Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5' flanking region and 478/477 nucleotides at the 3' flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish.

Feeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction. Members of the NG peptide family cause muscle contraction in other echinoderms (e.g. NGFFFamide in sea urchins and NGIWYamide in sea cucumbers), so we investigated NG peptides as candidate regulators of cardiac stomach retraction in starfish. Generation and analysis of neural transcriptome sequence data from A. rubens revealed a precursor protein comprising two copies of a novel NG peptide, NGFFYamide, which was confirmed by mass spectrometry. A noteworthy feature of the NGFFYamide precursor is a C-terminal neurophysin domain, indicative of a common ancestry with vasopressin/oxytocin-type neuropeptide precursors. Interestingly, in precursors of other NG peptides the neurophysin domain has been retained (e.g. NGFFFamide) or lost (e.g. NGIWYamide and human neuropeptide S) and its functional significance remains to be determined. Investigation of the pharmacological actions of NGFFYamide in starfish revealed that it is a potent stimulator of cardiac stomach contraction in vitro and that it triggers cardiac stomach retraction in vivo. Thus, discovery of NGFFYamide provides a novel insight into neural regulation of cardiac stomach retraction as well as a rationale for chemically based strategies to control starfish that feed on economically important shellfish (e.g. mussels) or protected marine fauna (e.g. coral).

Hyponatremia and the syndrome of inappropriate secretion of antidiuretic hormone (SIADH).

The syndrome of inappropriate ADH secretion (SIADH), also recently referred to as the "syndrome of inappropriate antidiuresis", is an often underdiagnosed cause of hypotonic hyponatremia, resulting for instance from ectopic release of ADH in lung cancer or as a side-effect of various drugs. In SIADH, hyponatremia results from a pure disorder of water handling by the kidney, whereas external Na+ balance is usually well regulated. Despite increased total body water, only minor changes of urine output and modest edema are usually seen. Renal function and acid-base balance are often preserved, while neurological impairment may range from subclinical to life-threatening. Hypouricemia is a distinguishing feature. The major causes and clinical variants of SIADH are reviewed, with particular emphasis on iatrogenic complications and hospital-acquired hyponatremia. Effective treatment of SIADH with water restriction, aquaretics, or hypertonic saline + loop diuretics, as opposed to worsening of hyponatremia during parenteral isotonic fluid administration, underscores the importance of an early accurate diagnosis and careful follow-up of these patients.

Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins.

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

Mutational Basin-Hopping: Combined Structure and Sequence Optimization for Biomolecules.

The study of energy landscapes has led to a good understanding of how and why proteins and nucleic acids adopt their native structure. Through evolution, sequences have adapted until they exhibit a strongly funneled energy landscape, stabilizing the native fold. Design of artificial biomolecules faces the challenge of creating similar stable, minimally frustrated, and functional sequences. Here we present a biminimization approach, mutational basin-hopping, in which we simultaneously use global optimization to optimize the energy and a target function describing a desired property of the system. This optimization of structure and sequence is a generalized basin-hopping method and produces an efficient design process, which can target properties such as binding affinity or solubility.

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I.

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.

[Small cell lung cancer associated with multiple paraneoplastic syndromes].

We report the case of a patient presenting with multiple severe electrolyte disturbances who was subsequently found to have small cell lung cancer. Upon further evaluation, she demonstrated three distinct paraneoplastic processes, including the syndrome of inappropriate antidiuretic hormone, Fanconi syndrome, and an inappropriate elevation in fibroblast growth factor-23 (FGF23). The patient underwent one round of chemotherapy, but she was found to have progressive disease. After 36 days of hospitalization, the patient made the decision to enter hospice care and later she expired.

Characterisation of two conopressin precursor isoforms in the land snail, Theba pisana.

Increased understanding of the molecular components involved in mollusc reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring. The neuropeptide conopressin, a member of the vasopressin/oxytocin-like peptide family, can modulate various reproductive activities in invertebrates. In this study, we used the hermaphroditic land snail, Theba pisana, to investigate the presence and tissue-specific distribution of a conopressin gene. Our transcriptomic analysis of T. pisana CNS sheath tissue has revealed two conopressin gene transcripts (Tpi-conopressin-1 and Tpi-conopressin-2), each encoding for precursors containing an identical conopressin nonapeptide and a variable neurophysin. T. pisana conopressins share high identity with other land snails and slugs, as well as other mollusc and vertebrate vasopressin/oxytocin, supported by phylogenetic analysis. Conserved residues in the T. pisana neurophysin are important for peptide binding, and we present molecular dynamic models demonstrating the most likely stable structure of the Tpi-conopressin-1 peptide when associated with neurophysin. RT-PCR shows that Tpi-conopressin-1 is additionally expressed in reproductive tissues, including the dart sac, where abundant spatial expression throughout the sac region is found; this implies a role in 'love' dart synthesis or dart injection during mating. The presence of a conopressin receptor in the CNS sheath indicates CNS neural excitation. In summary, this study represents a detailed molecular analysis of conopressin in a land snail.

Purification of 125I-labelled lysine-vasopressin by affinity chromatography on sepharose-bound neurophysins.

Purification of 125I-labelled lysine-vasopressin has been achieved by affinity chromatography on a Sepharose 4 B conjugate of porcine neurophysins. This affinity absorbent did not retain halogenated hormone, while native lysine-vasopressin was bound on the solid support. The specific activity of purified iodinated lysine-vasopressin was 1700-1800 Ci/g, corresponding to one iodine atom per mole. By comparison with an unpurified tracer, a five times increase in the minimum detectable dose was obtained in the vasopressin radioimmunoassay.

Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry.

Pro-vasopressin and pro-oxytocin are prohormones processed in the neurointermediate lobe pituitary to form the biologically active peptide hormones, arginine vasopressin (AVP) and oxytocin. Neurointermediate lobe pituitaries from normal (+/+), heterozygous (+/-), PC2-Null (-/-), PC1/3-Null and oxytocin-Null mice were analyzed by SELDI-TOF mass spectroscopy for the peptide hormone products, AVP, oxytocin and neurophysin I and II. Molecular ion species with masses characteristic of oxytocin, AVP, neurophysin I and II, i.e. 1009.41, 1084.5, 9677 and 9679 daltons respectively, were identified in all but the oxytocin-Null mice by comparison with synthetic standards or by C-terminal sequence analysis. Other ion species were found specifically in PC2-Null, heterozygote or normal mice. The results indicate that, in mice, both PC1/3 or PC2 enzyme activity are capable, but not required to correctly process pro-vasopressin or pro-oxytocin to their constituent active peptide hormones.

Crystallographic analysis of the neurophysin-oxytocin complex. A preliminary report.

Single crystals of a bovine neurophysin II-oxytocin complex have been obtained using (NH4)2SO4 as the precipitating agent. The crystals diffract to at least 2.7 A resolution, belong to Laue group 4/mmm and exhibit systematic absences consistent with either space group P4(1)2(1)2 or P4(3)2(1)2. The cell dimensions are a = b = 69.07 A and c = 113.26 A. The crystals contain one neurophysin-oxytocin dimer per asymmetric unit. Based on a Vm of 2.9 A3/Da, the solvent content is calculated to be 58%. Chromatographic analysis of the dissolved crystals suggests the presence of three oxytocin molecules per neurophysin dimer.

Crystals of a bovine neurophysin II tripeptide complex.

A bovine neurophysin II S-methyl-Cys-Tyr-Phe-NH2 complex has been crystallized using ammonium sulfate as the precipitating agent. The crystals are orthorhombic, the space group is either I222 or I2(1)2(1)2(1) with a = 124.9 A, b = 69.6 A and c = 151.5 A. The crystals diffract to at least 3.0 A resolution. Based on one neurophysin tetramer per asymmetric unit, the Matthews coefficient is calculated to be 3.92 with a solvent content of 69%.

Immunohistochemical detection of NRSA on small cell lung cancer with a monoclonal antibody (MAG-1) that recognizes the carboxyl terminus of provasopressin.

A single monoclonal antibody (MAG-1) directed against the C-terminal 18-amino acid region (VAGc18) of provasopressin was examined as an agent for recognizing the tumor-specific NRSA marker common to small cell lung cancer (SCLC) in formalin-fixed tissues with ABC immunohistochemistry. SCLC tumors were obtained from several tissue locations and included primary, metastatic, and recurrent disease. Positive staining was found in 91% of cases (53/58). All five of the unreactive tumors were of the lungs or chest wall, and there did not appear to be an association of this negativity with disease stage, age, or sex. Alternatively, almost all primary lesions, almost all metastatic lesions, and all recurrent lesions examined gave a positive reaction with MAG-1. For this study, vasopressin-producing cells of the human anterior hypothalamus served as a positive control, while negative controls comprised normal lung tissue, tumor that received MAG-1 in the presence of an excess of antigen (VAGc18 peptide), or tumor reacted with a commercial IgG1 isotype as primary antibody. All of the results indicate that MAG-1 can be effectively used to selectively identify the NRSA marker on almost all SCLC tumors, at all disease stages, and at all locations. Since all four tumors tested showing no reactivity with MAG-1 gave a positive reaction for synaptophysin, it is proposed that a combined use of MAG-1 with synaptophysin antibodies could allow all SCLC tumors to be detected by ABC immunohistochemistry.

Identification of a Novel Deletion in AVP-NPII Gene in a Patient with Central Diabetes Insipidus.

Central Diabetes Insipidus (CDI) is caused by a deficiency of antidiuretic hormone and characterized by polyuria, polydipsia and inability to concentrate urine. Our objective was to present the results of the molecular analyses of AVP-neurophysin II (AVP-NPII) gene in a large familial neurohypophyseal (central) DI pedigree. A male patient and his family members were analyzed and the prospective clinical data were collected. The proband applied to hospital for eligibility to be a recruit in Armed Forces. The patient had severe polyuria (20 L/day), polydipsia (20.5 L/day), fatique, and deep thirstiness. CDI was confirmed with the water deprivation-desmopressin test according to an increase in urine osmolality from 162 mOsm/kg to 432 mOsm/kg after desmopressin acetate injection. To evaluate the coding regions of AVP-NPII gene, polymerase chain reactions were performed and amplified regions were submitted to direct sequence analysis. We detected a heterozygous three base pair deletion at codon 69-70 (207_209delGGC) in exon 2, which lead to a deletion of the amino acid alanine. A three-dimensional protein structure prediction was shown for the deleted AVP-NPII and compared with the wild type. The three base pair deletion may yield an abnormal AVP precursor in neurophysin moiety, but further functional analyses are needed to understand the function of the deleted protein.

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.

Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms.

The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.

A de novo mutation in the coding sequence for neurophysin-II (Pro24-->Leu) is associated with onset and transmission of autosomal dominant neurohypophyseal diabetes insipidus.

The molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, a hereditary deficiency of vasopressin, was determined by nucleotide sequence analysis of the arginine vasopressin-neurophysin-II gene. A C-->T mutation at nucleotide 1761 was detected in one allele of this gene in each affected individual in three generations of one family. This mutant gene encodes a normal arginine vasopressin peptide, but predicts a substitution of leucine for proline at amino acid 24 of neurophysin-II, the arginine vasopressin carrier protein. This mutation was not detected 50 control individuals, thus demonstrating that it is not a common silent genetic polymorphism. The disease arose in the second generation of the studied family, and the chromosome 20 carrying this new mutation was identified by polymorphic CA microsatellite haplotype analysis. The first affected individual inherited this chromosome segment from her mother, who had neither the disease nor this mutation in her somatic cell DNA. Third generation individuals who subsequently inherited this mutation were affected. These data demonstrate that this amino acid substitution in neurophysin-II causes the disease. Two possibilities to explain the mechanism by which clinical deficiency of arginine vasopressin develops even in the presence of one normal arginine vasopressin-neurophysin-II allele are discussed.

Kinetics of precursor cleavage at the dibasic sites. Involvement of peptide dynamics.

The presence in the P'1 position relative to the LysArg doublet of either Phe, Tyr or Trp residues affects only pro-OT/Np(7-15) flexibility. This has a measurable effect on the dynamics of the peptide. Since the same modifications have a major influence on the K(m) and V(max) values of the peptide cleavage, these kinetic parameters should depend on the peptide substrate motions. Therefore, the primary kinetic contribution of substrate cleavage should arise from substrate dynamics rather than from the enzyme.

A diabetes insipidus vasopressin prohormone altered outside the central core of neurophysin accumulates in the endoplasmic reticulum.

Over 20 mutations affecting the neurophysin moiety of the vasopressin prohormone, have been identified in families suffering from familial neurohypophysial diabetes insipidus (FNDI). Only one of these, NP87E-->stop, is located outside the central conserved domain implicated in sorting of the vasopressin prohormone. To obtain clues about the mechanism of induction of FNDI by this atypical mutant we stably expressed wild type and NP87E-->stop vasopressin prohormones in (neuro)endocrine cell lines. Metabolic labeling and immunoprecipitation demonstrated reduced processing of the mutant prohormone to neurophysin. In addition, evoked secretion of neurophysin and vasopressin was diminished, suggesting that part of the mutant is retained in another intracellular compartment than the secretory granules. Indeed, immunofluorescence demonstrated accumulation of the truncated vasopressin prohormone in the endoplasmic reticulum. We conclude that the presence of the vasopressin moiety and the central conserved core of the neurophysin domain suffices for sorting and processing, but not for efficient endoplasmic reticulum exit of the vasopressin-neurophysin molecule.