Isolation and identification of a cAMP generating peptide from the flesh fly, Neobellieria bullata (Diptera: Sarcophagidae).

The Manduca sexta Malpighian tubule assay system, developed to monitor adenylate cyclase activity, was used in combination with HPLC to isolate a novel cAMP generating peptide from 350,000 whole flesh flies, Neobellieria bullata. Mass spectrometry revealed a molecular mass of 5,047 daltons, and Edman degradation the following sequence: AGAEAEKLSGLSKYFNGTTMAGRANVAKATYAVIGLIIAYNVMKPKKK. This 48-mer peptide, called Neb-cGP, does not belong to the corticotropin releasing factor family of insect diuretic peptides. Electrophoresis and subsequent immunoblotting of peptides immunoprecipitated from a homogenate of entire flies showed that one fly contained approximately 0.003 to 0.03 micrograms Neb-cGP and that 10 micrograms represents the lowest immunostainable amount on a Western blot.

Isolation of Ala1-proctolin, the first natural analogue of proctolin, from the brain of the Colorado potato beetle.

Methanolic head and brain extracts of the Colorado potato beetle contain several myotropins, active in the Locusta oviduct motility assay. Reversed phase high performance liquid chromatography (RP HPLC) gave evidence for the presence of three myotropic factors, with retention times close to that of proctolin. Both strongly stimulated the frequency, amplitude and tonus of the myogenic oviduct contractions. Gas phase sequencing and FAB-MS revealed that, besides proctolin (Arg-Tyr-Leu-Pro-Thr), two natural proctolin analogues were present. The first one is Ala-Tyr-Leu-Pro-Thr and is designed as Ala1-proctolin. The threshold concentration for biological activity of Ala1-proctolin was 10(-7) M, compared to 10(-10) M for proctolin itself. Ala1-proctolin is the first identified biological analogue of proctolin. The full nature of the first amino acid of a third proctolin-analogue (x-Tyr-Leu-Pro-Thr) is probably a modified amino acid of which the identity could as yet not be revealed. Our results suggest the existence of a family of proctolin-like peptides.

Neb-colloostatin, a second folliculostatin of the grey fleshfly, Neobellieria bullata.

During the purification of trypsin-modulating oostatic factor (TMOF) of the grey fleshfly Neobellieria bullata, a new factor with oostatic activity was discovered. We report herein its purification, primary structure and effects on oocyte development. Its amino acid sequence was determined as H-SIVPLGLPVPIGPIVVGPR-OH. Due to structural sequence similarities with parts of several known collagens and its oostatic activity, we named it Neb-colloostatin. The synthetic peptide inhibits yolk uptake by previtellogenic oocytes and might have a role in the absence of yolk deposition in penultimate oocytes. Neb-colloostatin does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands. It decreases vitellogenin concentrations in the hemolymph by an unknown mode of action. The role of extracellular matrix proteins in the feedback control of growth is discussed.

Identification, characterization, and immunological localization of a novel myotropic neuropeptide in the Colorado potato beetle, Leptinotarsa decemlineata.

A novel myotropic heptapeptide was isolated from an extract of 54,000 heads of adult Leptinotarsa decemlineata by means of high performance liquid chromatography (HPLC), using the Locusta migratoria oviduct motility bioassay as monitoring system. The full primary structure was established as H-Ala-Tyr-Asn-Gly-Pro-Leu-Ala-NH2. This peptide, designated as Led-MNP-I, has a unique structure and does not belong to any known vertebrate or invertebrate peptide family. Two adjacent Led-MNP-I-immunoreactive perikarya were found in each optic lobe and in each half of all thoracic ganglia. Its absence from the pars intercerebralis and neurohemal organs suggests that Led-MNP-I is not a neurohormone but a neurotransmitter or neuromodulator. Treatment of isolated oviducts with varying concentrations of Led-MNP-I did not elicit significant changes in the level of cAMP concentration, suggesting that cAMP does not act as a second messenger for Led-MNP-I. Instead, Led-MNP-I induces an elevation of IP3. Treatment with Led-MNP-I did not stimulate cAMP production in the Colorado beetle brain, but this could be due to the very small number of receptive cells present. Both tissues contained a forskolin-sensitive adenylate cyclase enzyme.

Distribution of beta-endorphin-like-immunoreactive structures in the chicken and quail brain as demonstrated with a new homologous antibody directed against a synthetic peptide.

A polyclonal rabbit antibody was raised against a synthetic peptide fragment located at the C-terminal end of turkey beta-endorphin (beta-END) and used to analyze the distribution of beta-END-immunoreactive-like structures in the quail and chicken brain. Three major groups of immunopositive cells were detected in the preoptic area-hypothalamus complex. A thin layer of immunopositive cells was parallel and adjacent to the ventral edge of the brain in the preoptic and anterior hypothalamic region, a more numerous group of immunoreactive perikarya was located along the walls of the third ventricle in these same regions, and, finally, a few scattered cells were found in a more lateral position on both the internal and external sides of the tip of the fasciculus prosencephali lateralis. The periventricular cell population extended in the caudal direction until the posterior hypothalamus. Labelled fibers were always associated with these immunoreactive perikarya, and they were also found in the adjacent hypothalamic regions. A dense innervation of the median eminence was also detected. These data are compared with previous studies in mammals and birds that had identified more restricted populations of immunoreactive cells and the possible sources of the observed discrepancy are discussed. The functional significance of the present data is also briefly analyzed.

A simple method for the immunocytochemical processing of large numbers of floating sections and its application in screening for monoclonal antibodies.

A technically simple modification of routine (non-adsorbent) multi-well plates, permitting the simultaneous immunocytochemical processing of hundreds of free-floating sections is described. The adaptations consist of (1) making a 1.5 mm wide perforation in the bottom of each well of the multi-well plate, (2) placing a 6 mm wide 50 microns mesh nylon filter on the bottom of each well and (3) preincubating the plate with excess inert protein in order to prevent adsorption of protein reagents. During the incubation of the floating sections with the immunocytochemical reagents, the fluid is retained in the well by capillarity, provided the detergent concentrations within the well do not exceed 0.005% (v/v). The wells can be emptied simply and quickly by blotting the plate bottom with a piece of laboratory paper toweling: the fragile sections are gently caught on the filter, without the risk of loss or damage. Sections start floating again as soon as the next reagent is added to the well. The present method drastically reduces the time needed for rinsing and reagent exchange, making immunocytochemistry on free-floating sections feasible as a primary screening method during hybridoma production.

Sequencing and characterization of trypsin modulating oostatic factor (TMOF) from the ovaries of the grey fleshfly, Neobellieria (Sarcophaga) bullata.

Injection of crude extracts of late vitellogenic ovaries into staged females of the grey fleshfly Neobellieria (Sarcophaga) bullata inhibited oocyte development and biosynthesis of trypsin-like enzymes in the gut. Trypsin synthesis in N. bullata is cyclic and is correlated with egg development, which is discontinuous. A trypsin modulating oostatic factor (Neb-TMOF) was purified from 10,000 vitellogenic ovaries and sequenced by mass spectrometry. Neb-TMOF is a hexapeptide (NH2-NPTNLH-COOH). Injection of the hormone at physiological concentrations (10(-9) M), inhibited trypsin-like synthesis by the midgut of liver-fed female flies, and caused a reduction of the vitellogenin concentration in the hemolymph and of oocyte growth. The role of Neb-TMOF in controlling egg development and the physiological similarities with Aedes-TMOF are discussed.

Distribution of luteinizing hormone-releasing hormones I and II (LHRH-I and -II) in the quail and chicken brain as demonstrated with antibodies directed against synthetic peptides.

Polyclonal antibodies were raised in rabbits against polypeptides corresponding to the N-terminal part (heptapeptides) of the two avian gonadotropin-releasing hormones, chicken (c) LHRH-I and -II. These peptides, which were synthesized by the continuous-flow technique, were selected because they contained the smallest number of common amino acid residues. The pGlu-His-Trp-Ser sequence at the C-terminal was suppressed to avoid possible cross-reactions between the antisera. The antisera generated in this way were tested for specificity by solid and liquid phase absorption as well as by antigen spot tests. The antiserum raised against cLHRH-I recognized this peptide preferentially though not exclusively. Some cross-reaction with cLHRH-II was observed in the absorption test, although spotting tests suggested a total specificity. The anti cLHRH-II appeared to be completely specific in all tests. These two antibodies were then used to study the distribution of cLHRH-I and -II immunoreactive structures in the quail and chicken brain. cLHRH-I immunoreactive perikarya were observed in a fairly wide area covering the preoptic-anterior hypothalamic and septal region. By contrast, cLHRH-II cells were confined to a single group located in the dorsal aspects of the occulomotor nuclei, at the junction of the di- and mesencephalon. A sex difference in the number of cLHRH-I cells was detected in the anterior lateral preoptic region of the quail. Fibers immunoreactive for either cLHRH-I or cLHRH-II were widely distributed in the telencephalon, diencephalon, and mesencephalon but showed a specific pattern of anatomical localization. In particular, a high density of cLHRH-I fibers were seen in the external layer of the median eminence, while cLHRH-II fibers were less prominent at this level. Contrary to previous reports, a significant amount of cLHRH-II fibers were however seen throughout the median eminence (mostly external layer). The extensive distribution of both cLHRH-I and -II fibers in the quail and chicken brain is consistent with the potential role played by these peptides in the gonadotropin secretion and in the control of reproductive behavior. The specific role of cLHRH-II remains however elusive at present.

Immunocytochemistry and immunoblotting of avian prolactins using polyclonal and monoclonal antibodies toward a synthetic fragment of chicken prolactin.

A major obstacle in the production of specific antibodies toward chicken prolactin (PRL) has been overcome by mimicking a putative epitope of the molecule using the synthetic decapeptide Lys-chPRL 59-67. This peptide represents the highest hydrophilicity peak of the amino acid sequence of chPRL that was recently derived from the nucleotide sequence. Polyclonal mouse antisera against the fragment specifically recognized the lactotropes in the cephalic lobe of the chicken pars distalis as illustrated by immunocytochemical double staining experiments. Monoclonal antibody production yielded antibodies that specifically labeled purified turkey PRL upon SDS-PAGE separation and immunoblotting. Turkey and chicken PRL showed a very similar polymorphism with respect to their apparent molecular weights, including the occurrence of a glycosylated variant of chicken PRL. The monoclonal antibodies were finally used to demonstrate the presence of PRL-like immunoreactivity both in the pituitary gland and in the brain of the quail. In the brain, immunoreactive neurons were in the nucleus accumbens and in the lateral parts of the ventro-medial hypothalamus, partly similar to those described in the rat.

Isolation, identification and synthesis of novel oviductal motility stimulating head peptide in the Colorado potato beetle, Leptinotarsa decemlineata.

A novel myotropic peptide was isolated from an extract of 10,000 heads of adult Leptinotarsa decemlineata by means of high performance liquid chromatography (HPLC). The peptide stimulates the contractions of the oviduct of Leptinotarsa as well as that of Locusta migratoria. Gas phase sequencing and comparison of candidate synthetic peptides in the amide and acid form revealed the following primary structure: Ile-Ala-Tyr-Lys-Pro-Glu-NH2. This new peptide has a molecular weight of 720 Da and has been named Led OVM. Led OVM does not exhibit significant sequence homology with any known vertebrate or invertebrate peptide. Sixteen additional myotropic factors were also separated by means of HPLC, but were as yet not recovered in amounts large enough for them to be sequenced.