Panagrellus redivivus and Caenorhabditis elegans: evidence for the absence of sialic acids

Complementary experiments were performed to indicate the presence or absence of sialic acids in axenically cultured Panagrellus redivivus and Caenorhabditis elegans. Competitive displacement experiments with radiolabeled Limax flavus agglutinin demonstrated the presence of sialic acid in nematodes grown in medium which contained liver extract as a growth factor but the absence of sialic acid when heme was substituted for liver extract. This finding suggested that sialic acid present in the liver medium was responsible for conflicting results of other studies. Transmission electron microscopy of thin sections from nematodes labeled with an LFA-ferritin conjugate revealed no label to the surface area of the cephalic chemosensilla. Fluorometric analysis with a modification of the thiobarbituric acid assay was negative for sialic acid. Analyses by gas chromatography-mass spectrometry, sensitive to the high picomole range, were also negative for sialic acid. Taken together the results provide evidence for the absence of sialic acid in P. redivivus and C. elegans using the most sensitive and diagnostic technique currently available.     

Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids

The parent sialic acid N-acetylneuraminic acid can be modified or substituted in various ways, giving rise to a family of more than 25 compounds. The definitive identification of these compounds has previously required isolation of nanomole amounts for mass spectrometry or NMR. We have explored the possibility of using the known metabolic precursors of the sialic acids, particularly N-acetyl-[6-3H]mannosamine, to label and identify various forms of sialic acids in tissue culture cells. Firstly, we defined several variables that affect the labeling of sialic acids with N-acetyl-[6-3H]mannosamine. Secondly, we have devised a simple screening method to identify cell lines that synthesize substituted or modified sialic acids. We next demonstrate that it is possible to definitively identify the natures of the various labeled sialic acids without the use of mass spectrometry, even though they are present only in tracer amounts. The methods used include paper chromatography, analytical de-O-acetylation, periodate release of the 9-3H as [3H]formaldehyde (which is subsequently converted to a specific 3H-labeled chromophore), acylneuraminate pyruvate lyase treatment with identification of [3H]acylmannosamines, gas-liquid chromatography with radioactive detection, and two new high-pressure liquid chromatography methods utilizing the amine-adsorption:ion suppression and ion-pair principles. The use of an internal N-acetyl-[4-14C]neuraminic acid standard in each of these methods assures precision and accuracy. The combined use of these methods now allows the identification of radioactive tracer amounts of the various types of sialic acids in well-defined populations of tissue culture cells; it may also allow the identification of hitherto unknown forms of sialic acids.     

Sterol biosynthesis in the free-living nematode Panagrellus redivivus

The fate of the known sterol precursor squalene 2,3-oxide was investigated in the free-living nematode Panagrellus redivivus. The nematodes were cultured axenically in the presence of [4-(3)H]squalene 2,3-oxide. Radioactivity was found in the total lipids of the isolated nematodes. Essentially all of the radioactivity encountered in the total lipids was found in the non-saponifiable fraction. The components present in the non-saponifiable fraction were separated and isolated by t.l.c. Three labelled components were identified by a combination of t.l.c., g.l.c. and mass spectroscopy. It is established that P. redivivus has the capacity for biosynthesis of lanosterol. No labelled C(27) sterols could be detected.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.     

O-acetylation and de-O-acetylation of sialic acids. 7- and 9-o-acetylation of alpha 2,6-linked sialic acids on endogenous N-linked glycans in rat liver Golgi vesicles

We have previously shown that radioactivity from [acetyl-3H]AcCoA is concentrated into isolated intact rat liver Golgi vesicles. The incorporated radioactivity occurred in acid-soluble and acid-insoluble components, and the acid-insoluble fraction included O-acetylated sialic acids (Varki, A., and Diaz, S. (1985) J. Biol. Chem. 260, 6600-6608). Nearly all of the protein-associated radioactivity was found to be in sialic acids alpha 2-6-linked to N-linked oligosaccharides on endogenous glycoproteins. Incubation of the vesicles with CMP-[3H]sialic acid resulted in labeling of a very similar group of glycoproteins. The 3H-O-acetyl groups were found at both the 7- and the 9-positions of N-acetylneuraminic acid residues at the end of the labeling reaction. Although 7-O-acetyl groups can undergo migration to the 9-position under physiological conditions, kinetic studies using O-acetyl-14C-labeled internal and O-acetyl-3H-labeled external standards indicate that during the labeling, release, and purification, negligible migration occurred. Studies with mild periodate oxidation provided further confirmation that O-acetyl esters are added directly to both the 7- and the 9-positions of the sialic acids in this system. The acid-soluble, low molecular weight component is released from the vesicles by increasing concentrations of saponin, and its exit parallels that of CMP-[14C]sialic acid taken up during the incubation. The vesicles themselves are impermeant to free acetate. However, even after short incubations, this saponin-releasable radioactivity was almost exclusively in [3H] acetate and not in [3H]acetyl-CoA. The apparent Km for accumulation of the [3H]acetate is almost identical with that for the generation of the acid-insoluble O-acetylated sialic acids. Most of this accumulation of free acetate is also blocked by coenzyme A-SH. Only a small portion arises from the action of an endogenous esterase on the 3H-O-acetylated sialic acids. Taken together, the results indicate that accumulation of free [3H]acetate occurs within the lumen of the vesicles in parallel with O-acetylation of sialic acids and is probably a product of abortive acetylation. It is not known if this reaction occurs in vivo. Permeabilization of Golgi vesicles to low molecular weight molecules with saponin does not alter the rate of acetylation substantially. Furthermore, double label studies suggest that the intact acetyl-CoA molecule does not gain access to the lumen of the vesicles. These results indicate that the acetylation reaction may have a different mechanism from previously described Golgi glycosylation reactions, wherein specific transporters concentrate sugar nucleotides for use by luminally oriented transferases.     

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita)

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.     

Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes

A cytosolic sialic acid-specific O-acetyl-esterase was previously described that can remove O-acetyl esters from the 9-position of sialic acids. We show that rat liver Golgi vesicles contain a distinct sialic acid-esterase located within the lumen of the same vesicles that add O-acetyl esters to sialic acids. Studies of a retinoblastoma cell line genetically deficient in the cytosolic enzyme also confirm the existence of distinct membrane-associated sialic acid esterase activity. We developed a sensitive, specific and facile assay, which measures release of [3H]acetyl groups from [3H-acetyl]9-O-acetyl-N-acetylneuraminic acid. Using this assay, we show that rat liver membranes may contain different sialic acid O-acetyl-esterases. The membrane-associated enzyme(s) bind to Concanavalin A Sepharose, whereas the cytosolic enzyme does not. Membrane-bound and cytosolic esterases are inactivated by di-isopropyl-fluorophosphate, showing they are serine-active-site enzymes.     

携带cip基因的酿酒酵母对自由生活线虫Panagrellus redivivus生长繁殖的影响

Photorhabdus luminescens细菌与昆虫病原异小杆属Heterorhabditis线虫专性共生。初生型共生细菌产生两种胞内晶体蛋白CipA and CipB,为共生线虫提供营养。为探索Cip蛋白是否对自由生活的全齿复活线虫Panagrellus redivivus具有类似的营养功能,建立了Cip蛋白的重组酿酒酵母表达体系,并用于饲喂无菌的P. redivivus线虫J1幼虫。重组酿酒酵母表达的Cip蛋白能为线虫所利用,表现为营养支持作用,体现为线虫生长发育速度的加快以及繁殖能力的提高,说明Cip蛋白能为此种自由生活线虫提供营养来源。     

The identification of a variant form of cystathionine beta-synthase in nematodes.

Characterization of the physical and catalytic properties of the enzyme responsible for nematode "activated L-serine sulfhydrase" activity (L-cysteine + R-SH-->cysteine thioether + H2S) has led to its identification as a novel, variant form (allelozyme) of cystathionine beta-synthase that is distinct from a mammalian-type synthase also present in nematodes. Additional work has demonstrated the ability of live Panagrellus redivivus to produce H2[35S] from exogenous L-[35S]cysteine and 2-mercaptoethanol, thus providing preliminary evidence for the in vivo operation of the activated L-serine sulfhydrase reaction in nematodes.     

Malaria parasites do not contain or synthesize sialic acids

The capacity of Plasmodia to synthesize sialic acids was investigated by adding radioactive acetate to short-term in vitro cultures of the intraerythrocytic asexual forms of three malaria parasites (the human malaria Plasmodium falciparum in Aotus trivirgatus erythrocytes; the simian malaria P. knowlesi in rhesus monkey erythrocytes; the rodent malaria P. berghei in mouse erythrocytes) and to cultures of extracellular zygotes of the avian malaria P. gallinaceum. Radioactive acetate was added to normal rhesus monkey erythrocytes and to cells of the murine myeloma NS-1 for comparison. Although [1-14C]-acetate labeled many proteins with each malaria parasite and the NS-1 cells, analysis of purified sialic acids revealed that only with the NS-1 cells was radioactivity incorporated into sialic acids. Furthermore, N-acetyl[6-3H]mannosamine was not incorporated into sialic acids or malarial glycoproteins when added to P. knowlesi cultures. All of the malaria parasites underwent growth or differentiation during these experiments as measured by [35S]methionine uptake into protein and by light microscopy. Extracellular parasites largely free of erythrocyte membranes were prepared to determine whether Plasmodia contain sialic acids that are not labeled by exogenous precursors. Purified merozoites of P. knowlesi and zygotes of P. gallinaceum did not contain detectable amounts of sialic acids on chemical analysis. Thus, although we could show that Plasmodia can incorporate radioactive sugars such as glucosamine, galactose and mannose into proteins, presumably glycoproteins, they do not synthesize sialic acids or sialo-glycoproteins, nor do they contain sialo-glycoconjugates of host origin.     

Evidence for glycoprotein components of the hepatocellular bile acid transporter

The hepatocellular transporter, responsible for the uptake of bile acids and some foreign substances, can be shown to contain carbohydrate moieties. The hepatocellular uptake of cholate and phallotoxin is immediately inhibited by addition of wheat-germ agglutinin. Concanavalin A and lentil lectin reduce the uptake in a time-dependent manner. Apparently sialic acids or N-acetylglucosamine residues are involved in the translocation process. Polypeptides (Mr 50,000, 54,000) of the above transport system, identified by affinity labeling with [3H]isothiocyanatobenzamido cholate and [3H2]diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid, are heterogenously glycosylated. Binding of 80-90% of the 54, 50 kDa polypeptides to all immobilized lectins tested suggests that both high-mannose and complex type oligosaccharides with fucose and terminal sialic acid residues occur as carbohydrate chains. A 67 kDa labeled polypeptide is not glycosylated. Pilot experiments for purification of the above glycosylated membrane proteins on concanavalin A, lentil lectin and wheat-germ lectin columns are described. However, lectin affinity chromatography is not suitable as a one-step purification procedure for the labeled polypeptides.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.     

Modification of sialyl residues of glycoconjugates by reductive amination. Characterization of the modified sialic acids

The sialic acid residues of alpha 1-acid glycoprotein and fetuin were modified by introduction of an amino residue, such as glycine and [3H]glycine. This modification involved (a) the selective periodate oxidation of the exocyclic carbon atoms of the sialic acid residue generating an aldehyde group at C-7, and (b) the reduction of the Schiff base formed with an amino compound by use of sodium cyanoborohydride. Thin layer chromatography, high pressure liquid chromatography, and amino acid composition data of the modified glycoprotein showed that the conversion was essentially quantitative. The glycine-modified sialic acids were isolated by mild acid hydrolysis and identified by g.l.c.-m.s. and n.m.r. spectroscopy, thus confirming that the quantitative modification produced a glycine-aminated C-7 sialic acid analog. Strong acid hydrolysis of the glycine-modified sialic acid yielded a fragment that had chromatographic characteristics similar to those of glycine.     

O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.     

Changes in cell surface anionogenic groups induced by propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10(-3) mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum.     

Development of a low-cost technology for mass production of the free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> as an alternative live food for first feeding fish larvae

The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm(3) sponge cubes) in autoclavable plastic bags (size range: 50 x 30 cm to 75 x 67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11-13 days) at 25 degrees C, the average yield (number of nematodes/g medium) was 241 x 10(3) for OM and 333 x 10(3) for PIM in 12-l bags (50 x 30 cm). The production scale has currently reached a bag volume of 50 l (75 x 67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3 x 10(9) nematodes/bag (291 x 10(3) nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259 x 10(3) and 290 x 10(3) nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.     

Differential Adhesion and Infection of Nematodes by the Endoparasitic Fungus Meria coniospora (Deuteromycetes)

The conidia of the endoparasitic fungus Meria coniospora (Deuteromycetes) had different patterns of adhesion to the cuticles of the several nematode species tested; adhesion in some species was only to the head and tail regions, on others over the entire cuticle, whereas on others there was a complete lack of adhesion. After adhesion, the fungus usually infected the nematode. However, adhesion to third-stage larvae of five animal parasitic nematodes, all of which carry the cast cuticle from the previous molt, did not result in infection. M. coniospora infected animal parasitic nematodes when this protective sheath was removed. Seven preparations of sialic acid (N-acetylneuraminic acid) gave three types of response in adhesion-infection of nematodes: (i) a significant reduction in conidial adhesions; (ii) no interference with adhesion, but a 10-day delay in infection; and (iii) a delay in infection by 2 to 3 days. The current results support previous findings indicating involvement of sialic acids localized on nematode cuticles in recognition of prey by M. coniospora.     

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.