A glia-derived acetylcholine-binding protein that modulates synaptic transmission.

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Family of prohormone convertases in Lymnaea: characterization of two alternatively spliced furin-like transcripts and cell-specific regulation of their expression.

The majority of neuropeptides in Lymnaea stagnalis are proteolytically processed from larger precursors at sites composed of single or multiple basic amino acid residues. Previous studies have identified several putative prohormone convertases in the brain of Lymnaea. To characterize the complete family, we undertook three independent approaches: reverse-transcribed polymerase chain reaction screening, and low-stringency cDNA and genomic library screenings. The central nervous system cDNA library screening yielded two cDNAs encoding Lfurin1 and its variant form, Lfurin1-X. Both proteins show the characteristic organization of (human) furin with a putative catalytic domain, a P domain, a Cys-rich domain, a transmembrane domain, and a cytoplasmic tail. Lfurin1 and Lfurin1-X are identical, apart from a putative alternatively spliced noncatalytic luminal protein domain, which is present exclusively in Lfurin1-X. In situ hybridization revealed that the Lfur1 gene is expressed throughout the Lymnaea brain, but that the level varies considerably from one neuron to another. Quantitative analysis of the expression level of the two alternatively spliced transcripts revealed that it is neuron type-specifically regulated. This probably indicates the functional importance of noncatalytic luminal protein domains in these enzymes. In addition, our findings suggest that apart from the identified convertases LPC2, Lfurin1/Lfurin1-X, and Lfurin2, additional prohormone convertase diversity is either not present or present only at low levels in the Lymnaea brain. Alternatively, additional prohormone convertases could exist with a lower degree of sequence conservation than the other Lymnaea prohormone convertase members. From our findings, it appears that the majority of prohormone processing in Lymnaea is carried out by the three thus far identified types of Kex2-related prohormone convertases despite the large number of neuropeptide precursors and diverse multiple basic cleavage sites hydrolyzed.

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Evolutionary conservation of the insulin gene structure in invertebrates: cloning of the gene encoding molluscan insulin-related peptide III from Lymnaea stagnalis.

Although insulins and structurally related peptides are found in vertebrates as well as in invertebrates, it is not clear whether the genes encoding these hormones have emerged from a single ancestral (insulin)-type of gene or, alternatively, have arisen independently through convergent evolution from different types of gene. To investigate this issue, we cloned the gene encoding the molluscan insulin-related peptide III (MIP III) from the freshwater snail, Lymnaea stagnalis. The predicted MIP III preprohormone had the overall organization of preproinsulin, with a signal peptide and A and B chains, connected by two putative C peptides. Although MIP III was found to share key features with vertebrate insulins, it also had unique structural characteristics in common with the previously identified MIPs I and II, thus forming a distinct class of MIP peptides within the insulin superfamily. MIP III is synthesized in neurones in the brain. It is encoded by a gene with the overall organization of the vertebrate insulin genes, with three exons and two introns, of which the second intron interrupts the coding region of the C peptides. Our data therefore demonstrate that in the Archaemetazoa, the common ancestor of the vertebrates and invertebrates, a primordial peptide with a two-chain insulin configuration encoded by a primordial insulin-type gene must have been present.

Quantification of proteins in the subnanogram and nanogram range: comparison of the AuroDye, FerriDye, and India ink staining methods.

The usefulness of three sensitive dyes, AuroDye, FerriDye, and India ink, for the quantification of proteins and peptides bound to nitrocellulose paper has been assessed. In general, the staining intensity varies linearly with the logarithm of protein concentrations. The detection limit of small peptides (Mr less than 5000) is higher than that of large peptides and proteins, but the sensitivity is independent of the molecular weight. Oligopeptides of four or less amino acids either stain with very high detection limits or do not stain at all. The detection limit of proteins stained by AuroDye is approximately 1 ng, and in a number of cases even lower. The useful range for quantification of proteins extends to around 100 ng. The FerriDye and India ink staining methods are less sensitive and can be used to quantify proteins over a wide nanogram range. Among the methods tested, the India ink staining method has the highest protein to protein variation in sensitivity.

Fixation increases sensitivity of India ink staining of proteins and peptides on nitrocellulose paper.

The detectability by India ink staining of proteins and peptides dot-blotted on nitrocellulose paper was assessed before and after fixation. Fixation considerably increased the detectability of proteins and peptides. Denaturation by KOH treatment or baking at 100 degrees C for 15 min gave the best results. Precipitation by isopropanol/acetic acid gave intermediate results, whereas crosslinking with glutaraldehyde improved the detectability of small peptides, but not of proteins. Ferridye and Aurodye were also tested after baking. Both dyes were more sensitive and stained more proteins and peptides than India ink. In all cases the detectability of peptides smaller than Mr 1500 was poor.

Schistosomin: a pronase-sensitive agent in the hemolymph of Trichobilharzia ocellata-infected Lymnaea stagnalis inhibits the activity of albumen glands in vitro.

The schistosome parasite, Trichobilharzia ocellata, nearly completely inhibits the reproductive activity of its intermediate host, Lymnaea stagnalis. The synthetic activity of albumen glands of infected snails at day 35 postinfection (p.i.) is only 1% of the control value. The parasite acts by humoral means. We tested the hypothesis that (a) specific humoral agent(s) is (are) involved and refer to this (these) agent(s) as schistosomin. The presence of schistosomin in the hemolymph of infected snails was investigated by using galactogen synthesis in albumen glands as an in vitro bioassay. The synthetic activity of albumen glands of noninfected snails decreased by about 50% during a 1-h incubation in the hemolymph of infected snails. This inhibition is attributed to schistosomin. Based on these results, with the present bioassay schistosomin appears in the hemolymph between days 28-36 p.i. onwards. Schistosomin is heat-stable (100 degrees C) and pronase-sensitive, and therefore it might have a peptide nature. Schistosomin suppresses the stimulating action of the female, gonadotrophic dorsal body hormone at relatively low doses, which suggests that it may compete with this hormone for the same receptors. The development of two other bioassays for schistosomin in our laboratory is discussed.

The effects of starvation and refeeding on egg laying and the synthetic activity of the albumen gland in Bulinus truncatus, a snail vector of urinary schistosomiasis.

1. The effects of starvation (for 1, 2, 3, 6, 9 and 12 days, respectively) and refeeding (12 days starvation and 1, 2 and 3 days refeeding, respectively) on egg laying and albumen gland activity in the freshwater snail, Bulinus truncatus were studied. 2. The egg laying of starving snails rapidly decreased and ceased by day 6 of starvation. Egg laying was restored 24 hr after refeeding. The recorded decrease in albumen gland wet weight was proportional to the starvation periods. The DNA contents of the glands of the different experimental groups was not statistically different from the controls. 3. Albumen gland synthetic activity expressed as 14C-glucose incorporation into galactogen/microgram DNA and 3H-amino acids into total protein was determined. The glands showed an abrupt decrease in synthetic activity after 1 day of starvation and gradually decreased further until days 9-12. The decrease in activity of the glands was more rapid than that of egg laying. Upon refeeding, the activity of the glands recovered rapidly, simultaneous with the increase in wet weight and egg laying. 4. In conclusion, there is a correlation between egg laying and the in vitro activity of albumen glands. The results show a short-term effect of starvation on the fecundity of the snails. Such studies could be useful in field studies as well as snail control by applying molluscicides under optimal conditions.

Trichobilharzia ocellata: physiological characterization of giant growth, glycogen depletion, and absence of reproductive activity in the intermediate snail host, Lymnaea stagnalis.

Giant growth, depletion of energy stores, and inhibition of reproductive activity are striking effects of many trematode parasites on their intermediate snail hosts. Two hypotheses have been put forward to explain these phenomena: (1) host and parasite compete for energy rich and other essential nutrients, with the parasite as the winner, and (2) the parasite intervenes in the endocrine control of reproduction of the snail. These hypotheses were tested in the present study with the Trichobilharzia ocellata/Lymnaea stagnalis association. The snails were infected at a juvenile stage, and release of cercariae started on Day 55 after exposure. It was shown that enhanced growth of infected snails is not paralleled by a greater increase in dry weight, but hemolymph volume does increase, being 35% greater than in the noninfected controls. Control snails, on the other hand, showed an increase in the percentage body dry weight during sexual maturation. The conclusion is that infected snails retain an essentially juvenile body structure. In control snails, glycogen was depleted from the mantle store at the start of egg laying but the onset of cercariae production marked a severe glycogen depletion from the headfoot and the mantle in infected snails, being nearly complete on Day 68 after exposure. The hemolymph glucose concentration was only slightly lower in infected than in control snails and it did not change (in both groups) during glycogen mobilization. This suggests that glycogen mobilization does not result from the snail and the parasite competing directly for metabolites within the hemolymph. Infection inhibited the maturation of the accessory sex organs: there was no increase in the relative wet weights nor in the amounts of DNA and secretion products in the albumin and prostate glands. Infected snails did not lay eggs. It is presumed that the parasite produces one or more agents which intervene in the action of the gonadotrophic hormones. The release of these agents commences at an early stage of infection.

Biosynthesis of tooth germ proteins in vitro: a fast quantitative extraction of amelogenins from intact hamster molar tooth germs.

A three step extraction procedure was carried out on intact hamster molar tooth germs in vitro labelled with 32PO4 and/or 3H-proline, in order to quantify separately the synthesis of dentine matrix (collagen) and the proline rich enamel matrix proteins. The extraction was based on the high solubility of the proline rich enamel matrix proteins compared with the relatively insoluble dentine matrix collagens. Pretreatment with 10% trichloroacetic acid (step 1) demineralized and removed the non-incorporated amino acids and/or small sized peptides. A consecutive water extraction (step 2) removed a large percentage of the phosphorylated amelogenins as assessed by SDS-urea-polyacrylamide-electrophoresis and amino acid analyses. Collagenase digestibility data showed that only small amounts of collagens were present in this extract. Further extraction with 10% formic acid (step 3) released only small amounts of amelogenins from the explants but also increased contamination with collagens and another predominantly low molecular components. Most of the 3H-activity remaining in the residues was found in the collagenase labile material and was considered to be an appropriate measure for production of dentine collagens. On the other hand, the residues also contained small amounts of 3H-labelled material with the same electrophoretic mobility as amelogenins but had much more 32P-activity than the amelogenins derived from the water and formic acid extracts. It is suggested that this material in the residues probably contains the crystal bound enamel matrix proteins.

Effects of two gonadotropic hormones on polysaccharide synthesis in the albumen gland of Lymnaea stagnalis, studied with the organ culture technique.

A medium for culturing organs of the basommatophoran freshwater snail Lymnaea stagnalis is described. One of the accessory sex glands, the albumen gland, which produces proteins and galactogen as nutritive substances for the embryos, was cultured along with parts of the central nervous system (CNS), including the female gonadotropic hormone-producing dorsal bodies (DB). Culture time was 4 days. The cerebral ganglia and the DB appeared to have a strong stimulating influence on polysaccharide synthesis in the albumen gland. The remaining part of the CNS has a weak stimulating effect. The use of extracts (culture time 6-26 hr) of parts of the cerebral ganglia and of the DB showed that not only the dorsal body hormone (DBH), but also the ovulation hormone (CDCH)--a neurohormone produced by the caudodorsal cells located in the cerebral ganglia--stimulates the synthetic activity of the albumen gland. The action of these hormones is direct; i.e., it is not exerted via the gonad. Dose- and time-response relations for the DBH and the CDCH are very similar. No additional rise in stimulation occurred when the two hormones were administered together. Experiments with albumen glands of adult snails demonstrated that the presence of large quantities of secretory material in the gland inhibits the response to DBH and CDCH.