Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis.

We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.     

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.     

Isolation and chemical characterization of a novel insulin-related neuropeptide from the freshwater snail, Lymnaea stagnalis.

A novel molluscan insulin-related peptide (MIP) III, has been isolated from alcohol extracts of the neurohaemal area of the cerebral neuroendocrine light-green neurones of Lymnaea stagnalis. MIP III was purified by sequential high-performance gel-permeation chromatography followed by reverse-phase HPLC. MIP III is a heterodimer connected by disulphide bonds. Edman degradation analysis and subsequent alignment with the A and B chains of the previously identified MIP I and II showed that the 24-amino-acid peptide with the sequence pQSRPSIVC(E)CCFNQCTVQ(E)LLAYC represents the MIP III A chain, and the 37-amino-acid peptide sequence TTQHTCSILSRPHPRGLCGSTLANMVQWLCSTYTTSS the B chain. The overall amino acid sequence of MIP III shows about 50% similarity with those of MIP I and II, and only 20-40% similarity with other peptides of the insulin superfamily. Important structural features, e.g. disulphide bridges and the hydrophobic core, are conserved in MIP III.     

Structural and functional characterization of neuropeptides involved in the control of male mating behavior of Lymnaea stagnalis.

Mating as a male in the simultaneous hermaphrodite freshwater snail, Lymnaea stagnalis, comprises a series of complex behaviors that are a prelude to copulation. Copulatory behavior itself is assumed to be controlled by various types of peptidergic neurons as well as serotonergic cells. Here we report the primary structure of two peptides that were extracted from a cluster of neurons that innervates the penial complex and that is located in the anterior lobe of the right cerebral ganglion. The sequences of the peptides were determined as: Ala-Pro-Gly-Trp-amide and Ser-Gly-Ser-Asp-Tyr-Cys-Glu-Thr-Leu-Lys-Glu-Val-Ala-Asp-Glu-Tyr-Ile-Leu- Leu- Ser-Tyr-Lys-Ile-Glu-Glu-Gln-Arg-Ala-Ala-Asp-Cys-Gly-Gly-Glu-Pro-Pro-Asn- Ser- Gln(amide), respectively. The longer peptide is a homodimer. Both peptides are processed from the recently identified Ala-Pro-Gly-Trp-amide prohormone, which is expressed in the neurons of the anterior lobe of the right cerebral ganglion. Ala-Pro-Gly-Trp-amide could also be recovered from the penial complex. This peptide, when applied in vitro, inhibits the contractions of the penis retractor muscles evoked by serotonin in a dose-dependent fashion.     

Thermography imaging during static and controlled thermoregulation in complex regional pain syndrome type 1: diagnostic value and involvement of the central sympathetic system

Background  

Complex Regional Pain Syndrome type 1 (CRPS1) is a clinical diagnosis based on criteria describing symptoms of the disease.     

Purification and chemical characterization of caudodorsal cell hormone-II from the egg-laying controlling caudodorsal cells of Lymnaea stagnalis.

Caudodorsal cell hormone-I (CDCH-I), a 36 amino acid peptide synthesized by the caudodorsal cells (CDCs), is one of the major neuropeptides present in the cerebral ganglia of the freshwater snail, Lymnaea stagnalis. This peptide induces ovulation and egg-mass formation, and, in addition, acts on neuronal circuits involved in the control of overt egg-laying behavior. cDNA cloning studies revealed that there is a CDCH gene family comprising two genes coding for CDCH-I and the related yet distinct CDCH-II, respectively. In the present study, using sequential high performance gel permeation and reversed phase high performance liquid chromatography, we have isolated and sequenced the peptide CDCH-II. This neuropeptide has the following sequence: Ser-Ile-Thr-Asn-Asp-Leu-Arg-Ala-Ile-Ala-Asp-Ser-Tyr-Leu-Tyr-Asp-Gln-His- Lys-Leu - Arg-Glu-Gln-Gln-Glu-Glu-Asn-Leu-Arg-Arg-Arg-Phe-Tyr-Glu-Leu-Ser-Leu-Arg- Pro-Tyr - Pro-Asp-Asn-Leu. The sequence differs from those predicted by the cDNA sequence encoding preproCDCH-II at two positions, suggesting the polymorphism of CDCH-II exists in L. stagnalis.     

Characterization of a cDNA clone encoding molluscan insulin-related peptide II of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.     

Interaction of Photoperiod,Grouping and Isolation in Female Reproduction of Lymnaea stagnalis

Summary

A study was made of the effects of the photoperiod on female reproductive activities in grouped and isolated specimens of the hermaphrodite freshwater snail Lymnaea stagnalis. Isolation of snails rapidly increased (< 3 days) egg production. The effect was long-lasting, and was probably caused by deprivation of copulation. Changes of photoperiod had similar effects in isolated and grouped snails. Compared to the effects of isolation, the photoperiodic effects were relatively slow in appearing (> 14 days). In comparison with a medium day (MD) (12 hr light/12 hr dark), a long day (LD; 16 hr light/8 hr dark) stimulated egg production whereas a short day (SD) (8 hr light/16 hr dark) lowered egg production. The LD effects were clearer in grouped than in isolated snails, but SD effects were clearer in isolated than in grouped snails. It is argued that the effects of grouping, isolation and photoperiod on egg production are mediated by the neuroendocrine system. It is suggested that the photoperiod plays an important role in the control of the egg laying season. Mating is probably important in the control of fecundity in relation to population density.