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.

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.

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).

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.