Poh Kheng Loi

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu‐Leu‐Tyr‐Ala‐Phe‐Pro‐Arg‐Val‐amide, has a molecular weight of 974.6 and is blocked at both the amino and car☐yl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.

Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl‐terminus is amidated. The structure of this peptide, Pro‐Phe‐Cys‐Asn‐Ala‐Phe‐Thr‐Gly‐Cys‐NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.

From behavior to molecules: an integrated approach to the study of neuropeptides

Despite extensive information on many aspects of peptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory peptides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes in target sensitivity and the distribution of the CAP-containing neurons, including a set of peptidergic neurons that alter their transmitter phenotype postembryonically. Studies show that inositol 1, 4, 5-trisphosphate (IP3), linked to intracellular Ca2+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.

Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta.

SUMMARY The gene coding for cardioacceleratory peptide 2b (CAP2b; pELYAFPRV) has been isolated and sequenced from the moth Manduca sexta (GenBank accession #AY649544). Because of its significant homology to the CAPA gene in Drosophila melanogaster, this gene is called the Manduca CAPA gene. The Manduca CAPA gene is 958 nucleotides long with 29 untranslated nucleotides from the beginning of the sequence to the putative start initiation site. The CAPA gene has a single open reading frame, 441 nucleotides long, that codes for a predicted precursor protein of 147 amino acids. The predicted prepropeptide encodes a single copy of each of three deduced propeptides, a CAP2b propeptide, with a Q substituted for an E at the N-terminus (QLYAFPRVa), and two novel CAP2b-related propeptides (DGVLNLYPFPRVa and TEGPGMWFGPRLa). To reduce confusion and to adopt a more standardized nomenclature, we rename pELYAFPRVa as Mas-CAPA-1 and assign the names of Mas-CAPA-2 to DGVLNLYPFPRVa and Mas-PK-1 (Pyrokinin-1) to TEGPGMWFGPRLa. The spatial and temporal expression pattern of the CAPA gene in the Manduca central nervous system (CNS) was determined in all major post-embryonic stages using in situ hybridization techniques. The CAPA gene is expressed in a total of 27 pairs of neurons in the post-embryonic Manduca CNS. A total of 16 pairs of cells is observed in the brain, two pairs in the sub-esophageal ganglion (SEG), one pair in the third thoracic ganglion (T3), one pair in each unfused abdominal ganglion (A1–A6) and two pairs in the fused terminal ganglion. The mRNA from the CAPA gene is present in nearly every ganglion in each post-embryonic stage. The number of cells expressing the CAPA gene varies during post-embryonic life, starting at 54 cells in first-instar larvae and declining to a minimum of 14 cells midway through adult development.

Roles of glutamate and FMRFamide-related peptides at the chromatophore neuromuscular junction in the cuttlefish, Sepia officinalis

Body patterning behavior, the expression of highly intricate patterns, is ubiquitous among all unshelled cephalopods. These body patterns are in part generated by the coordinated activity of millions of skin chromatophore organs, each of which is regulated by a set of chromatophore muscles directly innervated by centrally located chromatophore motoneurons. This study addresses the question of the identity and function of the transmitter(s) at the chromatophore neuromuscular junction (NMJ) in the European cuttlefish Sepia officinalis. Glutamate application causes a rapid contraction of the chromatophore muscles, resulting in chromatophore expansion. Pharmacological studies demonstrate that the chromatophore muscles contain receptors blocked by glutamate‐specific antagonists. Glutamate‐like immunoreactivity is also present in the somata of putative chromatophore motoneurons. These findings suggest that glutamate likely acts as a neurotransmitter at the chromatophore NMJ. Evidence is also presented suggesting that FMRFamide‐related peptides (FaRPs) also function as neurotransmitters at the Sepia chromatophore NMJ. FMRFamide application causes contraction of chromatophore muscles; however, the FMRFamide effect is slower and longer lasting than that of glutamate. Pharmacological data show that FMRFamide acts directly on the chromatophore muscles. FMRFamide‐immunopositive cells are present in the posterior chromatophore lobe, the putative location of the chromatophore motoneuron somata. A combination of immunocytochemistry and in situ hybridization shows that some putative chromatophore motoneurons express FaRP‐like immunoreactivity and an FaRP‐coding mRNA transcript. Many FMRFamide‐immunopositive cells in the posterior chromatophore lobes also express glutamate‐like immunoreactivity. We conclude that glutamate and FaRPs likely function as fast and slow transmitters, respectively, at the Sepia chromatophore NMJ. J. Comp. Neurol. 420:499–511, 2000.

PEPTIDE DETECTION IN SINGLE CELLS USING A DOT IMMUNOBLOT ASSAY

A highly sensitive dot immunoblot assay (DIA) for the detection and quantitative measurement of small peptides in single cells is presented. This DIA protocol is simple, rapid, and produces no radioactive waste. Its femtomole sensitivity is 100 fold greater than previously described DIAs. This DIA method is sufficiently sensitive to allow reliable peptide measurements to be obtained from a single cell in a manner than is faster and easier than other peptide detection procedures. This method can also be used for several other purposes, including assessing antibody specificity and peptide quantification.

Neural regulation of a complex behavior: body patterning in cephalopod molluscs

Unshelled cephalopods have a remarkable ability to alter their appearance, using textural, postural, and chromatic elements to generate a myriad of body patterns. Of the unshelled cephalopods, it is generally acknowledged that cuttlefish express the most detailed and widest range of body patterns, including static and dynamic patterns. In this paper we present data on the neuronal mechanisms underlying this amazing behavior, focusing on the neuroregulation of the chromatic elements, the chromatophore organs, in the European cuttlefish Sepia officinalis. Cephalopod chromatophore organs, including those in Sepia, are unlike those in any other animal taxa; each consists of a pigment-containing chromatophore cell that expands in response to the coordinated activation of a set of radial muscles which are directly attached to the chromatophore cell. We show that the chromatophore muscles are regulated by 2 different excitatory transmitters, glutamate and the family of FMRFamide-related peptides (FaRPs). Glutamate mediates rapid and transient chromatophore cell expansion whereas the FaRPs are responsible for slower, more sustained responses. Using retrograde dye filling, immunocytochemical and in situ hybridization techniques, we demonstrate that the cell bodies of the glutamatergic and FaRPs-containing motoneurons innervating the fin chromatophore muscles are primarily localized to the posterior chromatophore and fin lobes in the posterior subesophageal mass of the Sepia brain. Data are also presented showing that some fin chromatophore motoneurons have multiple axons in different nerve branches, which accounts for overlapping chromatophore motor fields by adjacent peripheral nerves.

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP2) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.