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R H Scheller

Publications and source records attributed to R H Scheller.

At least 199 records · Page 11Linked to original sources
PubMed1988

Multiple neuropeptides derived from a common precursor are differentially packaged and transported.

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.

Animals↗
PubMed1988

Processing of the egg-laying hormone (ELH) precursor in the bag cell neurons of Aplysia.

Egg laying in Aplysia is mediated by a battery of neuropeptides released from the bag cell neurons. Predominant intermediates in the proteolytic processing of the Aplysia egg-laying hormone neuropeptide precursor were characterized using biochemical and immunological techniques. Following removal of the signal peptide, a rapid cleavage at the tetrabasic sequence Arg-Arg-Lys-Arg separates the amino and carboxyl regions of the prohormone. Processing of the carboxyl-terminal portion of the precursor then proceeds rapidly via two further cleavages at dibasic residues, resulting in a well defined product mixture within 4 h of chase. By contrast, processing of the amino-terminal side of the molecule proceeds only partially to completion after 20 h of chase and a well defined set of intermediates is not observed. Molecular genetic, physiological, and behavioral studies in conjunction with the biochemical investigations presented here are defining the information flow which governs the egg-laying behavior of Aplysia.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of a Drosophila neuropeptide gene.

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.

Amino Acid Sequence↗
PubMed1988

VAMP-1: a synaptic vesicle-associated integral membrane protein.

Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters.

Amino Acid Sequence↗
PubMed1988

Egg-laying hormone, serotonin, and cyclic nucleotide modulation of ionic currents in the identified motoneuron B16 of Aplysia.

We have used the 2-electrode voltage-clamp technique to analyze the effects of the neuropeptide, egg-laying hormone (ELH), and the biogenic amine 5-HT on ionic currents in the buccal motoneuron B16 of Aplysia. When B16 is voltage-clamped near resting membrane potential, bath-applied ELH induces a prolonged inward shift in holding current. The ELH-induced inward current is not due to a decrease in the transient, the delayed, or the calcium-activated potassium currents. Current-voltage measurements, along with ion substitution and channel-blocking experiments, indicate that ELH primarily induces or increases a voltage-dependent slow inward current carried by sodium. Serotonin also causes a prolonged inward shift in holding current in B16. Like ELH, 5-HT induces or enhances the voltage-dependent inward current carried by sodium. In addition, 5-HT increases an inwardly rectifying potassium current, and, in some preparations, decreases an outward current that is activated when the cell is depolarized to -40 mV and above. None of the currents modulated by ELH or by 5-HT are affected by 200 microM ouabain or by reducing extracellular chloride concentration. Extracellular application of isobutylmethylxanthine (IBMX), forskolin, 8-bromo-cAMP and 8-bromo-cGMP, and intracellular injection of cAMP elicits the slow inward current carried by sodium. The inward current response to ELH is blocked by prior application of 8-bromo-cAMP, while, under these conditions, 5-HT continues to elicit the increase in inward-rectifier potassium current, but decreases the slow inward sodium current. Serotonin also reduces the slow inward sodium current when applied after ELH. These results suggest that the modulation of B16 by ELH may be mediated entirely by either cAMP or cGMP, while at least a portion of the response to 5-HT may involve an unidentified second messenger in addition to cyclic nucleotides.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.

We used an antiserum against purified cholinergic synaptic vesicles from Torpedo and expression screening to isolate a cDNA clone encoding synuclein, a 143 amino acid neuron-specific protein. A cDNA clone was also isolated from a rat brain cDNA library that encodes a highly homologous 140 amino acid protein. The amino terminal 100 amino acids of both proteins are comprised of an 11 amino acid repeating unit that contains a conserved core of 6 residues. The synuclein gene is expressed only in nervous system tissue, not in electric organ, muscle, liver, spleen, heart, or kidney. In the electric organ synapse Torpedo synuclein-immunoreactive proteins are found in 3 major molecular-weight classes of 17.5, 18.5, and 20.0 kDa. In the neuronal cell soma the 17.5 kDa species is predominant and immunoreactivity is localized to a portion of the nuclear envelope.

Amino Acid Sequence↗
PubMed1987

Cellular and synaptic morphology of a feeding motor circuit in Aplysia californica.

The cellular and synaptic morphology of a component of the feeding motor circuit in Aplysia californica was examined with light and electron microscopic techniques. The circuit consists of a pair of inhibitory premotor interneurons, B4 and B5, as well as two motoneurons, B15 and B16, which innervate the accessory radula closer muscle. The neurons have wide, varicose arborizations in the buccal ganglion neuropil. All four of these neurons are cholinergic, and in addition, B15 contains immunoreactivity to sera raised against small cardioactive peptide B. Varicose processes in the accessory radula closer muscle are immunoreactive with antisera against several neuropeptides. We identified specific neuromuscular junctions by visualizing horseradish peroxidase uptake in recycled synaptic vesicles. Direct innervation of the accessory radula closer muscle by B15 and B16 is demonstrated by experiments in which horseradish peroxidase is transported from motoneuronal soma to the terminals on muscle fibers. In addition, specific synaptic contacts between B4 and B5 and each of the motoneurons are observed in the buccal ganglion neuropil. Finally, multiple contacts consistent with peptidergic, serotoninergic, and cholinergic synapses are made onto the neurons, suggesting that a variety of transmitters modulate motor output at each level of the hierarchical circuit. These results support the physiological evidence suggesting the involvement of neuropeptides as well as "classical" transmitters in the modulation of circuitry governing feeding behavior in Aplysia.

Animals↗
PubMed1987

Aplysia californica neurons express microinjected neuropeptide genes.

Neuropeptide genes are expressed in specific subsets of large polyploid neurons in Aplysia californica. We have defined the transcription initiation sites of three of these neuropeptide genes (the R14, L11, and ELH genes) and determined the nucleotide sequence of the promoter regions. The genes contain the usual eucaryotic promoter signals as well as other structures of potential regulatory importance, including inverted and direct repeats. The L11 and ELH genes, which are otherwise unrelated, have homology in the promoter regions, while the R14 promoter was distinct. When cloned plasmids were microinjected into Aplysia neurons in organ culture, transitions between supercoiled, relaxed circular, and linear DNAs occurred along with ligation into high-molecular-weight species. About 20% of the microinjected neurons expressed the genes. The promoter region of the R14 gene functioned in expression of the microinjected DNA in all cells studied. When both additional 5' and 3' sequences were included, the gene was specifically expressed only in R14, suggesting that the specificity of expression is generated by a multicomponent repression system. Finally, the R14 peptide could be expressed in L11, demonstrating that it is possible to alter the transmitter phenotype of these neurons by introduction of cloned genes.

Animals↗
PubMed1987

Histidine-rich basic peptide: a cardioactive neuropeptide from Aplysia neurons R3-14.

We have previously demonstrated that neurons R3-14 of the Aplysia abdominal ganglia specifically express a gene encoding a 108-amino acid neuropeptide precursor. This precursor is postranslationally processed by cleavage of a signal sequence and two internal dibasic residues resulting in three peptides. The peptide products are colocalized in dense core granules throughout the R3-14 processes that innervate the efferent vein of the gill and the auricle. Gel filtration and reverse phase high-pressure liquid chromatography (rpHPLC) were used to purify a 4.9-kDa peptide produced by the R3-14 neurons. We call this peptide the histidine-rich basic peptide (HRBP), which reflects its primary structure. In vitro tension measurements of cannulated Aplysia hearts revealed dose-dependent cardioexcitatory actions of HRBP. HRBP increased both beat frequency and amplitude with a threshold of 10(-7) M. HRBP increased the amplitude of ventricular contractions in a dose-dependent manner, whereas the frequency of contraction is unaffected. In contrast both the amplitude and frequency of auricular contractions were enhanced. High concentrations of HRBP also had a positive tonotropic effect on the auricle. HRBP was also demonstrated to have actions on tissue of the gut. Circular muscles of the crop adjacent to the anterior gizzard showed infrequent spontaneous contractions. Both HRBP and acetylcholine (ACh) induced repetitive contractions of this muscle. Circular muscles of the posterior gizzard had a high degree of spontaneous activity when continually perfused. Contraction amplitude and frequency was increased by HRBP and ACh, whereas contractility was inhibited by Phe-Met-Arg-Phe-amide (FMRFamide).

Amino Acid Sequence↗
PubMed1987

Peptidergic modulation of neuronal circuitry controlling feeding in Aplysia.

We examined the effects of 3 neuropeptides and the bioactive amine 5-HT on identified motoneurons (B15 and B16) and interneurons (B4, B5) involved in the control of feeding behavior in Aplysia californica. The application of egg-laying hormone (ELH), small cardioactive peptide b (SCPb), and 5-HT elicits distinct patterns of synaptically induced bursting in the neurons, while PheMetArgPheamide (FMRFamide) inhibits firing due to synaptic activity. Repetitive IPSPs recorded in B15 and B16 are induced by 5-HT and SCPb and inhibited by FMRFamide. The substances also may act directly: In solutions that block synaptic transmission SCPb excites B15, ELH excites B16, 5-HT excites B15, B16, and B4, and FMRFamide both inhibits B15 and B16 and excites B4. We suggest that the output of a buccal ganglion central pattern generator may be modulated to produce distinct patterns of motoneuron activity by these candidate transmitters. We also noted differences in the intrinsic properties of the 2 motoneurons. B15 contains SCPb immunoreactivity while B16 does not. This finding suggests that B15 may be the source for the SCPb immunoreactivity previously found at the ARC muscle and that SCPb may be acting in an autocrine mode. Also, B15 has a significantly lower resting potential than B16 and contains a large transient outward (Ia-like) current. The candidate transmitters act by exciting or inhibiting elements at every level within the hierarchically organized motor system that controls feeding. This expands the diversity of behavioral repertoires that may be elicited from a particular neural circuit.

Animals↗
PubMed1987

Proteolytic processing of the Aplysia egg-laying hormone and R3-14 neuropeptide precursors.

A number of animal behaviors are influenced by the actions of neuropeptides that arise from the processing of complex protein precursors. In this report we investigate the proteolytic processing of neuropeptide precursors expressed in the Aplysia californica bag cells, which govern egg-laying, and neurons R3-14, which mediate aspects of cardiac output. Peptides were purified by fractionation on 2 high-pressure liquid chromatography systems followed by determination of amino acid compositions. Most of these compositions are indicative of processing products derived from the egg-laying hormone (ELH) and R3-14 precursors by cleavage at basic residues. We characterized 9 peptides that arise from the ELH precursor by cleavage of the signal sequence, as well as 7 out of 8 dibasic residues and at least 1 single Arg residue. The peptides range in size from 5 to about 60 amino acids. The R3-14 neuropeptide precursor is cleaved at 2 internal dibasic residues in addition to the signal sequence, resulting in 3 peptides. Shortened forms of several peptides probably result from amino- and carboxy-terminal peptidase action. It is likely that the complex mixtures of neuropeptides arising from these single protein precursors are co-secreted.

Amino Acid Sequence↗
PubMed1986

Proteolytic processing of a peptide precursor in Aplysia neuron R14.

The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.

Amino Acid Sequence↗
PubMed1986

Expression of the egg-laying hormone gene family in the head ganglia of Aplysia.

RNA blotting and cDNA cloning techniques were used to study expression of the egg-laying hormone (ELH) gene family in the head ganglia of Aplysia californica. All head ganglia were found to express a 1800 nucleotide (nt) mRNA homologous to the ELH gene family. The nucleotide sequence of a clone isolated from a ring ganglia cDNA library demonstrates that this message encodes the ELH precursor. Further studies demonstrate the presence of a smaller, 1500 nt, transcript which encodes the peptide A precursor. However, the level of this mRNA is at least 10-fold lower than the ELH encoding message.

Animals↗
PubMed1986

Egg-laying hormone of Aplysia induces a voltage-dependent slow inward current carried by Na+ in an identified motoneuron.

This report presents studies on ionic currents in Aplysia motoneuron B16 that are modulated by the neuropeptide egg-laying hormone (ELH) of Aplysia. ELH induces an inward current that persists in the presence of the peptide and that decays slowly after ELH is removed from the bath. The effect is not due to a decrease in the delayed potassium current, the calcium-activated potassium current, or the transient potassium current. Current-voltage measurements indicate that ELH produces increased inward currents from -80 mV to approximately equal to 0 mV. The effect is particularly enhanced in the region from -40 mV to -25 mV where a negative slope conductance due to voltage-dependent slow inward current is observed. The slow inward current and the response to ELH persist in saline solutions in which Ca2+ is replaced with Co2+ but are eliminated when Na+ is replaced with equimolar concentrations of either Tris or N-methyl-D-glucamine. The response to ELH is unaffected by replacing chloride with equimolar acetate; by increasing the potassium concentration; or by adding tetraethylammonium chloride, CsCl, 4-amino-pyridine, or tetrodotoxin to the saline bath. In addition, the reversal potentials for the ELH response (range, -28 to +46 mV), obtained from difference current-voltage relationships, are consistent with an increase in the Na+-dependent slow inward current. We conclude that at least one of the effects of ELH on B16 is to increase a slow inward current carried by Na+.

Animals↗
PubMed1986

Localization of Aplysia neurosecretory peptides to multiple populations of dense core vesicles.

Many neurons in the mollusc Aplysia are identifiable and provide a useful model system for investigating the cellular mechanisms used by the neuroendocrine system to mediate simple behaviors. In this study we determined the subcellular localization of eight Aplysia neuropeptides using immunogold labeling techniques, and analyzed the size distribution of dense core and granular vesicles in peptidergic neurons. Recent observations demonstrate that many neurons use multiple chemical messengers. Thus, an understanding of the functional significance of cotransmitters requires an analysis of their relative subcellular distributions. The peptides are expressed in a subset of neurons, or the exocrine atrial gland, and are primarily localized to dense core vesicles. Multiple regions of precursors which are cleaved into several components are co-localized. Each neuron has a distinct size distribution of peptide-containing dense core vesicles ranging in size from 65 to 600 nm. The atrial gland contains very large (up to 2 micron) peptide-containing granules. Single neurons have multiple populations of granules whose quantal sizes agree with predictions based on physical constraints. Some cells contain very large peptide-containing granules which are found in the cell soma and not in processes. Thus, the genetic determination of neuronal cell type includes not only transmitter choices but also multiple modes of packaging the intercellular messengers.

Animals↗
PubMed1986

The Aplysia FMRFamide gene encodes sequences related to mammalian brain peptides.

We have characterized a precursor protein which gives rise to the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) by determining the nucleotide sequence of a genomic and five cDNA clones. The 597-amino-acid protein contains 28 copies of the tetrapeptide FMRFamide, a single Phe-Leu-Arg-Phe-amide (FLRFamide), and other sequences flanked by paired basic residues, some of which have homologies to mammalian brain peptides. The data presented suggest the genes encoding pro-opiomelanocortin, pre-pro-enkephalin, and the hypothalamic releasing factor, cortico-releasing factor (CRF), arose from a common ancestral gene.

Animals↗