We have developed a method for dissecting single neurons from the nematode Ascaris suum, in order to determine their peptide content by mass spectrometry (MS). In this paper, we use MALDI-TOF MS and tandem MS to enumerate and sequence the peptides present in the two neurons, ALA and RID, that comprise the dorsal ganglion. We compare the peptide content determined by MS with the results of immunocytochemistry and in situ hybridization of previously isolated peptides AF2, AF8 and 6 peptides encoded by the afp-1 transcript. We find complete agreement between the three techniques, which validates single neuron MS as a method for peptide localization. We also discovered and sequenced 6 novel peptides in the ALA neuron. Cloning of cDNAs and database searching of Genomic Survey Sequences showed that transcript afp-12 encodes peptide AF36 (VPSAADMMIRFamide), and afp-13 encodes AF19 (AEGLSSPLIRFamide), AF34 (DSKLMDPLIRFamide), AF35 (DPQQRIVTDETVLRFamide), and 3 non-amidated peptides (PepTT, PepTL, and PepGE). We have found no similarities with reported peptide expression in the nematode Caenorhabditis elegans. This method promises to be ideally suited for determining the peptide content of each of the 298 neurons in the nervous system of this nematode.

We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.

Patients with Crohn's disease on recommended systemic medications may show a delayed and altered wound healing indicating that periodontal surgery must be closely monitored. Treatment planning should take into account the potential wound healing promoting effects of enamel matrix derivative as well as adverse healing effects of chlorhexidine gluconate administration.