K.D. Buchanan

NEUROPEPTIDE-F - A NOVEL PARASITIC FLATWORM REGULATORY PEPTIDE FROM MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

Using a C-terminally directed pancreatic polypeptide (PP) antiserum and immunocytochemical methods, PP-immuno-reactivity (IR) was localized throughout the central (CNS) and peripheral nervous systems (PNS) of the cestode, Moniezia expansa . In the CNS, immunostaining was evident in the paired cerebral ganglia (primitive brain), connecting commissure, and the paired longitudinal nerve cords that are cross-linked by numerous regular transverse connectives. The PNS was seen to consist of a fine anastomosing nerve-net of immunoreactive fibres, many of which were closely associated with reproductive structures. Radioimmunoassay of this peptide IR in acid-alcohol extracts of the worm measured 192·8 ng/g of PP–IR. HPLC analyses of the M. expansa PP–IR identified a single molecular form which was purified to homogeneity. Plasma desorption mass spectrometry (PDMS) of purified parasite peptide resolved a single peptide with a molecular mass of 4599±10 Da. Automated gas-phase Edman degradation identified a 39-amino acid peptide with a C-terminal phenyl-alaninamide. Examination of its primary structure shows that it displays significant sequence homology with the vertebrate neuropeptide Y superfamily, suggesting that this platyhelminth-derived peptide is the phylogenetic precursor. Neuropeptide F ( M. expansa ) is the first regulatory peptide to be fully sequenced from the phylum Platyhelminthes and may represent a member of an important new class of invertebrate neuropeptide.

Neuropeptide F : primary structure from the tubellarian, Artioposthia triangulata

1. A neuropeptide exhibiting vertebrate pancreatic polypeptide immunoreactivity has been isolated and sequenced from extracts of the terrestrial turbellarian, Artioposthia triangulata. 2. This neuropeptide, designated neuropeptide F, consists of 36 amino acid residues terminating in a phenylalaninamide. 3. The full primary structure was established as: KVVHLRPRSSFSSEDEYQIYLRNVSKYIQLYGRPRF.NH2. The molecular mass, deduced from this sequence, was 4433 Da. 4. This neuropeptide exhibits C-terminal homology with neuropeptide F (Moniezia expansa) and with the vertebrate neuropeptide Y/pancreatic polypeptide superfamily of which it may represent a phylogenetic precursor.

The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera.

SummaryThe distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELATE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.

Isolation and primary structure of a novel chromogranin A‐derived peptide, WE‐14, from a human midgut carcinoid tumour

The primary structure of a novel human chromogranin A‐derived tetradecapeptide, WE‐14, possessing N‐terminal tryptophanyl(W) and C‐terminal glutamyl (E) residues was isolated from a hepatic metastasis of an human ileal carcinoid tumour. Human and bovine WE‐14 are structurally identical, while rat, mouse and porcine analogues exhibit 93 % homology. WE‐14 is flanked by paired basic residues (KR) in all known chromogranin A sequences.

The complete primary structure of pancreatic polypeptide from the European common frog, Rana temporaria

Using an antiserum directed against the highly-conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), numerous immunoreactive endocrine cells were identified within the pancreas of the European common frog, R. temporaria. An acidified ethanolic extract of pancreatic tissue (0.859 g, n = 35) contained 26.2 nmol equivalents/g of tissue. Gel permeation chromatography of the extract resolved a single peak of immunoreactivity co-eluting with synthetic bovine PP standard. Reverse phase HPLC of this material resolved a single peak of immunoreactivity which was purified to homogeneity following chromatography on a semipreparative C-18 column and an analytical C-8 column. Plasma desorption mass spectrometry (PDMS) of the purified peptide resolved a single component with a molecular mass of 4240.9 Da. Direct gas phase sequencing established the sequence of the first 26 residues. Following incubation of the peptide with endopeptidase Asp-N and direct application of the digest to the sequencer, the entire primary structure of the peptide was established as: APSEPHHPGDQATQDQLAQYYSDLYQYITFVTRPRF. The derived molecular mass of this peptide, incorporating a C-terminal amide, was 4240.6 Da which is entirely consistent with that obtained by PDMS.

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida, Oligochaeta)

SummaryThe localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.

Intact neurotensin (NT) in human plasma: response to oral feeding

Neurotensin-like immunoreactivity (NTLI) increases in human plasma postprandially. Intact neurotensin (NT) however, has been found to be a minor component of NTLI, the major components being the N-terminal fragments 1-11 and 1-8. Intact NT is the only known biologically-active form. A radioimmunoassay (RIA) has been developed which employs an antiserum unreactive to 1-11 or smaller N-terminal NT fragments. Using this RIA, intact NT response to a mixed meal has been assessed in 10 healthy humans. Intact NT levels were significantly elevated over basal 15 min after ingestion of the meal and remained so for the duration of the experiment (120 min). The suggestion that intact NT is a circulating hormone has been substantiated. Due to the rapidity of the rise in plasma NT after feeding it is proposed that the initial NT response is mediated by neural or hormonal means, rather than by direct luminal stimulation of the N cell-rich jejunoileum.

Differential processing of the neurotensin/neuromedin N precursor in the mouse.

Posttranslational processing of the neurotensin/neuromedin N (NT/NN) precursor has been investigated in mouse brain and small intestine by means of region-specific radioimmunoassays coupled to chromatographic fractionations. In brain, total NT/NN immunoreactivity measured with a common C-terminal antiserum was 15.72 pmol/g. NT measured with an N-terminal antiserum was 9.74 pmol/g and NN measured with an N-terminal antiserum was 5.98 pmol/g. In small intestine, combined NT/NN immunoreactivity was 108.55 pmol/g, consisting of 66.37 pmol/g NT but only 0.96 pmol/g NN. Gel permeation chromatography and reverse phase HPLC revealed that the large discrepancy in the NT and NN values obtained in small intestinal extracts was due to the presence of a high molecular weight, hydrophobic peptide, which was reactive only with the common C-terminally directed antiserum. Pepsinization of this generated an immunoreactive peptide with similar chromatographic characteristics to NN. In mouse intestine, NN is only partially cleaved from the common NT/NN precursor, resulting in the presence of an N-terminally extended molecular species. This novel molecular species of neuromedin N may be the physiological mediator of certain peripheral biological effects hitherto attributed to neurotensin or neuromedin N.

Tachykinin immunoreactivity in the parasitic flatworm Diclidophora merlangi and its fish host the whiting (Merlangius merlangus): Radioimmunoassay and chromatographic characterisation using region-specific substance P and neurokinin a antisera

1. Tachykinin immunoreactivity has been quantified and chemically characterised in extracts of the monogenean parasite, Diclidophora merlangi and its fish host, Merlangius merlangus, by means of four tachykinin radioimmunoassays interfaced with gel permeation chromatography and reverse-phase HPLC. 2. Of the two tachykinins identified in parasite tissue, one was SP-like and the other was NKA-like, although neither was identical to previously identified tachykinins. 3. Three tachykinins were identified in extracts of whiting GI tract, one of which was a neuropeptide and also occurred in whiting brain. 4. The parasite and fish tachykinins had different molecular weights and elution profiles in HPLC analyses, and were therefore chemically distinct.

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in human eccrine sweat glands: demonstration of innervation, specific binding sites and presence in secretions.

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) immunoreactivities have been detected in alcohol extracts of human axillary skin using sensitive and specific radioimmunoassays. VIP immunoreactivity (7.63+2.33 pmol/g, x+SE, n=9) was more abundant than PHM immunoreactivity (3.86+0.56 pmol/g, x+SE, n=9). Immunocytochemistry of sections of skin revealed a network of VIP/PHM immunoreactive nerve fibres around the perimeter of eccrine but not apocrine sweat glands. In vitro autoradiography of skin sections using 125I‐labelled VIP and PHM, demonstrated binding sites on the membranes of eccrine gland secretory cells. The binding of each radiolabelled ligand was eliminated by the presence of a large molar excess of appropriate cold peptide but was unaffected when incubated with related peptide, indicating the presence of specific binding sites for both VIP and PHM. Radioimmunoassay of Sep‐pak concentrated human sweat identified the presence of both VIP immunoreactivity (30.6 pmol/1) and PHM immunoreactivity (43.4 pmol/1). Reverse‐phase HPLC analysis of axillary skin extracts and sweat, followed by radioimmunoassay of fractions, identified single resolved peaks of VIP and PHM immunoreactivity with identical retention times to synthetic standards. Eccrine sweat glands in human axillary skin have VIP and PHM peptidergic innervation and possess specific binding sites for each peptide which are both secreted to the surface of the skin.