A high recovery microsampling device based on a microdialysis probe for peptide sampling.

A high recovery microsampling probe based on microdialysis was devised. The new probe showed a high recovery (100%) of peptides in vitro at different perfusion flow rates (0.1-1.0 microl/min). At a high flow rate, 1.0 microl/min, a 10-fold increased in recovery of peptides compared to the conventional microdialysis probe was achieved. A probe made of a low molecular weight cutoff membrane is suitable for filtering off proteins. The new probe can be a useful tool for high recovery of peptides from living tissues.

Isolation, cloning, and expression mapping of a gene encoding an antidiuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus.

After a blood meal, Rhodnius prolixus undergoes a rapid diuresis to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas' disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresis in R. prolixus is under the neurohormonal control of serotonin and peptidergic diuretic hormones, and thus, diuretic hormones play an important role in the transmission of Chagas' disease. Although diuretic hormones may be degraded or excreted, resulting in the termination of diuresis, it would also seem appropriate, given the high rates of secretion, that a potent antidiuretic factor could be present and act to prevent excessive loss of water and salts after the postgorging diuresis. Despite the medical importance of R. prolixus, no genes for any neuropeptides have been cloned, including obviously, those that control diuresis. Here, using molecular biology in combination with matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry, we determined the sequence of the CAPA gene and CAPA-related peptides in R. prolixus, which includes a peptide with anti-diuretic activity. We have characterized the expression of mRNA encoding these peptides in various developmental stage and also examined the tissue-specific distribution in fifth-instars. The expression is localized to numerous bilaterally paired cell bodies within the central nervous system. In addition, our results show that RhoprCAPA gene expression is also associated with the testes, suggesting a novel role for this family of peptides in reproduction.

Continuous and selective measurement of oxytocin and vasopressin using boron-doped diamond electrodes.

The electrochemical detection of oxytocin using boron-doped diamond (BDD) electrodes was studied. Cyclic voltammetry of oxytocin in a phosphate buffer solution exhibits an oxidation peak at +0.7 V (vs. Ag/AgCl), which is attributable to oxidation of the phenolic group in the tyrosyl moiety. Furthermore, the linearity of the current peaks obtained in flow injection analysis (FIA) using BDD microelectrodes over the oxytocin concentration range from 0.1 to 10.0 µM with a detection limit of 50 nM (S/N = 3) was high (R(2) = 0.995). Although the voltammograms of oxytocin and vasopressin observed with an as-deposited BDD electrode, as well as with a cathodically-reduced BDD electrode, were similar, a clear distinction was observed with anodically-oxidized BDD electrodes due to the attractive interaction between vasopressin and the oxidized BDD surface. By means of this distinction, selective measurements using chronoamperometry combined with flow injection analysis at an optimized potential were demonstrated, indicating the possibility of making selective in situ or in vivo measurements of oxytocin.

Characterization of neurophysin-vasopressin prohormones in human posterior pituitary tissue.

To better characterize putative neurophysin-vasopressin prohormones in human posterior pituitary tissue, we extracted human posterior pituitary glands in 0.1 M HCl and isolated the higher molecular weight neurophysin-immunoreactive proteins. Sephadex G-75 gel filtration in 0.1 M formic acid with 6 M urea showed four distinct peaks of neurophysin immunoreactivity. Analysis of isolated lyophilized fractions of these peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed neurophysin-immunoreactive proteins at molecular weights of 10,000 daltons (79-87% of the total neurophysins), 19,000-20,000 daltons (10-16%), 26,000-30,000 daltons (1-2%), and a broad range of 30,000- to 100,000-dalton immunoreactivity from the void volume (V0) peak (2-3%). The 19,000- to 20,000-dalton and 26,000- to 30,000-dalton proteins were stable after both heating and treatment with reducing agents, but could be converted by chymotrypsin proteolysis to 10,000-dalton neurophysins and 3,000- to 5,000-dalton AVP-immunoreactive proteins. In contrast, the neurophysin immunoreactivity in the V0 peak was broken down to lower molecular weight neurophysin- and AVP-immunoreactive proteins by heating alone. Extraction of human posterior pituitaries in the presence of either [125I]human AVP-neurophysin or [35S] cysteine-labeled monkey neurophysin showed that no labeled neurophysin eluted in the areas of the 19,000- to 20,000- or 26,000- to 30,000-dalton proteins, but a significant fraction of the [35S]monkey neurophysin eluted in the V0. These data suggest that the 19,000- to 20,000- and 26,000- to 30,000-dalton human neurophysins represent stable proteins which are probably common precursor molecules for neurophysin and AVP, but the greater than 30,000-dalton neurophysins found in the V0 appear to be aggregates of neurophysins, neurophysin precursors, AVP, oxytocin, and probably other proteins and lipids as well, rather than very high molecular weight precursor proteins.

Human neuropeptide Y, somatostatin and vasopressin precursors identified in cell-free translations of hypothalamic mRNA.

Messenger mRNA has been prepared from post mortem human hypothalami and translated in a cell-free system. Using specific antibodies, biosynthetic precursors have been identified to neuropeptide Y (12 kDa), somatostatin (15 kDa) and vasopressin/neurophysin (19 kDa).

The distribution of vasopressin and oxytocin in the hypothalamoneurohypophysial system of the guinea-pig.

1. The ratio of the content of vasopressin to that of oxytocin (V/O ratio) was estimated in the supraoptic nucleus (SON), paraventricular nucleus (PVN) and posterior pituitary gland (PIT) of guinea-pigs.2. Extracts were assayed for antidiuretic activity to estimate vasopressin and for milk-ejecting activity to estimate oxytocin. In assays for milk-ejecting activity, trypsin was used to inactivate vasopressin in the extracts.3. The mean V/O ratios in the SON, PVN and PIT were 28, 8.5 and 7.0 respectively in male guinea-pigs, 6.8, 7.4 and 6.9 in non-lactating females, and 5.1, 3.3 and 6.6 in lactating females.4. The distribution of the hormones within the hypothalamus is discussed in relation to their independent release in response to electrical stimulation of the SON and PVN.

Two vasopressin-like peptides in the pig testis?

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

Ganglioneuroblastoma of the thymus: an adult case with the syndrome of inappropriate secretion of antidiuretic hormone.

A 61-year-old woman was admitted to the hospital because of general fatigue. Laboratory examinations showed hyponatremia, plasma hypo-osmolarity, and inappropriate increased concentration of the plasma antidiuretic hormone (ADH) in the presence of concentrated urine. Magnetic resonance imaging revealed a mass lesion in the anterior mediastinum. An extended thymectomy was performed under the diagnosis of thymoma with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Histologically the tumor was located in the thymic tissue and was diagnosed as ganglioneuroblastoma. Immunohistochemical studies showed the existence of ADH in the tumor cells. To the knowledge of the authors, this is the first case of ganglioneuroblastoma of the thymus with SIADH.

A critique of the vasopressin-memory hypothesis.

During the past 20 years, evidence has accumulated to suggest that the neuropeptide vasopressin (VP) enhances memory by acting on central mechanisms, and that oxytocin (OT) has amnestic effects. In this review, the evidence for the memory hypothesis with respect to VP is considered and alternative interpretations evaluated. A critical approach has been adopted; negative findings, design considerations and problems with the various hypotheses are given prominence. It is concluded that the memory hypothesis fails to provide an adequate account, and some alternative theories and suggestions are discussed. It is speculated that the peptide may affect behaviour by two distinct mechanisms: peripheral action may involve reinforcement mechanisms, but its central role may be to modulate arousal level, especially in stressful situations.

Single-step isolation and sequencing of vasopressin and oxytocin precursors.

The pre- and post-Golgi processing of preprovasopressin and prepro-oxytocin was evaluated by microsequencing for incorporated radiolabel. 35S-Cysteine and 3H-fucose were microinjected into rat supraoptic nuclei (SON), and proteins and peptides related to the biosynthesis of vasopressin (VP) and oxytocin (OT) were isolated at various times from the supraoptic nuclei and neural lobe by employing a one-step procedure of high performance liquid chromatography (HPLC). These proteins and peptides were recognized through their binding to specific antibodies against VP, OT, and rat neurophysins (RNPs), and by their binding to ConA-Sepharose. Two immunoreactive glycoproteins related to VP biosynthesis were recovered from the SON and both contained fucose and had a 35S-cysteine placement consistent with the location of the hormone sequence at the N-terminus. SDS-electrophoresis revealed the major protein form to be 21,000 daltons and the minor protein form to be 19,000 daltons. One nonglycosylated protein of 16,000 daltons related to oxytocin biosynthesis was recovered from the SON, and this protein also had a 35S-cysteine placement consistent with an N-terminal OT sequence. These data provide the first sequential evidence that prior to, or shortly after, packaging in the Golgi the preprohormones of VP and OT have lost their entire leader-peptide structures.

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.

HPLC separation of vasopressin-like hormones in chicken neurohypophysial extracts.

HPLC techniques were used to identify peptides that possess vasopressin-like immunoreactivity in the chicken neurohypophysis. The presence of arginine vasotocin (AVT) was confirmed together with arginine vasopressin (AVP). That the presence of AVP may be confined to the chicken, and not other avian species, was concluded from the absence of the hormone in the neurohypophysis of the duck and turkey. The chicken thus resembles some members of the suiformes and metatheria in possessing two vasopressin-like peptides.

Biochemical characterization of a vasopressin-like neuropeptide in Locusta migratoria. Evidence of high molecular weight protein encoding vasopressin sequence.

The biochemical characterization of a diuretic neurohormone, immunologically related to the mammalian vasopressin (AVP) and present in Locusta migratoria has been performed. The results have been obtained using an AVP radioimmunoassay as method of detection and quantification. The "AVP like" molecule exhibits the same C terminal moiety: the tetrapeptide 1/2 Cys-PrO-Arg-Gly NH2. 125I-radiolabelling allows us to demonstrate the presence of a tyrosyl residue. The molecular weight of this molecule is estimated by gel filtration to 2500 +/- 400 Daltons. The isoelectric point is 7.5 and the electrophoretic migration lead to conclude to the presence of amino acid residues lacking in the vasopressin hormone. We have demonstrated the presence of a vasopressin sequence included in high molecular weight protein which have been quantified in suboesophageal ganglion (biosynthetic site) and in the nervous ventral cord (release site).

[Radio-immunoassay of antidiuretic hormone. Choice of a technic of extraction, Normal and pathological values (author's transl)]. / Dosage radio-immunologique de l'hormone antidiurétique. Valeurs usuelles et pathologiques. Choix d'une technique d'extraction.

The low serum concentration of ADH requires extraction prior to assay. Various methods are compared (direct extraction by acetone, adsorption on talcum powder. Florisil, silicilic acid, Fuller's earth, QUSO, carbon-dextran, adsormone, then elution with hydrochloric acetone). The technic finally used was extraction with Florisil. The antibody used was commercially accessible. The separation of bound from free hormone required a second antibody fixed on Sephadex (DASP). The usual values found in 31 normal subjects from 22 to 89 years were: 7,4 . 4,1 pmol/l (M 2 sigma) 1 pmol/l = 1,08 pg/ml. These assays were carried out on tissue extracts prepared by mashing tumour fragments or lymph nodes removed from a patient with the Schwartz Bartter syndrome. They showed the existence in these tissues of high concentrations of ADH immunologically identical with that present in the serum of normal subjects.

Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis.

Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called pro-sepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs.