My life with Sydney, 1961-1971.

During the 1961-1971 decade, Sydney Brenner made several significant contributions to molecular biology-showing that the genetic code is a triplet code; discovery of messenger RNA; colinearity of gene and protein; decoding of chain terminating codons; and then an important transition: the development of the nematode Caenorhabditis elegans into the model eucaryote genetic system that has permeated the whole of recent biology.

Different Bioactive Neuropeptides are Expressed in Two Sub-Classes of GABAergic RME Nerve Ring Motorneurons in Ascaris suum.

Neuropeptides can have significant effects on neurons and synapses, but among the ∼250 predicted peptides in nematodes, few have been characterized functionally. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by five genes), nine of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle. Using peptide sequences obtained by tandem MS, we cloned the peptide-encoding transcripts and synthesized riboprobes for in situ hybridization (ISH). This complementary technique corroborated the results from single-cell MS, showing that the dissections were not contaminated with adhering tissue from other cells. We also synthesized a multiple antigenic peptide to raise a highly specific antibody against one of the endogenous peptides, which labeled the same cells detected by MS and ISH. Our results show that the RMEs can be divided into two subsets: RMED/V (expressing afp-2, afp-15, Asu-nlp-58, and high levels of afp-16) and RMEL/R (expressing afp-15 and low levels of afp-4 and afp-16). Almost all of these peptides are bioactive in A. suum.

Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum.

Neuromodulators have become an increasingly important component of functional circuits, dramatically changing the properties of both neurons and synapses to affect behavior. To explore the role of neuropeptides in Ascaris suum behavior, we devised an improved method for cleanly dissecting single motorneuronal cell bodies from the many other cell processes and hypodermal tissue in the ventral nerve cord. We determined their peptide content using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The reduced complexity of the peptide mixture greatly aided the detection of peptides; peptide levels were sufficient to permit sequencing by tandem MS from single cells. Inhibitory motorneurons, known to be GABAergic, contain a novel neuropeptide, As-NLP-22 (SLASGRWGLRPamide). From this sequence and information from the A. suum expressed sequence tag (EST) database, we cloned the transcript (As-nlp-22) and synthesized a riboprobe for in situ hybridization, which labeled the inhibitory motorneurons; this validates the integrity of the dissection method, showing that the peptides detected originate from the cells themselves and not from adhering processes from other cells (e.g., synaptic terminals). Synthetic As-NLP-22 has potent inhibitory activity on acetylcholine-induced muscle contraction as well as on basal muscle tone. Both of these effects are dose-dependent: the inhibitory effect on ACh contraction has an IC50 of 8.3 × 10(-9) M. When injected into whole worms, As-NLP-22 produces a dose-dependent inhibition of locomotory movements and, at higher levels, complete paralysis. These experiments demonstrate the utility of MALDI TOF/TOF MS in identifying novel neuromodulators at the single-cell level. Graphical Abstract ᅟ.

Different neuropeptides are expressed in different functional subsets of cholinergic excitatory motorneurons in the nematode Ascaris suum.

Neuropeptides are known to have dramatic effects on neurons and synapses; however, despite extensive studies of the motorneurons in the parasitic nematode Ascaris suum, their peptide content had not yet been described. We determined the peptide content of single excitatory motorneurons by mass spectrometry and tandem mass spectrometry. There are two subsets of ventral cord excitatory motorneurons, each with neuromuscular output either anterior or posterior to their cell body, mediating forward or backward locomotion, respectively. Strikingly, the two sets of neurons contain different neuropeptides, with AF9 and six novel peptides (As-NLP-21.1-6) in anterior projectors, and the six afp-1 peptides in addition to AF2 in posterior projectors. In situ hybridization confirmed the expression of these peptides, validating the integrity of the dissection technique. This work identifies new components of the functional behavioral circuit, as well as potential targets for antiparasitic drug development.

Three independent techniques localize expression of transcript afp-11 and its bioactive peptide products to the paired AVK neurons in Ascaris suum: in situ hybridization, immunocytochemistry, and single cell mass spectrometry.

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.

Changes in cyclic nucleotides, locomotory behavior, and body length produced by novel endogenous neuropeptides in the parasitic nematode Ascaris suum.

Recent technical advances have rapidly advanced the discovery of novel peptides, as well as the transcripts that encode them, in the parasitic nematode Ascaris suum. Here we report that many of these novel peptides produce profound and varied effects on locomotory behavior and levels of cyclic nucleotides in A. suum. We investigated the effects of 31 endogenous neuropeptides encoded by transcripts afp-1, afp-2, afp-4, afp-6, afp-7, and afp-9-14 (afp: Ascaris FMRFamide-like Precursor protein) on cyclic nucleotide levels, body length and locomotory behavior. Worms were induced to generate anteriorly propagating waveforms, peptides were injected into the pseudocoelomic cavity, and changes in the specific activity (nmol/mg protein) of second messengers cAMP (3'5' cyclic adenosine monophosphate) and cGMP (3'5' cyclic guanosine monophosphate) were determined. Many of these neuropeptides changed the levels of cAMP (both increases and decreases were found), whereas few neuropeptides changed the level of cGMP. A subset of the peptides that lowered cAMP was investigated for effects on the locomotory waveform and on body length. Injection of AF19, or AF34 (afp-13), AF9 (afp-14), AF26 or AF41 (afp-11) caused immediate paralysis and cessation of propagating body waveforms. These neuropeptides also significantly increased body length. In contrast, injection of AF15 (afp-9) reduced the body length, and decreased the amplitude of waves in the body waveform. AF30 (afp-10) produced worms with tight ventral coils. Although injection of neuropeptides encoded by afp-1 (AF3, AF4, AF10 or AF13) produced an increased number of exaggerated body waves, there were no effects on either cAMP or cGMP. By injecting peptides into behaving A. suum, we have provided an initial screen of the effects of novel peptides on several behavioral and biochemical parameters.

A specific antibody to neuropeptide AF1 (KNEFIRFamide) recognizes a small subset of neurons in Ascaris suum: differences from Caenorhabditis elegans.

A monoclonal antibody, AF1-003, highly specific to the Ascaris suum neuropeptide AF1 (KNEFIRFamide), was generated. This antibody binds strongly to AF1 and extremely weakly to other peptides with C-terminal FIRFamide: AF5 (SGKPTFIRFamide), AF6 (FIRFamide), and AF7 (AGPRFIRFamide). It does not recognize 35 other AF (A. suum FMRFamide-like) peptides at the highest concentration tested, nor does it recognize FMRFamide. When crude peptide extracts of A. suum are fractionated by two-step HPLC, the only fractions recognized by AF1-003 are those comigrating with synthetic AF1. By immunocytochemistry, antibody AF1-003 recognizes a small subset of the 298 neurons of A. suum: these include the paired URX and RIP neurons, two pairs of lateral ganglion neurons in the head, and the unpaired PQR and PDA or -B tail neurons that send processes to the head along the dorsal and ventral nerve cords, respectively. AF1 immunoreactivity is also seen in three pairs of pharyngeal neurons. Mass spectroscopy (MS) shows the presence of AF1 in the head, pharynx, and dorsal and ventral nerve cords. In A. suum, the neurons that contain AF1 show little overlap with neurons that express green fluorescent protein constructs targeting the flp-8 gene, which encodes AF1 in Caenorhabditis elegans (Kim and Li [2004] J. Comp. Neurol. 475:540-550); the URX neurons express AF1 in both species, but, in C. elegans, flp-8 expression was not detected in RIP, PQR, and PDA or -B or in the pharynx. Other, less specific monoclonal antibodies recognize AF1, as well as other peptides to differing degrees; these antibodies are useful reagents for determination of neuronal morphology.

Discovery of neuropeptides in the nematode Ascaris suum by database mining and tandem mass spectrometry.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs) or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1× coverage.

Mapping neuropeptide expression by mass spectrometry in single dissected identified neurons from the dorsal ganglion of the nematode Ascaris suum.

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.

In situ hybridization of neuropeptide-encoding transcripts afp-1, afp-3, and afp-4 in neurons of the nematode Ascaris suum.

The gene transcripts encoding both the AF8 and AF2 neuropeptides of the nematode Ascaris suum have been identified, cloned, and sequenced. The AF8 transcript (afp-3) encodes five identical copies of AF8; each peptide-encoding region is flanked by the appropriate dibasic or monobasic cleavage processing sites. The AF2 transcript (afp-4) encodes three identical copies of AF2 along with the appropriate cleavage sites. In contrast, the afp-1 transcript (Edison et al. [1997] Peptides 18:929-935) encodes six different AF peptides (AF3, 4, 10, 13, 14, 20) which all share a -PGVLRFamide C-terminus but have different N-terminal sequences. By using in situ hybridization, gene transcript expression patterns of afp-1, afp-3, and afp-4 (As-flp-18, As-flp-6, and As-flp-14, respectively, in the naming convention proposed by Blaxter et al. [1997] Parasitol Today 13:416-417) were determined in the adult A. suum anterior nervous system. Each gene transcript can be localized to a different subset of neurons. These subsets of neurons are different from the subsets of Caenorhabditis elegans neurons that were shown to express identical or similar peptides by the use of promoter GFP constructs (Kim and Li [2004] J Comp Neurol 475:540-550).

Peptide products of the afp-6 gene of the nematode Ascaris suum have different biological actions.

Matrix-assisted laser desorption/ionization time-of-flight and tandem time-of-flight (MALDI-TOF and MALDI-TOF/TOF) mass spectrometry were used to sequence and localize three novel, related neuropeptides in the nervous system of the nematode Ascaris suum, AMRNALVRFamide (AF21), NGAPQPFVRFamide (AF22), and SGMRNALVRFamide (AF23). The amino acid sequences were used to clone a novel neuropeptide gene (afp-6) that encodes a precursor bearing a single copy of each of the peptides. In situ hybridization and immunocytochemistry revealed that both the transcript and the peptides are expressed in a single cell in the ventral ganglion. Pharmacological studies of intact nematodes injected with these peptides, as well as physiological studies of responses to them in muscle tissue, motor neurons, and the pharynx, reveal that these peptides have potent bioactivity in the locomotory and feeding systems. Further exploration of their effects may contribute to our understanding of neuropeptide modulation of behavior and also to the development of compounds with anthelmintic relevance.

Mass spectrometric map of neuropeptide expression in Ascaris suum.

A mass spectrometric method was used for the localization and sequence characterization of peptides in the nervous system of the parasitic nematode Ascaris suum. Mass spectrometric techniques utilizing MALDI-TOF, MALDI-TOF/TOF, and MALDI-FT instruments were combined with in situ chemical derivatization to examine the expression of known and putative neuropeptides in the A. suum nervous system. This first attempt at peptidomic characterization in A. suum mapped the expression of 39 neuropeptides, 17 of which are considered to be novel and whose expression has not been previously reported. These analyses also revealed that the peptide expression profile is unique to each nervous structure and that the majority of peptides observed belong to the RFamide family of neuropeptides. In addition, four new peptide sequences with a shared C-terminal PNFLRFamide motif are proposed based on in situ sequencing with mass spectrometry.

De novo sequencing of novel neuropeptides directly from Ascaris suum tissue using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight.

Direct analysis of tissue by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows for the rapid profiling of biological molecules with minimal loss of sample or degradation and reduced likelihood of chemical modification. However, there are still considerable challenges to overcome due to the complexity of tissue and the low quantity of endogenous peptide in a single cell. These problems are exacerbated in the nematode Ascaris suum because of the small size of individual neurons and the paucity of peptide per cell. In an effort to address these difficulties, the recently developed matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) technology was used in combination with an on-target derivatization in order to sequence novel neuropeptides directly from Ascaris nervous tissue. Direct MALDI-TOF/TOF analysis of Ascaris tissue provided the complete amino acid sequences for a previously characterized neuropeptide as well as for three novel peptides with homologues found in other nematodes. These results demonstrate a method for the rapid characterization of sub-femtomolar amounts of peptide directly from tissue using MALDI-TOF/TOF.

A strategy for isolating rare peptides: isolation and sequencing of a large peptide present in a single neuron of the nematode Ascaris suum.

Monoclonal antibody G15-6A was generated by immunizing mice with Ascaris head extracts. It recognizes an antigen present in a single neuron, with a cell body in the dorsal rectal ganglion, that projects along the ventral cord to the nerve ring. Ascaris extracts were fractionated by HPLC and ammonium sulfate precipitation, and fractions assayed by dot-blotting with antibody G15-6A. A single immunoreactive polypeptide was purified; mass spectrometry showed a molecular weight of 11,542 Da. Partial N-terminal sequencing, followed by cloning of the transcript encoding the peptide, revealed a predicted peptide product comprising 109 amino acids, and a molecular mass of 11,863 Da. The N-terminus of the predicted peptide includes four more amino acids than are found in the isolated product.