Oxytocin-like peptide: a novel epitope colocalized with the FMRFamide-like peptide in the supernumerary neurons of the sex segmental ganglia of leeches—morphological and biochemical characterization; putative anti-diuretic function

Salzet, Michel; Wattez, Christian; Verger-Bocquet, Martine; Beauvillain, Jean-Claudé; Malecha, Jean

doi:10.1016/0006-8993(93)91708-z

Cited by 26 publications

(24 citation statements)

References 25 publications

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“…This hypothesis is reinforced by our findings that in a more primitive animal, the annelid leech E. octoculutu, only one molecule related to the oxytocidvasopressin superfamily, the lysine-conopressin, has been isolated, the molecule immunoreactive to an anti-oxytocin present in the central nervous system [18] possessing a structure different from peptides of the oxytocidvasopressin superfamily (unpublished data). To conclude, it can be suggested that lysineconopressin is the ancestral molecule of the oxytocidvasopressin superfamily in coeomates.…”

Section: Biological Assaymentioning

confidence: 78%

Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata

Salzet

Bulet

Dorsselaer

et al. 1993

European Journal of Biochemistry

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Several neuropeptides are suspected to act on the control of hydric balance in leeches. One of these peptides, a peptide immunoreactive to an antibody against oxytocin, was previously characterized from the central nervous system of the leech Erpobdella octoculata [Salzet, M., Wattez, C., J. (1993) Brain Res. 601, 173-1841 This paper reports the isolation from the central nervous system of E. octoculata of another peptide of the oxytocidvasopressin family ; a lysine-vasopressin-like molecule. Its purification was performed by reverse-phase high-performance liquid chromatography combined with both dot immunobinding assay and enzyme-linked immunosorbent assay for lysine-vasopressin. The amino acid sequence was established by Edman degradation and confirmed by electrospray-mass-spectrometry measurement. The nonapeptide obtained corresponded to the lysine-conopressin previously isolated The advent of immunocytochemical techniques has strongly influenced thinking in invertebrate endocrinology. With antisera directed against various vertebrate neuropeptides, a large number of immunoreactivities have been demonstrated at the level of fibres and neurons in invertebrate nervous systems [l]. The improvement of chromatographical, immunological and molecular-biology techniques has greatly facilitated the purification and the determination of the sequences of neuropeptides in invertebrates. A comparison of the sequences of neuropeptides isolated from vertebrates and invertebrates has shown the existence of neuropeptides families, e.g. RFamides, tachykinins, opioids, gastridcholecystokinin peptides, oxytocinhasopressin peptides.In this investigation, we focused our interest on peptides of the oxytocidvasopressin superfamily which are widely distributed throughout the animal kingdom [2, 31. The peptides belonging to this superfamily are structurally related nonapeptides, having five invariant amino acid residues including two Cys that form a disulphide bridge and an amidated carboxy-terminal Glyc. These molecules control hydric Correspondence to M. Salzet, Laboratoire de PhylogCnie molCculaire des AnnClides, ERS

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Section: Biological Assaymentioning

confidence: 78%

Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata

Salzet

Bulet

Dorsselaer

et al. 1993

European Journal of Biochemistry

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“…Paraffin was removed by 2 washes in xylene for 5 min and lightly rehydrated with a graded ethanol series (100%, 96%, and 70%) before drying at room temperature. 25 FFPE Tissues Stored for 6 Months. 2,4 Dinitrophenylhydrazin (2,4-DNPH) or a mix of 2,4-DNPH and HCCA was used as matrix.…”

Section: Methodsmentioning

confidence: 99%

“…As expected, specific profiles were obtained depending upon the enzyme used ( Figure 6). Other fixation procedures were studied (e.g., Bouin's protocol 25 ) and successfully analyzed after enzymatic digestion (data not shown). It is thus possible to obtain and detect peptides/proteins directly from fixed tissues, by MALDI direct analysis after enzymatic digestion with various types of enzyme regardless of the fixation process used.…”

Section: Analysis Of Formalin-fixed Paraffin-embedded Tissue Sectionmentioning

confidence: 99%

“…Briefly, after dehydratation in successive graduated ethanol baths, tissue was fixed with 4% formalin in Tris-HCL buffer, pH 7.4, for 24 h. Tissues were then embedded in paraffin using xylene and strored in a box at room temperature until use. 25 Tissue Dewaxing and Preparation. Tissue sections of 10 µm were applied onto ITO-coated conductive glass slides.…”

Section: Methodsmentioning

confidence: 99%

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Direct Analysis and MALDI Imaging of Formalin-Fixed, Paraffin-Embedded Tissue Sections

et al. 2007

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Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffinembedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

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“…Among the 21 segmental ganglia (SG) of the ventral nerve cord of leeches, SG5 and SG6, which innervate the sexual organs, are designated as sex SG. These sex SG contain, compared to the nonsex SG [lo], an additional population of neurons immunostained with anti-FMRFamide in H. medicinalis [ll] cently, we demonstrated immunocytochemically and biochemically, in another leech, Erpobdella octoculata, the presence in these supernumerary cells of the sex SG of an RFamide epitope [12]. This RFamide epitope is colocalized with an epitope immunoreactive to an antiserum against oxytocin and exerting an anti-diuretic effect when injected in leeches [121.…”

mentioning

confidence: 81%

FMRFamide‐related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata

Salzet¹,

Bulet

Wattez³

et al. 1994

European Journal of Biochemistry

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Using enzyme-linked immunosorbent assays, a dot-immunobinding assay and a three-step reverse-phase HPLC separation, four Arg-Phe-amide (RFamide) peptides were purified from sex segmental ganglia extracts of the leech Erpobdella octoculata ; FMRFamide, FM(O)RFamide, FLRFamide and GDPFLRFamide. Their amino acid sequences were elucidated by means of a combined approach using antiserum specificity, synthetic-peptide coelution, automated Edman degradation and electrospray mass spectrometry. One of these peptides, GDPFLRFamide, is a novel leech RFamide neuropeptide.Two of the above RFamide peptides are involved in the control of leech hydric balance; one (GDPFLRFamide) is diuretic, the other (FMRFamide) is anti-diuretic.Titration of each purified RFamide peptide indicated a similar amount of each tetrapeptide and of tetrapeptides and heptapeptides. A comparison between RFamide peptides of E. octoculata and molluscs reveals structural similarities supporting the hypothesis for the existence of an ancestral RFamide peptide gene common to leeches and molluscs.The family of Arg-Phe-amide (RFamide) peptides is a group of peptides having, in common, the carboxy-terminal sequence Arg-Phe-amide. These peptides are found throughout the metazoans (for a review, see [l]) and notably in annelids.In this phylum, authentic FMRFamide is present in the polychaetes Nereis virens [2] and Nereis diversicolor [3]. In this last species, two other RFamide peptides [FM(O)RFamide and FTRFamide] have been isolated. Immunohistochemical and pharniacological data suggest that RFamides are involved in the control of the heartbeat and the body-wall tone of the polychaete Sabellastarte magnijka [4] as in the oligochaete Eisenia foetida [5]. In hirudinae, an immunoreactivity to anti-FMRFamide has been reported in cell bodies and neuronal processes throughout the central nervous system of Hirudo medicinalis [6, 71. In the segmental ganglia of the ventral nerve cord, this immunoreactivity is localized to heart-excitator motor neurons, heart-accessory modulatory neurons and several motor neurons innervating longitudinal and medio dorso ventral muscles [7-91. Among the 21 segmental ganglia (SG) of the ventral nerve cord of leeches, SG5 and SG6, which innervate the sexual organs, are designated as sex SG. These sex SG contain, compared to the nonsex SG [lo], an additional population of neurons immunostained with anti-FMRFamide in H. medicinalis [ll] cently, we demonstrated immunocytochemically and biochemically, in another leech, Erpobdella octoculata, the presence in these supernumerary cells of the sex SG of an RFamide epitope [12]. This RFamide epitope is colocalized with an epitope immunoreactive to an antiserum against oxytocin and exerting an anti-diuretic effect when injected in leeches [121.The physiological roles of RFamide peptides are well known in H. medicinalis where they act in the control of heartbeat [ 131 and in the contraction of longitudinal muscles [9]. Bath application of FMRFamide increases the strength and accelerates the rate of m...

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Oxytocin-like peptide: a novel epitope colocalized with the FMRFamide-like peptide in the supernumerary neurons of the sex segmental ganglia of leeches—morphological and biochemical characterization; putative anti-diuretic function

Cited by 26 publications

References 25 publications

Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata

Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata

Direct Analysis and MALDI Imaging of Formalin-Fixed, Paraffin-Embedded Tissue Sections

FMRFamide‐related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata

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