An unrecognized species of the Culicoides obsoletus complex feeding on livestock in The Netherlands.

In studies on Culicoides attacking livestock in the Netherlands, we chanced upon a species of the Obsoletus complex that we do not recognize, but whose dark wing pattern is distinctive. Nine cytochrome c oxidase (CO1) sequences of our so-called 'dark obsoletus' support its status as a separate species, the sequences differing significantly from those representing Culicoides obsoletus (Meigen) (90-91% homology) and Culicoides scoticus Downes & Kettle (87-88% homology). In the last decade, several research groups in Europe have encountered 'mystery species' related to C. obsoletus and in some instances have made their sequences for various genetic loci available in GenBank. These include a CO1 series submitted from Sweden in 2012 (annotated as 'obsoletus 01, 02, or 03 MA-2012') and of which some share a 99% identity with our sequences for 'dark obsoletus'. Without doubt, the series from the Netherlands, along with a portion of the Swedish submissions, together represent a single species ('dark obsoletus'). Whether this species is referable to the Russian Culicoides gornostaevae Mirzaeva recorded recently from Norway, Sweden and Poland, and based solely upon the external morphology of the male, is not clear. The presence in Western Europe of multiple undescribed species related to C. obsoletus means that the taxonomy of this important vector complex is not fully resolved; consequently, we know little about these cryptic species with regard to seasonality, geographic range and host preference. This is undesirable given that Culicoides-borne arboviruses causing disease in livestock are moving more regularly out of the tropics and spreading north into temperate latitudes.

The Hormone Domain of the Vasopressin Prohormone is Required for the Correct Prohormone Trafficking Through the Secretory Pathway.

It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct prohormone folding in the endoplasmic reticulum.

Neuropeptides in the therapy of Alzheimer's disease.

Owing to the heterogeneity of cognitive dysfunctions in patients with Alzheimer's disease (AD), a single drug may not suffice. Combination therapy may be the most appropriate solution, but the cognitive networks in the brain have yet to be unraveled. In the years ahead, more comprehensive and precise knowledge of neurological function will allow scientists to pinpoint ever more sophisticated and effective therapeutic options for AD patients. In this review, the author discusses the currently known advantages and disadvantages of neuropeptides and how they may provide yet another drug target for AD treatment.

Trafficking of the vasopressin and oxytocin prohormone through the regulated secretory pathway.

The trafficking of prohormones and of regulated secretory proteins in general has been studied extensively in the last decades of the last century. Prohormone trafficking starts with correct folding and subsequently efficient sorting into the secretory granule of the regulated secretory pathway. The vasopressin/oxytocin prohormone is particularly interesting for studying protein trafficking, because the physicochemical properties and three-dimensional structure have been largely elucidated. In the case of pro-vasopressin and pro-oxytocin, folding and sorting depend completely on both intramolecular and intermolecular interactions. Proper folding is guided by the hormone-neurophysin association and the sorting event relies on the aggregative properties of the neurophysin domain in the prohormone, as well as a specific sorting signal, which is revealed when the aggregative property of the neurophysin domain is deleted. A comprehensive mechanism for trafficking of the vasopressin/oxytocin prohormone from the endoplasmic reticulum to the secretory granule is proposed.

Processing of frameshifted vasopressin precursors.

Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones.

Sorting of the vasopressin prohormone into the regulated secretory pathway.

The sorting of soluble proteins into the regulated secretory pathway (RSP) involves aggregation, but whether an additional sorting domain is also required is not clear. By fusing vasopressin prohormone (proVP) fragments to green fluorescent protein (eGFP) we have determined whether a sorting domain can function independently of the aggregative neurophysin domain. Although eGFP itself can be immunolocalised in the RSP of Neuro2A and AtT20 cells, most of the protein enters the constitutive pathway, and is found in the culture medium. In contrast, the N-terminal 27 residues of proVP promote residence in the RSP. Furthermore, only the processed form of this fusion was secreted when stimulated. We suggest a sorting mechanism based on the recognition of a sorting motif, the efficiency of which is enhanced by neurophysin aggregation.

Novel opioid peptides endomorphin-1 and endomorphin-2 are present in mammalian immune tissues.

Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides, reported within the central nervous system, which have very high specificity and affinity for the mu-opioid receptor. We have used newly developed and well-characterised radioimmunoassays (RIAs) in combination with reversed-phase high-performance liquid chromatography (HPLC) to detect EM-1 and EM-2 immunoreactivity (ir) in rat immune tissues. Endomorphins were detectable in extracts of rat spleen (total EM-1-ir/spleen: 440+/-73 pg, mean+/-SEM, a=group of eight rats; EM-2-ir: 150+/-12 pg) and thymus (EM-1-ir: 152+/-18 pg, mean+/-SEM n=8; EM-2-ir: 156+/-28 pg). EM-2-ir was detectable in extracts of human spleen (338+/-196 pg/g tissue, n=3). Multiple peaks of EM-1-ir and EM-2-ir were observed in rat spleen and thymus extracts, and multiple peaks of EM-2-ir were observed in extracts of human spleen, following reversed-phase HPLC and RIAs. This is the first report of endomorphin immunoreactivity in tissues of the rat and human immune systems.

Preparation and characterization of the recombinant selenomethionine analogue of insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I), a single-chain polypeptide consisting of 70 amino acids and 3 disulfide bridges, is a member of a class of growth factors that are involved in many proliferative and metabolic processes. To assist in solving the crystallographic three-dimensional structure, we have expressed a recombinant fusion protein precursor of IGF-I in a methionine auxotrophic strain of Escherichia coli grown in the presence of selenomethionine. An homogeneous preparation of selenomethionyl-IGF-I was then obtained by chemical cleavage of the fusion protein. The selenomethionine analogue of IGF-I was characterized by electrospray mass spectrometry, peptide mapping, analytical chromatography, and electrophoresis as well as by biological assays. The final preparation of IGF-I was found to incorporate about 90% of selenium and fully retained the functional activity.

Structure-function relationships of the vasopressin prohormone domains.

1. In this review the structure-function relationships of the different vasopressin prohormone domains are dated and discussed, with special reference to the neurophysin and glycopeptide domains. 2. The primary structures of the currently known neurophysins and glycopeptide sequences are compared and discussed. 3. The hormone-binding and aggregational properties of neurophysin are reviewed and related to a possible function within the regulated secretory pathway. 4. It is proposed, based on the properties reviewed here as well as our own data shown here, that the sorting of the vasopressin prohormone is initiated by hormone binding, which triggers aggregation of the prohormone into the characteristic dense cores of the regulated secretory pathway. 5. This may suggest that prohormone sorting into the regulated secretory pathway is, in general, determined by noncovalent, intramolecular interactions that promote aggregation.

Functional domains in the oxytocin gene for regulation of expression and biosynthesis of gene products.

In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.

Heterologous biosynthesis and processing of preprovasopressin in Neuro2A neuroblastoma cells.

To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.

Properties of aminopeptidase activity involved in the conversion of vasopressin by rat brain membranes.

Previously it has been shown that vasopressin (VP) and oxytocin are converted by aminopeptidase activity in brain membranes into fragments with potent CNS activities. This report concerns the properties of this enzyme activity, addressed as VP-converting aminopeptidase (VP-AP) activity, in membranes of the rat brain. The VP-AP activity had a pH optimum at pH 7.0 and had a Km of 17 microM for its action on VP. Amastatin was the most potent aminopeptidase inhibitor. Enzyme activity was inhibited by relatively low concentrations of metal chelators. Treatment of brain membranes by EDTA resulted in loss of enzyme activity that was completely reversed by 10 microM Zn2+, indicating that VP-AP activity is a metallopeptidase. Several VP analogues and fragments, in particular VP(1-8), inhibited the action of enzyme activity on VP. Among peptides unrelated to VP, angiotension I, somatostatin, and porcine ACTH(1-39) markedly inhibited enzyme activity. Solubilization of VP-AP activity from brain membranes and gel filtration on Sephadex G200 showed two peaks of activity, one eluting with an apparent mass of about 140 kDa, the other in the void volume. Gel filtration fractions were able to convert [3H][Phe3]VP in a step-wise fashion. The VP-AP-like activity was found in many tissues outside the brain. Highest activity was present in lung, kidney, parts of the gastrointestinal tract, ovary, and uterus. The results indicate that VP-AP activity is a widely distributed enzyme with probably multiple functions, one of which involves the metabolism of vasopressin in the brain.

Testicular oxytocin gene expression in seminiferous tubules of cattle and transgenic mice.

We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha-inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans-acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue.