Molecular cloning, mRNA expression and biological activity of the pheromone biosynthesis activating neuropeptide (PBAN) from the European corn borer, Ostrinia nubilalis.

Pheromone biosynthesis activating neuropeptide (PBAN) is a member of the pyrokinin (FXPRLamide) insect neuropeptides. Here, we report the cloning of the gene Ostnu-PBAN from the E and Z pheromone strains of the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae), a major pest of maize. The Ostnu-PBAN genomic sequence is > 5 kb in length and consists of six exons. The deduced amino acid sequence revealed a 200-residue precursor protein including a signal peptide, a 24-amino acid (aa) diapause hormone, a 37-aa PBAN and three other FXPRLamide neuropeptides. Our in vivo assays suggest that the 37-aa synthetic Ostnu-PBAN is hormonally active in the pheromone gland. It restores sex pheromone production to normal levels in mated females and decapitated virgins of both E and Z cultures. The results of a real-time PCR analysis indicated that Ostnu-PBAN mRNA levels reached a plateau in the brain-suboesophageal ganglion complexes 1 day after eclosion, and mating did not affect the mRNA expression. Three size classes of Ostnu-PBAN mRNA (1.9, 2.0 and 2.1 kb) were obtained, differing only in the length of the 3' untranslated region. However, there was no correlation between sequence divergence and the pheromone composition, voltinism or geographical origin (Hungary, Slovenia, Sweden, Turkey) of ECB moths.

Chemical characterization of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori.

Accumulation of lipid droplets within the cytoplasm is a common feature of the pheromone gland cells of many lepidopteran species. The cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori, were effectively extracted by dipping the trimmed glands in acetone for 10 min. In order to analyze the components originating from the lipid droplets, we separated the acetone extracts prepared before and after adult eclosion using HPLC, and specified the peaks showing a similar pattern of stage-dependence to that in the morphological change of the lipid droplets previously reported by Fónagy et al. (Arthropod Struct. Dev. 30 (2001) 113). Finally, we specified the peaks #1-5 and #1a-4a separated by reversed-phase HPLC as lipid droplet contents. Structure elucidation using FAB-MS and MS-MS analyses confirmed that they were triacylglycerols (TGs), and 12 species of TGs were identified as lipid droplet contents. Fatty acyl groups contained in these TGs were limited to five unsaturated C16 and C18 fatty acyl groups (delta 11-hexadecenoate, delta 10,12-hexadecadienoate, delta 9-octadecenoate, delta 9,12-ocatadecadienoate, and delta 9,12,15-ocatadecatrienoate), including the pheromone precursor delta 10,12-hexadecadienoate as a major component. Digestion with porcine pancreatic lipase confirmed that three major TGs eluted in the peaks #3-5 all contained C18 fatty acyl groups at the sn-2 position, indicating that the pheromone precursor is sequestered preferentially at the sn-1 and/or sn-3 position. Present results combined with the fact that the morphological change of the lipid droplets is under the control of PBAN indicate that the role of the cytoplasmic lipid droplets in the pheromone-producing cells is to store the pheromone precursor in the form of TGs and to provide it for pheromone production in response to the external signal of PBAN.

Physiological status and change of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori (Lepidoptera, Bombycidae).

Changes in size and number of cytoplasmic lipid droplets were quantified in the pheromone gland (PG) of Bombyx mori before and after adult eclosion. Two days before eclosion, size and number of droplets are small (diameter is 2-7 microm) and few. The formation and significant proliferation of larger droplets (5-12 microm) take place between 2 days and 1 day before eclosion. From the day of emergence until day 3 a fluctuation in size and number of lipid droplets during the photophase (4h intervals) is observed. The changes are more characteristic and dramatic on the day of emergence and first day, while attenuation of these changes can be observed from the second day and seems to disappear by day 4. Bombykol content, at each respective time, is in good correlation with the observed fluctuation in lipid droplet parameters. Highest bombykol production daily is observed towards the early evening, when lipid droplets are the smallest (2-4 microm) and most numerous. By day 4, however, this regularity also ceases. In 24h old mated females PG cell structure is quite similar to newly emerged ones. In glands of 72 h old decapitated females the formation of 'extra' large lipid droplets is remarkable. In vivo pheromone biosynthesis activating neuropeptide (PBAN) treatment, however, induced the formation of many small droplets, although numerous larger ones also remained. The morphological changes in lipid droplets and cellular dynamics associated with the external signal of PBAN in the PG suggest a storage-pool function of the lipid droplets.

Pheromone-producing cells in the silkmoth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli.

A method to isolate functional clusters of viable pheromone gland cells of Bombyx mori was developed. The 8th-9th intersegmental invaginated membrane corresponding to the pheromone gland was dissected, trimmed and separated into two distinct layers, the outer and inner layers, by enzymatic digestion with papain. The outer layer mainly consists of cuticle, while the inner layer consists of homogeneous cells with many refractile granules. The solubilized microsome fraction prepared from the inner layer retained the ability to produce bombykol in vitro, whereas the outer layer fraction did not produce bombykol. Moreover, in tissue incubations, the inner layer - but not the outer layer - produced bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. These results indicate that the inner-layer cells are indeed the pheromone-producing cells, which retain their functional integrity after separation with papain. These cells could be cultured successfully in Grace's medium for at least 5days.The presence or absence of pheromonotropic stimuli prior to dissection greatly influenced the size, number and distribution of refractile granules in the cytoplasm of the pheromone-producing cells. Staining with Nile Red proved that these refractile granules were lipid droplets. When pheromone production was studied under normal conditions or stimulated in decapitated females with pheromone-biosynthesis-activating neuorpeptide (PBAN) charge, the size of lipid droplets observed in the pheromone-producing cells reduced prominently and their number increased dramatically with time. By contrast, when pheromone production was suppressed by decapitation, the size and number of the lipid droplets remained constant. Lipid droplets observed in the pheromone-producing cells could be carriers of pheromone precursors and/or the pheromone bombykol. The present results suggest that the isolated cell preparation can be used for quantitative visualization of the cellular dynamics during pheromone production in B. mori.

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera).

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.

Isolation and characterization of calmodulin in the pheromone gland of the silkworm, Bombyx mori.

Production of the sex pheromone bombykol in the silkworm, Bombyx mori, is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN). It has been suggested that the external signal of PBAN in this species is transmitted to the intracellular cascade reactions consisting of Ca2+/calmodulin (CaM) complex and phosphoprotein phosphatase. To demonstrate the molecular mechanisms regulated by PBAN, we attempted to characterize CaM in the pheromone gland of B. mori. By using ion-exchange and RP-HPLC, B. mori CaM was purified from the cytosolic fraction of the pheromone gland. The primary structure was deduced by composition/sequence analysis and mass spectrometric analysis of the fragment peptides obtained from enzymatic and chemical fragmentations. The amino acid sequence of B. mori CaM was identical with Drosophila CaM deduced from the CaM gene of D. melanogaster, suggesting that insects have well conserved the molecule of CaM.

Effects of arginine vasopressin and atriopeptin on chloride uptake in cultured astroglia.

Ion and water homeostasis in the CNS is subjected to a neuroendocrine control exerted by neuropeptides formed within the brain. In order to gain information on this neuroendocrine control of Cl- homeostasis, 36Cl- uptake was measured in cultured Type-I astrocytes exposed to the neuropeptides [Arg8]Vasopressin (AVP), and atriopeptin (AP) and to various Cl- transport modifiers. AVP increased while AP decreased 36Cl- uptake of cultured astrocytes in a dose-dependent manner. Both effects became statistically significant at greater than 10(-9) M concentration of the peptides. For the appearance of the effects at least 30-min exposure was necessary. AVP and AP extinguished each other's effect by almost stochiometric manner. When administered together with AVP, the VIA vasopressin receptor antagonist "Manning compound" inhibited, while V2 vasopressin receptor agonist did not influence the 36Cl- uptake-increasing effect of AVP. However, bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransport, inhibited the effect of vasopressin and also inhibited the 36Cl- uptake of AVP non-treated, control cells. Our findings suggest that brain Cl- homeostasis is controlled by neuroendocrine system in the CNS.

Progesterone in Periplaneta americana and Neobellieria bullata adults from the procuticle phase until first progeny production.

A significant amount of progesterone-like immunoreactive material (150 ng/g) was measured by EIA in the procuticle phase of adult of both sexes of Periplaneta americana. This peak markedly decreased to 1-10 ng/g during sclerotization and was unlikely to be of dietary origin. In the case of 0-hr-old P. americana adults 96-98% of progesterone-like material was localized in the digestive tract and Malpighian tubules. In contrast, a relatively low level of progesterone-like immunoreactive material was measured in 0-hr-old Neobellieria bullata adults. Activity of 3beta-HSD/isomerase converting pregnenolone to progesterone was high (22-43 fmol/mg protein/20 min) in 0-hr-old P. americana adults and significantly fell during sclerotization. High progesterone levels (13-16 ng/g), measured by HPLC-RIA, coexist with high levels of 3beta-HSD/isomerase activity. Orally active human contraceptives (ethisterone, ethynodiol, ethynodiol diacetate, lynestrenol, mestranol, norgestrel, norethynodrel, tamoxifen citrate, and mifepristone) which act on mammalian steroid receptors had no significant effects on progeny production in either polytrophic or meroistic insect ovaries even at concentration of 5000 mg/kg.

A putative tetrapeptide antagonist prevents beta-amyloid-induced long-term elevation of [Ca2+]i in rat astrocytes.

Comparative fluorimetric studies on the long-term (8-hour) action of beta[1-42]amyloid and its shorter fragments beta[1-40], beta[25-35] and beta[31-35] on the steady-state intracellular Ca2+ concentration in primary cultures of rat astroglial cells using the Ca2+-sensitive fluorescent probe Fura-2 AM revealed higher 340/380 fluorescence excitation ratios in the treated cells as compared to the untreated controls. All the peptides were found to induce similar cellular effects, suggesting the [31-35] region as the putative active centre of the molecule. No significant alteration was detectable in Fura-2 fluorescence using the Ca2+-insensitive excitation wavelength of 367 nm, indicating that the observed changes reflect a real alteration in the Ca2+ concentration of the cells. Moreover, no considerable difference was observed in the total protein content of treated and untreated cells. Co-treatment of the cells with Pr-Ile-Ile-Gly-Leu-NH2 (Pr-IIGL) peptide, an analogue of the [31-34] region of beta[1-42]-amyloid, was found to effectively antagonize the beta[1-42]-amyloid-induced elevation of the fluorescence excitation ratio, leaving the 367-nm fluorescence unaffected. To the best of the authors' knowledge, this is the first report on an analogue of beta-amyloid peptide capable of blocking one of its physiological effects, thereby raising the possibility that this sequence could prove to be a lead compound for designing effective beta-amyloid antagonists.

Intracellular free Ca2+ elevations in cultured astroglia induced by neuroligands playing a role in cerebral ischemia.

For better understanding of glial participation in cerebral ischemia, spectrofluorimetric analysis using the calcium indicator Fura-2AM was applied to examine the role of intracellular free Ca2+ ([Ca2+])i elevation induced by different neuroactive substances in cultured rat brain astrocytes. The activation by the general receptor agonist glutamate resulted in a biphasic cell response in [Ca2+]i. We couldn't observe N-methyl-D-aspartate-evoked [Ca2+]i response at all. Quisqualate triggered a complex [Ca2+]i response in astrocytes consisting of mobilization of Ca2+ from the intracellular stores and also Ca2+ influx from the extracellular space. Kainate elicited a markedly different Ca2+ signal an external Ca(2+)-dependent sustained [Ca2+]i rise resulting from the activation of the ionotropic glutamate receptor. According to our results two types of glutamate receptors, the quisqualate-specific metabotropic and kainate-specific ionotropic receptor, are involved in [Ca2+]i elevation in these cultures. We could monitor agonist-specific cell response to noradrenaline, serotonin, vasopressin and ATP as well in these cultured rat astrocytes.

Nucleolar p120 is expressed as a delayed early response gene and is inducible by DNA-damaging agents.

Regulation of the expression of the growth-related nucleolar p120 protein was examined in serum-deprived and stimulated nontransformed and SV40-transformed WI-38 human fibroblasts. In quiescent cells, transcriptional activity of the p120 gene was very low or undetectable, and the steady-state levels of the p120 mRNA and the p120 protein were also negligible. The transient expression of the p120 gene in the cell cycle was detected in middle G1-phase after the expression of the early response genes and before the expression of the DNA-synthesis genes. Protein synthesis was required for the induction of p120 expression in serum-stimulated cells. The increased level of p120 mRNA in middle G1-phase was attributed to an increased transcription rate of the p120 gene, and not to a change in p120 mRNA stability. The calculated half-life of p120 mRNA was unchanged (1.8 +/- 0.2 hr) in all four cell conditions tested; i.e., in middle G1- or S-phase cells and in exponentially growing normal or transformed cells. Transcription rate of the p120 gene was correlated with the steady-state levels of either p120 protein or p120 mRNA. A sharp increase in p120 mRNA level occurred in both normal and transformed cells treated with actinomycin D used to examine p120 mRNA stability. This induction of p120 mRNA expression was seen in early G1-phase, but not in quiescent cells, or in middle to late G1-phase when cells expressed the highest level of p120 mRNA. The same expression pattern was seen by treatment with chlorambucil, another DNA-damaging agent. The conclusions of these studies are that the expression of p120 (1) is serum inducible in a fashion characteristic of the delayed early response gene products, (2) requires the presence of newly synthesized proteins, (3) is regulated transcriptionally, and (4) can be induced by DNA-damaging agents.

Altered transcription control is responsible for the increased level of proliferation-associated P120 in rapidly growing breast carcinoma.

Transcriptional and post-transcriptional regulation of proliferation-associated nucleolar P120 protein expression was examined in 1 normal and 5 malignant breast cell lines. The 6 breast cell lines could be placed into 3 categories on the basis of in vitro growth rate. BT549 and HBL100 grew rapidly; MCF-7/6, MCF-7/AZ and Hs578T grew at a moderate rate; and Hs578N normal epithelia grew slowly. There was a significant correlation between the growth rate, measured by percentage of S-phase fraction of cells or doubling time of cell numbers and the steady-state levels of either P120 protein or P120 mRNA. Next, the mechanisms responsible for the increased level of P120 expression, which is associated with rapidly growing breast carcinoma, were examined. The stability of P120 mRNA was measured by densitometry of quantitative Northern blots of mRNAs from cells treated with actinomycin D. Before the expected decay, a sharp increase in P120 mRNA level was detected shortly after inhibiting the overall RNA or protein synthesis. The calculated half-life of P120 mRNA was very similar (1.8 +/- 0.2 hr) in all 6 cell lines examined. The transcription rate of the P120 gene was determined by densitometric analysis of quantitative nuclear run-off assays. A significant positive correlation was found between the transcription rate of the P120 gene and the steady-state levels of either P120 protein or P120 mRNA. Our conclusion is that the expression of P120 protein is transcriptionally regulated in these breast cells. Therefore, the characteristically high level of P120 protein and mRNA found in most breast carcinomas is due to the altered transcription rate of the P120 gene in transformed cells.

Effect of nucleolar P120 expression level on the proliferation capacity of breast cancer cells.

Steady-state level of nucleolar P120 protein and P120 mRNA was compared to the doubling time and S-phase fraction in human breast cancer cell lines growing exponentially and in similar cells treated with a single dose of P120 antisense oligodeoxynucleotides. The study included six breast cancer cell lines and one nontransformed breast cell line with doubling times from 1.1 to 5.5 days and with S-phase fractions from 35 to 9%. P120 expression level was determined by densitometric computerized evaluation of protein and mRNA blots and with a quantitative 32P-reverse transcriptase-polymerase chain reaction method developed for small-scale samples. In the slowest growing normal cell line, P120 expression level was only about 10% of the level found in the most rapidly growing cancer cell line. The amount of P120 mRNA was highly correlated with the amount of P120 protein (P = 0.0001), indicating that P120 accumulation is regulated in these cells primarily at a transcriptional level. There was also a significant positive correlation between the level of P120 protein/mRNA and doubling time of cell lines (P = 0.0008) or percentage of S-phase cells (P = 0.210). P120 antisense oligomer treatment decreased the growth rate of cells in a dose-dependent manner, and the inhibition reached 70% at 100 microM concentration. Both P120 mRNA and P120 protein levels were also decreased by approximately 70% in cells treated with 100 microM P120 antisense oligomer. Slowly growing cells exhibited 50% inhibition by treatment at a proportionally lower concentration of P120 antisense oligomer than fast growing cells. This study shows that the expression of P120, measured either at the protein or the mRNA level, correlates with proliferation rate, identifying P120 as a cell proliferation marker.

Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts.

Normal and SV40 virus-transformed WI-38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [3H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non-synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G1-phase cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G1 and P120 protein synthesis initiated in middle G1. A dramatic increase of P120 protein level occurred in S-phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI-38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G1 to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation.

Antisense-mediated specific inhibition of P120 protein expression prevents G1- to S-phase transition.

A pentadecadeoxyribonucleotide (5'-AAAGCCCCCCACCAC), complementary to a splice junction site of mRNA for human proliferation-associated nucleolar protein P120, inhibited expression of the P120 gene and the mitogen-induced proliferation of human lymphocytes. The inhibition of P120 gene expression and proliferation was concentration dependent and reached 90% at 200 microM, as measured by [3H]thymidine uptake and by densitometric scanning of Northern (mRNA) and Western (protein) blots of P120. Inhibition was not observed in cells treated with the correspondent nonsense oligomer. P120 antisense oligomer treatment prevented S-phase entry of mitogen-stimulated lymphocytes, as determined by flow cytometric analysis, but did not block G0-G1 transition assessed by morphological blast transformation and induction of [3H]uridine incorporation. Results of this study suggest that P120 expression may be required for the upregulation of nucleolar function necessary for cell proliferation.

Isolation and primary structure of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata.

1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. A series of four, high-performance liquid chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis. 3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-II). 4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides. 5. Here we provide the first evidence for the expression of such peptides present in Dipterans. 6. Insect sulfakinins show structural identities with the hormonally-active portion of vertebrate gastrin II-, cholecystokinin- and caerulin-related peptides and they share common carboxy terminal sequences with invertebrate/vertebrate peptides of the FMRFamide peptide family.

Isolation, primary structure and synthesis of neomyosuppressin, a myoinhibiting neuropeptide from the grey fleshfly, Neobellieria bullata.

1. An amidated decapeptide, showing strong inhibitory activity of spontaneous visceral muscle movement was isolated, from head extracts of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. Amino acid sequencing and verification by peptide synthesis revealed the following primary structure: Thr-Asp-Val-Asp-His-Val-Phe-Leu-Arg-PheNH2. 3. The novel peptide was termed neomyosuppressin or Neb-MS. 4. During the process of consecutive high performance liquid chromatography (HPLC) purifications the biological activity of the samples was monitored using heterologous bioassay system. 5. The threshold level of synthetic Neb-MS was found to be 8.6 +/- 0.5 x 10(-11) M on the Leucophaea hindgut and 3.4 +/- 0.5 x 10(-10) M on the Locusta oviduct bioassay, respectively.

Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata.

A novel pheromonotropic neuropeptide has been isolated from a head extract of the armyworm larvae, Pseudaletia separata, by a seven step purification procedure using an in vivo assay with decapitated female moths of Bombyx mori. Amino acid sequence analysis and comparison with synthetic peptides established the primary structure of the peptide, termed Pseudaletia pheromonotropin (Pss PT), as H-Lys-Leu-Ser-Tyr-Asp-Asp-Lys-Val-Phe-Glu-Asn-Val-Glu-Phe-Thr-Pro-Arg-Le u-NH2. Pss PT is structurally related to leucopyrokinin, an insect myotropic neuropeptide, and possesses the C-terminal pentapeptide, Phe-Thr-Pro-Arg-Leu-NH2, responsible for the biological activity.