Biomedical subjects

C R Jiménez

Publications and source records attributed to C R Jiménez.

15 recordsLinked to original sources

PubMed2005

Auto-inhibitory control of peptidergic molluscan neurons and reproductive senescence.

We recently, characterized a novel peptide of the egg-laying controlling caudodorsal cells (CDC) of Lymnaea stagnalis. Here, we show that the novel peptide has autoinhibitory actions and its expression is significantly up-regulated in reproductively senescent animals. Intracellular recordings show that when bath-applied to the central nervous system in vitro, the peptide reduces the depolarizing after potential (DAP) in CDCs without affecting the action potential-threshold and -amplitude and the resting membrane potential. Moreover, peptide application can terminate an ongoing after discharge in the CDCs or, when electrical stimulation fails to induce an after discharge, can terminate the long-lasting depolarization. Semiquantitative peptide profiling by mass spectrometry demonstrated correct processing and targeting of peptides in the CDC somata and axon terminals of reproductively senescent animals. Interestingly, the level of the autoinhibitory peptide was selectively increased in the CDCs of reproductively senescent animals. Our results indicate that a shift in balance between excitatory and inhibitory auto-transmitter peptides in the CDC system of old non-egg-laying animals, plays a role in after discharge failure in CDCs of reproductively senescent Lymnaea.

Action Potentials↗

PubMed2004

Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach.

The activity-dependent release of peptides from the neuro-endocrine caudodorsal cell (CDC) system of the freshwater snail Lymnaea stagnalis regulates egg laying and related behaviors. In this study, we optimized a mass spectrometry-based approach to study the spatio-temporal dynamics of peptides that are largely derived from the CDC hormone precursor during an egg-laying cycle and a CDC discharge in vitro. Semi-quantitative peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) indicated a massive depletion of peptides from the neurohemal area in the cerebral commissure (COM) during egg laying and the existence of a reserve pool of peptides in the CDC somata that were transported to the COM to restore peptide levels. The depletion of CDC peptides from the COM was correlated to their release during an induced electrical discharge in vitro. Moreover, MALDI-MS of the releasate revealed extensive truncation of the carboxyl terminal peptide. Finally, two novel peptides of 1788 and 5895 Da, not encoded by the CDC hormone precursor, also exhibited temporal quantitative changes similar to those of CDC peptides. Sequencing of the peptide of 1788 Da by tandem mass spectrometry yielded the novel sequence HF(FH)FYGPYDVFQRDVamide. Together, this implicates a more complex set of CDC peptides for the regulation of egg laying than previously anticipated.

Amino Acid Sequence↗

PubMed2003

Peptidergic modulation of male sexual behavior in Lymnaea stagnalis: structural and functional characterization of -FVamide neuropeptides.

In the simultaneous hermaphrodite snail Lymnaea stagnalis, copulation as a male is controlled by neurons that send axons to the male copulatory organs via a single penis nerve. Using direct mass spectrometry of a penis nerve sample, we show that one of the molecular ions has a mass corresponding to GAPRFVamide, previously identified from the buccal ganglia, and named Lymnaea inhibitory peptide (LIP). The identity of this peptide is confirmed by partial peptide purification from the penis nerve, followed by post source decay mass spectrometry. We cloned the LIP-encoding cDNA, which predicts a prohormone that gives rise to five copies of LIP (now re-named LIP A), two other -FVamide peptides (LIPs B and C), and five structurally unrelated peptides. The LIP gene is expressed in neurons of the right cerebral ventral lobe that send their axons into the penis nerve. We show that the LIP A peptide is present in these neurons and in the penis nerve, and confirmed the presence of LIP B and C in the penis nerve by post source decay mass spectrometry. Finally, we demonstrate that LIP A, B and C inhibit the contractions of the penis retractor muscle, thereby implicating their role in male copulation behavior.

Amino Acid Sequence↗

PubMed2002

Protein synthesis in synaptosomes: a proteomics analysis.

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Amino Acid Sequence↗

PubMed1998

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis.

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.

Animals↗

PubMed1998

Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network.

Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in vitro.

Amino Acid Sequence↗

PubMed1997

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling.

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Amino Acid Sequence↗

PubMed1997

Intracellular degradation of C-peptides in molluscan neurons producing insulin-related hormones.

Single Light Green Cells (LGC) of Lymnaea stagnalis, expressing four genes encoding insulin-related peptides (MIPs) and C-peptides, and sections from the median lip nerve (MLN) were subjected to MALDI-MS. Mass spectra of LGCs and MLNs were almost identical. Masses corresponding to those of the MIPs and some C alpha-peptides could be distinguished. ProMIP III C alpha-peptide and C beta-peptides were not found. The spectra showed additional masses matching those of carboxyterminally truncated C alpha-peptides. Peptides with similar masses were isolated from MLN extracts by HPLC, using electrospray-MS screening. Amino acid sequence analysis revealed intact proMIP I, II and V C alpha-peptides and I, II C alpha-peptide 1-24, 1-22 and 1-15.

Amino Acid Sequence↗

PubMed1996

Various isoforms of myomodulin identified from the male copulatory organ of Lymnaea show overlapping yet distinct modulatory effects on the penis muscle.

Male copulatory behavior in the snail Lymnaea stagnalis is controlled by several types of peptidergic neurons, including a cluster of neurons in the ventral lobe of the right cerebral ganglion that show immunoreactivity to myomodulin-A of Aplysia and innervate the penis complex. We identified structurally myomodulin-A and three related peptides from Lymnaea and showed that they are present in a characteristic ratio in both the penis nerve and penis complex, suggesting that they are processed from a single precursor and transported from the ventral lobe to the penis complex. All four peptides decreased the relaxation time of electrically evoked contractions of the penis retractor muscle. However, their effects on the amplitude of contraction were different, ranging from no effect to an increase or a decrease in the amplitude. A mixture of the peptides in a ratio as determined by direct mass spectrometry of the penis nerve decreased the contraction time, the relaxation time, and the amplitude. These effects resemble those of one particular peptide in the mixture. The direct mass spectrometry determinations of the peptide profile in the penis nerve suggest that many more, as yet unidentified, neuropeptides are involved in modulation of muscle activities of the penis complex.

Amino Acid Sequence↗

PubMed1994

Direct peptide profiling by mass spectrometry of single identified neurons reveals complex neuropeptide-processing pattern.

A novel strategy combining peptide fingerprinting of single neurons by matrix-assisted laser desorption ionization mass spectrometry, molecular cloning, peptide chemistry, and electrospray ionization mass spectrometry was used to study the intricate processing pattern of a preprohormone expressed in identified neurons, the neuroendocrine light yellow cells (LYCs) of the gastropod mollusc, Lymnaea stagnalis. The cDNA encoding the precursor, named prepro-LYCP (LYCPs, light yellow cell peptides), predicts a straightforward processing into three peptides, LYCP I, II, and III, at conventional dibasic processing sites flanking the peptide domains on the precursor. However, matrix-assisted laser desorption ionization mass spectrometry of single LYCs revealed trimmed variant peptides derived from LYCP I and II. The variants were much more abundant than the intact peptides, indicating that LYCP I and II serve as intermediates in a peptide-processing sequence. Using the molecular masses of the peptides as markers to guide their isolation by well established purification methods, the structural identities of the peptides could be confirmed by amino acid sequencing. Furthermore, matrix-assisted laser desorption ionization mass spectrometry could detect colocalization of a novel peptide with the LYCPs.

Amino Acid Sequence↗

PubMed1994

Processing, axonal transport and cardioregulatory functions of peptides derived from two related prohormones generated by alternative splicing of a single gene in identified neurons VD1 and RPD2 of Lymnaea.

The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and -B preprohormones. Here, we report the isolation and structural characterization of alpha 1, alpha 2 and beta peptides from dissected neurons VD1 and RPD2. The alpha 1 and alpha 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas beta peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the alpha 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic alpha 1 and alpha 2 peptides on isolated auricle preparations of the Lymnaea heart were examined. The two alpha peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, through non-identical, sets of peptides.

Amino Acid Sequence↗

PubMed1994

Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons.

Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI-MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI-MS study of direct peptide fingerprinting in the single neuron, these experiments demonstrate that MALDI-MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single-cell studies in vertebrates, including humans.

Animals↗

PubMed1994

Processing and targeting of a molluscan egg-laying peptide prohormone as revealed by mass spectrometric peptide fingerprinting and peptide sequencing.

The neuroendocrine cerebral caudodorsal cells of Lymnaea stagnalis initiate and coordinate ovulation and egg mass production and associated behaviors through the release of a complex set of peptides that are derived from the caudodorsal cell hormone-I (CDCH-I) precursor. We have previously characterized the CDCH-I peptide. In the present study, we isolated and amino acid sequenced by conventional peptide chemistry five additional peptides, epsilon-peptide, calfluxin, alpha-caudodorsal cell peptide, delta-peptide, and carboxyl-terminally located peptide, from the cerebral commissure, the neurohemal area of the caudodorsal cells. Fingerprinting by matrix-assisted laser desorption mass spectrometry of peptides in the commissure demonstrated the presence of all sequenced peptides and, in addition, could identify two other peptides derived from pro-CDCH-1, the beta 1- and beta 3-peptides. These findings together with previous immunocytochemical studies enabled us to define cleavage sites and major processing events of pro-CDCH-1. Pro-CDCH-1 is initially cleaved in the Golgi apparatus into carboxyl- and amino-terminal parts, each of which is sorted into distinct vesicle classes that traffic to different intracellular sites. As a result, in the commissure, peptides derived from the carboxyl-terminal part, including CDCH-1, are present at a many-fold higher concentration than those derived from the amino-terminal part.

Amino Acid Sequence↗

PubMed1992

Characterization of a cDNA clone encoding multiple copies of the neuropeptide APGWamide in the mollusk Lymnaea stagnalis.

Male mating behavior of the simultaneous hermaphrodite freshwater snail Lymnaea stagnalis is controlled by a neuronal network that consists of various types of peptidergic neurons, as well as serotonergic cells. In the present article, we describe the isolation and characterization of a cDNA clone that encodes a multipeptide preprohormone expressed in the anterior lobe of the right cerebral ganglion, in a group of neurons that principally innervate the penial complex. The preprohormone is 219 amino acids in length and contains 10 copies of the peptide Ala-Pro-Gly-Trp-Gly. Posttranslational processing of the prohormone may lead to the generation of the amidated neuropeptide Ala-Pro-Gly-Trp-amide (APGWamide), an amidated C-terminal anterior lobe peptide, and four connecting peptide sequences, C1-C4. We show by in situ and filter hybridizations that neurons of the right anterior lobe comprise the major site of expression of the APGWamide gene. Expression of the APGWamide gene is detected in the CNS of both adult animals and noncopulating juveniles. Peptides derived from the APGWamide prohormone are probably involved in the control of a part of the male mating behavior and have both central and peripheral targets.

Amino Acid Sequence↗

PubMedDate not supplied

Ultramicroanalysis of peptide profiles in biological samples using MALDI mass spectrometry.

In the past few years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has emerged as a powerful tool for the direct analysis of peptide profiles contained in single neuroendocrine cells, tissue biopsies, as well as in releasates (after a simple sample clean-up). These studies were performed in both invertebrate (the pond snail) and vertebrate (xenopus and rat) species. The present article provides an overview of these data with a special emphasis on the sample handling required for each application.

Animals↗