Cowden C et al. (1986) Worm Breeder's Gazette "a CCK-like neuropeptide in Ascaris."

Stretton AOW, Cowden C

[Worm Breeder's Gazette, 1986]

Our aim is to obtain peptides of known sequence from Ascaris in order to determine their mode of action using electrophysiological methods. Since McIntire and Horvitz (C. elegans CSH Abstracts 1985) showed that cholecystokinin-like immunoreactivity (CCK-LI) is present in certain neurons in C. elegans, we are initially investigating the role of a CCK-like peptide (CCK-LP) in the nervous system of Ascaris using anti-CCK8 antisera to detect and localize CCK-LI. Using the methods developed by Johnson (see Johnson and Stretton, Soc. Neurosci. Abstr. 9: 302; Sithigorngul, Johnson and Stretton, C. elegans CSH Abstracts 1985) we find that in Ascaris, CCK-LI is concentrated in 2 cells (AVF cells) in the ventral nerve cord, in 3 cells in the ventral ganglion, in 4 processes in the ventral cord, and in 2 processes in the lateral line. Thus the CCK-LP is concentrated in a small minority of the 180 neurons present in the anterior region of adult Ascaris.We have developed a procedure for extracting the CCK- LP from C. elegans and fractionating the extract on a C18 cartridge. High voltage paper electrophoresis shows that added radioiodinated CCK8 is chemically intact after this extraction and after fractionation. The CCK-LP was separated from more hydrophilic components with a 20-40% gradient of acetonitrile on reversed phase HPLC. RIA's detected CCK-LI associated with a peak of optical density. Addition of authentic CCK8 (non-sulfated) to the sample showed that the RIA-positive peak was close to, but distinctly separate from, CCK8. Assuming that the specific immunoreactivity of nematode CCK-LP and mammalian CCK8 is the same, we can obtain 100 pmoles from 60g of C. elegans. From this crude estimate, it seems that the levels of recoverable peptide are sufficient for amino acid sequence determination which is now our top priority.

Cowden C et al. (1987) International C. elegans Meeting "neuropeptide diversity in Ascaris."

Guastella J, Cowden C, Stretton AOW, Sithigorngul P

[International C. elegans Meeting, 1987]

Cowden C et al. (1991) International C. elegans Meeting "ASCARIS FLPs FOREVER."

Stretton AOW, Cowden C

[International C. elegans Meeting, 1991]

Two FMRFamide-like peptides (FLPs), AFl (KNEFIRFamide, Cowden et al., 1989) and AF2 (KHEYLRFamide, Cowden and Stretton, 1990) have been isolated from Ascaris heads. Both AFl and AF2 have multiple effects on muscle tension including relaxation, contraction, and generation of rhythmic activity. A different set of FLPs, having the common C-terminus PNFLRFamide, have been predicted from a C. elegans gene (Li, 1990). Two hypotheses related to the observed differences in Ascaris and C. elegans FLPs have been tested. The first is that the FLPs are differentially distributed in Ascaris such that a restricted subset of the FLPs are localized in Ascaris heads. The second is that FLPs in Ascaris and C. elegans are differentially extracted by acetone and acid methanol. Both hypotheses appear to be

Cowden C et al. (1989) International C. elegans Meeting "diversity of Ascaris FMRFamide-like peptides."

Stretton AOW, Cowden C

[International C. elegans Meeting, 1989]

Cowden C et al. (1988) Worm Breeder's Gazette "AF1, a sequenced bioactive neuropeptide isolated from the nematode, Ascaris suum."

Stretton AOW, Cowden C, Davis RE

[Worm Breeder's Gazette, 1988]

AF1 was isolated from an acid methanol extract of 10,000 Ascaris heads. The purification procedure consisted of a two-step fractionation with C18 cartridges, reverse phase HPLC (RP-HPLC) using three solvent systems, and HPLC gel filtration. Separations were monitored with a FMRFamide radioimmunoassay (RIA), using an antiserum provided by R. Calabrese. Material eluted from C18 cartridges with 50% acetonitrile in aqueous trifluoroacetic acid (TFA) was separated into several immunoreactive peaks by gradient RP-HPLC with butanol in aqueous TFA. Further purification was carried out by gradient RP-HPLC with acetonitrile in aqueous TFA. Subsequent HPLC gel filtration yielded several peaks of optical density not corresponding to the peak of immunoreactivity; in separate runs the active peak eluted earlier than FMRFamide, suggesting that the Ascaris peptide is bigger. The final step was isocratic RP-HPLC with acetonitrile in aqueous heptafluorobutyric acid (HFBA) yielding several peaks of optical density, one of which corresponded to the active peak. Half (55 pmoles) of the purified peptide was taken for amino acid sequence determination on a liquid phase sequenator, and gave the sequence KNEFIRF. Amidation of the C-terminus was indicated by the specificity of the antiserum used for RIA. Synthetic KNEFIRFamide ( sequence determination and peptide synthesis were performed by the Biotechnology Center, University of Wisconsin) coeluted with the natural peptide by HPLC gel filtration and by isocratic RP-HPLC. Since FMRFamide was first described, more than 20 FMRFamide-like neuropeptides have been isolated or predicted from a gene sequence. Only one of these, MDSNFIRFamide, predicted from the Drosophila FMRFamide gene, also has a C-terminal IRFamide. Four additional FMRFamide-like neuropeptides have been isolated from Ascaris, yielding two complete sequences and two partial sequences; these four peptides are also structurally different from previously known FMRFamidelike neuropeptides. Hence, it is clear that there exists a family of FMRFamide-like neuropeptides in Ascaris and it will now be possible to assess their physiological roles. In the cases from other phyla that have been analyzed, different isoforms of FMRFamide had quantitatively or qualitatively different physiological activities. There may also be diversity of action of the Ascaris FMRFamidelike neuropeptides. One action of AF1 seems to be inhibition of locomotory movements. Injection of 0.1 ml 10+E-6 M AF1 into the anterior portion of Ascaris ( N=15) blocks locomotory movements in the region of the injection. The physiological effects of AF1 on the Ascaris motornervous system were explored using intracellular recording techniques. AF1 exerts a dramatic effect on the electrical properties of the ventral inhibitory (VI) motorneurons: slow oscillatory potentials, whether spontaneous or induced by depolarization, were rapidly and reversibly blocked. Consistent block was obtained at a concentration of 10+E-7 M (5 of 5 preparations) with some preparations sensitive at a concentration as low as 10+E-9 M (5 of 12). AF1 will be useful in analyzing the physiological mechanisms underlying membrane potential oscillations in Ascaris.

Cowden C et al. (1993) J Comp Neurol "Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum."

Guastella J, Brackley P, Stretton AOW, Cowden C, Sithigorngul P

[J Comp Neurol, 1993]

By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extracts, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans.

Sithigorngul P et al. (1991) International C. elegans Meeting "DIFFERENTIAL DISTRIBUTION OF FMRFamide-LIKE IMMUNOREACTIVITY IN ASCARIS REVEALED BY MONOCLONAL ANTIBODIES GENERATED FROM AF1 AND AF2 PEPTIDES."

Stretton AOW, Sithigorngul P

[International C. elegans Meeting, 1991]

Monoclonal antibodies were generated from mice immunized with AFl ( KNEFIRFamide; Cowden et al., 1989) or AF2 (KHEYLRFamide; Cowden and Stretton,1990) peptides conjugated to BSA or OA. One monoclonal antibody binds to both AFl and AF2 peptides, but only very slightly to FMRFamide. In whole mount preparations of Ascaris, this antibody recognizes a subpopulation of neurons recognized by other monoclonal antibodies that bind to FMRFamide, as well as to AFl and AF2. This evidence suggests that FMRFamide-like peptides may be differentially localized in different subpopulations of neurons in the Ascaris nervous system.

Lee DA et al. (2005) J Med Genet "Genetics of skin appendage neoplasms and related syndromes."

Schneiderman P, Grossman ME, Celebi JT, Lee DA

[J Med Genet, 2005]

In the past decade the molecular basis of many inherited syndromes has been unravelled. This article reviews the clinical and genetic aspects of inherited syndromes that are characterised by skin appendage neoplasms, including Cowden syndrome, Birt-Hogg-Dube syndrome, naevoid basal cell carcinoma syndrome, generalised basaloid follicular hamartoma syndrome, Bazex syndrome, Brooke-Spiegler syndrome, familial cylindromatosis, multiple familial trichoepitheliomas, and Muir-Torre syndrome.

Sithigorngul P et al. (1989) J Comp Neurol "Generation of monoclonal antibodies against a nematode peptide extract: another approach for identifying unknown neuropeptides."

Stretton AOW, Guastella J, Sithigorngul P, Cowden C

[J Comp Neurol, 1989]

Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.

Edison AS et al. (1995) International C. elegans Meeting "Cloning and Sequencing of Multiple Transcripts of a FMRFamide-like Neuropeptide Gene in the Nematode Ascaris suum"

Messinger LA, Edison AS, Stretton AOW

[International C. elegans Meeting, 1995]

Five transcripts of a gene encoding a novel sub-family of six FMRFamide-like neuropeptides in the nematode Ascaris suum have been cloned and sequenced. The translated product of these transcripts is a precursor protein containing two main halves: a relatively hydrophobic region with no obvious peptides and a series of peptides separated by characteristic processing sites. The mature peptides share the C-terminal sequence PGVLRFamide but have different N-terminal sequences. Three of the peptides were previously isolated by immunocytochemistry [Cowden and Stretton, Peptides, in press] and three others are novel sequences. Of the transcripts, four have identical translated regions but differ in the 5' or 3' untranslated regions. A fifth transcript encodes a precursor protein with only the peptide-containing C-terminal domain.