmacological features distinguishing them from the corresponding CR of vertebrates. The MCR of these neurons must be classed as a special subtype differing from the well-known M₁ and M₂ subtypes.M. V. Lomonosov State University of Moscow. Translated from Neirofiziologiya, Vol. 24, No. 1, pp. 77–86, January–February, 1992.