Immunochemical and biological analysis of pheromone biosynthesis activating neuropeptide in Heliothis peltigera.

This study describes the preparation and characterization of a highly specific antiserum to Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN), and the use of this antiserum, in an enzyme linked immunosorbent assay (ELISA), to determine: a) the content of endogenous PBAN in head extracts of male and female Heliothis peltigera; b) the level of PBAN at different developmental stages; and c) the content of PBAN in four different moth species. Cross-reactivity studies revealed that the antiserum is directed mainly toward the N-terminal region of the neuropeptide, and that it exhibits similar binding affinities toward the oxidized and reduced forms of PBAN. Analysis of PBAN content in head extracts of male and female H. peltigera, at scotophase, revealed the presence of 4.97 and 4.58 pmol, respectively, in 3-day-old moths, and 5.33 and 4.78 pmol, respectively, in 7-day-old moths. The similarity in the content of PBAN at both ages and sexes was in accordance with the amount of pheromonotropic activity in these extracts which stimulated pheromone biosynthesis to a similar level. Analysis of PBAN-like immunoreactivity (IR) in head extracts of H. peltigera larvae and pupae demonstrated the existence of the neuropeptide in the 4th larval instar and continued to increase as a function of development. No IR could be detected in the first three larval instars. The larval and pupal extracts also exerted pheromonotropic activity which followed a similar pattern. The activity in these extracts, however, was considerably lower than that found in adult male and female heads. IR was also detected in head extracts of three other Noctuidae moths: Helicoverpa armigera, Cornutiplusia circumflexa and Spodoptera littoralis, indicating a high degree of chemical and structural similarity of PBAN in these moths.     

Spontaneous inactivation of enkephalin.

The role of isoleucyl-, valyl-, and leucyl-tRNA synthetases in attenuation of the $\text{ilvEDA}$ operon was examined. The results indicate that the activities of isoleucyl- and valyl-tRNA synthetases are necessary to maintain attenuation of the $\text{ilvEDA}$ operon. Leucyl-tRNA synthetase activity is nonessential for attenuation. These studies imply that uncharged tRNA^Ile and tRNA^Val each may cause deattenuation.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [(3)H]tyrosyl-PBAN28-33NH(2) to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO(3)(-) ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K(d) of 5.73 +/- 1.05 x 10(-6) M and a Bmax of 1.85 +/- 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH(2) and PBAN28-33NuEta(2) with a K(i) of 4.3 +/- 1.1 x 10(-6) M and 4.9 +/- 2.6 x 10(-6) M, respectively.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists.

A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C-terminal hexapeptide sequence Tyr-Phe-Ser-Pro-Arg-Leu-NH2 of PBAN1-33NH2; whereas library II (D-Phe library) was based on the sequence of the PBAN lead linear antagonist Arg-Tyr-Phe-d-Phe-Pro-Arg-Leu-NH2. In both libraries the Pro residue was replaced by the BBC building unit Nalpha-(omega-aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N-terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl3I for selective deprotection of the Boc group from the building unit prior to on-resin amino-end to backbone-nitrogen (AE-BN) cyclization, during solid-phase synthesis with Fmoc chemistry.     

PBAN-induced sex pheromone biosynthesis in Heliothis peltigera: Structure,dose, and time dependent analysis

A structure-activity relationship study of Hez-PBAN was performed with respect to its pheromonotropic activity, using Heliothis peltigera as the test animal. The activity of N- and C-terminally derived sequences was examined in a time- and dose-dependent mode. Using a variety of Hez-PBAN-derived fragments at two doses (1 and 10 pmol) and at different times post-injection (5–120 min), we were able to demonstrate that peptides lacking 12 and 16 amino acids from their N-terminus are as potent as the full length PBAN, and that the C-terminally derived hexapeptide was capable of stimulating sex pheromone production to a similar extent as PBAN 1–33NH₂, when its activity was analyzed at shorter post-injection times. Within the C-terminal sequence, the amide was found to play a crucial role. In addition, it was observed that the region between amino acids 9 and 13 is important for the biological activity of the full length PBAN. The fact that the pheromonotropic activity of the hexapeptide was similar to that of the full length PBAN, under specific conditions, suggests that this sequence constitutes the biologically active site of the neuropeptide. The discovery that PBAN-derived peptides reacted in a time- and dose-dependent mode, strengthens the assumption that proteolytic enzymes interfere with the pheromonotropic activity of the PBAN-derived fragments. The ability of a variety of peptides to stimulate sex pheromone biosynthesis suggests two possible mechanisms: (1) Existence of multiple pheromonotropic mechanisms which may be mediated by multiple PBAN receptors that are activated at different kinetics; (2) Existence of only one mechanism mediated by short C-terminally derived peptides. In the first case, the C-terminally derived sequences fulfill the conformational requirement of only one class of receptors, and other regions in the PBAN molecule (e.g., 9–13) fulfill the conformational requirements of a second (or other) class of receptors. In the second case, the C-terminally derived sequence is the only conformationally important sequence, and other sequences, which were found to be essential for the biological activity, serve other non-conformational purposes (e.g., protection against proteolytic degradation). © 1995 Wiley-Liss, Inc.     

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.