Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

Functional vasoactive intestinal polypeptide (VIP)-system in salt glands of the Pekin duck

Summary In saltwater-acclimated ducks with fully specialized supraorbital salt glands, intracarotid application of acetylcholine (5 nmoles/min/kg b.w.) or porcine vasoactive intestinal polypeptide (pVIP) (240 pmoles/min/kg b.w.) induced secretion from the salt glands at threshold conditions of secretory activity. pVIP-like immunoreactivity could be localized in fibers of the postganglionic secretory nerve ramifying throughout the glandular parenchyma. Both middle-sized arterioles and secretory tubules were innervated, and pVIP-immunoreactive varicose fibers formed peritubular baskets around the basal region of secretory tubules indicating direct innervation of the secretory tissue. pVIP-specific staining could be abolished by preabsorption of the antiserum with peptide extracts of salt-gland tissue. Synthetic pVIP and endogenous VIP from salt glands of the duck co-eluted on the HPLC system, suggesting structural similarity of the peptides. Membrane-binding studies with radioiodinated pVIP revealed the presence of high-affinity binding sites in salt-gland tissue. Affinities of unlabeled pVIP analogues to compete for these binding sites were as follows: pVIP > PHI > pVIP antagonist > secretin > pVIP (10–28) > chicken VIP (16–28). Peptide extracts of salt glands had affinities similar to pVIP. Binding sites could be localized mainly at the apical end of the radially arranged secretory tubules, as demonstrated by receptor autoradiography.It is concluded that, in addition to the classical parasympathetic transmitter acetycholine, VIP serves as neuromodulator/transmitter in cranial parasympathetic control of avian salt-gland secretion by acting on both the arteriolar network and the secretory tubules of the gland.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

Induction of transverse polarity by blue light: An all-or-none response

Phototropic stimulation induces a spatial memory. This was inferred from experiments with maize (Zea mays L.) coleoptiles involving opposing blue-light pulses, separated by variable time intervals, and rotation on a horizontal clinostat (Nick and Schafer, 1988b, Planta 175, 380-388). In those experiments, individual seedlings either curved towards the first or towards the second pulse, or they remained straight. Bending, if it occurred, seemed to be an all-or-none response. Intermediates, i.e. plants, bending only weakly, were not observed. In the first part of the present study it was attempted to create such intermediates. For this purpose the strength of the first, inducing, and the second, opposing, pulse was varied. The result was complex: (i) Individual seedlings maintained the all-or-none expression of spatial memory. (ii) However, on the level of the whole population, the time intervals at which a given response type dominated depended on the fluence ratio. (iii) Furthermore, the final curvature was determined by the fluence ratio. These results are discussed in terms of a blue-light-induced transverse polarity. This polarity initiates from a labile precursor, which can be reoriented by an opposing stimulation (indicated by the strong bending towards the second pulse). The strong curvatures towards the first pulse over long time intervals reveal that, eventually, the blue-light-induced transverse polarity becomes stabilised and thus immune to the counterpulse. In the second part of the study, the relation between phototropic transduction and transverse polarity was characterised by a phenomenological approach involving the following points: (i) Sensory adaptation for induction of transverse polarity disappears with a time course similar to that for phototropic sensory adaptatation. (ii) The fluence response for induction of transverse polarity is a saturation curve and not bell-shaped like the curve for phototropism (iii) For strong counterpulses and long time intervals the clinostat-elicited nastic response (Nick and Schafer 1989, Planta 179, 123-131) becomes manifest and causes an "aiming error" towards the caryopsis. (iv) Temperature-sensitivity of polarity induction was high in the first 20 min after induction, then dropped sharply and rose again with the approach of polarity fixation. (v) Stimulus-summation experiments indicated that, for different inducing fluences, the actual fixation of polarity happened at about 2 h after induction. These experiments point towards an early separation of the transduction chains mediating phototropism and transverse polarity, possibly before phototrophic asymmetry is formed.     

Stimulation by auxin of phospholipase A in membrane vesicles from an auxin-sensitive tissue is mediated by an auxin receptor

Microsomal vesicles were prepared from zucchini (Cucurbita pepo L.) hypocotyls containing radioactive phosphatidylethanolamine or phosphatidylcholine, and these lipids were used as substrates by phospholipase A which is activated by auxins. Phospholipase D and phospholipase C hydrolysed the same substrates but were not influenced by auxin. Phospholipase A was activated by the auxins indolyl-3-acetic acid, 2,4-dichlorophenoxyacetic acid and, to a lesser extent, by -naphthaleneacetic acid whereas the weak auxins 2,3-dichlorophenoxyacetic acid and -naphthaleneacetic acid were almost inactive. This hormone specificity was also found in growth tests with etiolated zucchini hypocotyls. Phospholipase A activation by auxin was blocked by a polyclonal antibody against the maize auxin-binding protein. We propose that phospholipase A activation is a primary reaction in the signal transduction leading from hormone-binding to the growth response.Abbreviations IAA indolyl-3-acetic acid - 2,3-D, 2,4-D 2,3- and 2,4-dichlorophenoxyacetic acid - -NAA; -NAA - and -naphthaleneacetic acid This work was supported by the Deutsche Forchungsgemeinschaft. We thank D. Klämbt (Botanical Institute, University of Bonn, FRG) for a generous gift of polyclonal antibody (IgG fraction) against auxin-binding protein and U. Kutschera (Botanical Institute, University of Bonn, FRG) for advice with the growth tests.     

Nystatin and killer toxin sensitivity of free and immobilizedSaccharomyces cerevisiae

The inhibitory effect of nystatin and killer toxin on the growth of free and covalently-immobilizedSaccharomyces cerevisiae cells was studied. The resistance of immobilized cells to both agents was accompanied by increased amounts of phospholipids and sterols. The possible relationship between these changes in the membrane composition and the transduction of a signal across the cytoplasmic membrane is discussed.     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae

Summary Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the P_L promoter of bacteriophage , and expression was controlled using a cI_ts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.