Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum.

Aphids respond to environmental changes by developing alternative phenotypes with differing reproductive modes. Parthenogenetic reproduction occurs in spring and summer, whereas decreasing day lengths in autumn provoke the production of sexual forms. Changing environmental signals are relayed by brain neuroendocrine signals to the ovarioles. We combined bioinformatic analyses with brain peptidomics and cDNA analyses to establish a catalogue of pea aphid neuropeptides and neurohormones. 42 genes encoding neuropeptides and neurohormones were identified, of which several were supported by expressed sequence tags and/or peptide mass analyses. Interesting features of the pea aphid peptidome are the absence of genes coding for corazonin, vasopressin and sulfakinin and the presence of 10 different genes coding insulin related peptides, one of which appears to be very abundantly expressed.

Localization of the phase-related 6-kDa peptide (PRP) in different tissues of the desert locust Schistocerca gregaria--immunocytochemical and mass spectrometric approach.

A 6-kDa phase-related peptide (PRP) was recently identified from the hemolymph of the desert locust Schistocerca gregaria. Its presence in much higher concentrations in the crowd-reared (gregarious) phase than in the isolated-reared (solitarious) one suggests a role in phase polyphenism. However, when tested in a variety of classical bioassays, no activity could be found. We hoped that uncovering its site(s) of synthesis might yield hints as to possible functions. An antiserum was raised against the C-terminal 16 aa part of PRP for use in immunocytochemistry. No immunoreactivity was recorded in the fat body, midgut, or Malpighian tubules. The strongest positive immunostaining was observed in the follicle cells of the ovary and in the seminal vesicle tubes of the male accessory gland complex. Also, positive were a pair of large neurosecretory cells in the subesophageal ganglion, the storage part of the corpora cardiaca and some nerve fibers in the brain- and abdominal regions. An additional mass spectrometric analysis was successfully done in combination with a BLAST search to detect possible false positive staining. This confirmed the presence of genuine PRP in most of the immunopositive tissues. Additional experiments are needed to unravel the role of PRP.

Melatonin-induced neuropeptide release from isolated locust corpora cardiaca.

A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.

Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry.

The pars intercerebralis-corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.

Peptidomics of the locust corpora allata: identification of novel pyrokinins (-FXPRLamides).

The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.

New insights in Adipokinetic Hormone (AKH) precursor processing in Locusta migratoria obtained by capillary liquid chromatography-tandem mass spectrometry.

After translation, the AKH I and AKH II precursors form three dimeric constructs prior to further processing into the respective AKHs and three dimeric Adipokinetic Hormone Precursor Related Peptides or APRPs (two homodimers and one heterodimer). By capillary liquid chromatography-tandem mass spectrometry we demonstrate that the APRPs in Locusta migratoria are further processed to form two smaller neuropeptides: DAADFADPYSFL (residue 36 to 47 of the AKH I precursor) and YADPNADPMAFL (residue 34 to 45 of the AKH II precursor). The peptides are designated as Adipokinetic Hormone Joining Peptide 1 (AKH-JP I) and 2 (AKH-JP II) respectively. Within the AKH I and AKH II precursor molecules, the classic KK and RR processing sites separate the AKH-JPs from the AKH I and II respectively. At the carboxyterminus, both AKH-JP I and II are flanked by Tyr-Arg, a cleaving site not described before. Such an unusual cleavage site suggests the presence, in the corpora cardiaca, of specific convertases. The AKH-JP-II does not stimulate lipid release from the fat body nor does it stimulate glycogen phosphorylase activity, both key functions of AKH.

A double-blind study of neurotropin in patients with acute ischemic stroke.

Neurotropin was found to reduce brain oedema in an experimental model of brain infarction in the guinea-pig. A randomized double-blind controlled trial with Neurotropin was performed in 220 patients admitted within 24 h after an acute ischemic stroke. 35 of the neurotropin and 41 of the placebo-randomized patients had to be excluded. 10 included patients in the neurotropin and 13 in the placebo-treated group died within the study period of 15 days. A better clinical outcome was observed in the 65 included surviving neurotropin compared with the 56 placebo-treated patients. The size of the infarct and of the oedema zones was significantly more decreased on CT scans from Day 11 compared with Day 3 after stroke in the neurotropin than in the placebo treated group. Neurotropin is helpful in treating brain oedema, related to acute ischemic stroke.

Resistance patterns between cis-diamminedichloroplatinum(II) and ionizing radiation.

Cross-resistance between cis-diamminedichloroplatinum(II) (CDDP) and radiation resistance has been suggested from clinical and experimental data (C. T. Coughlin and R. C. Richmond, Semin. Oncol., 16: 31-43, 1989). To determine whether cross-resistance patterns between both cytotoxic approaches exist, resistance against CDDP and ionizing radiation was induced separately in human ovarian cancer cells in a cross-over design. Subsequently sensitivity changes were determined for both treatment modalities. CDDP resistance was induced previously (P. J. Kuppen et al., Cancer Res., 48: 3355-3359, 1988), and resistant cells were grown at three different levels of CDDP:0 ng/ml; 250 ng/ml; and 500 ng/ml. Resistance with resistance factor (RF) 3.4 to 5.1 proved to be stable, since withdrawal of CDDP pressure for at least 6 mo did not alter resistance patterns. CDDP-resistant cells also demonstrated stable resistance against ionizing radiation, with RF ranging from 1.7 to 2.0. The resistance patterns could not be explained by differences in growth kinetics and DNA content. Resistance to ionizing radiation was induced in the same human ovarian cancer cells as used for CDDP resistance studies. Exposure with 1.5 Gy of intermittent irradiation during 6 mo, at time intervals of 48 h, resulted in cells which were able to grow under chronic ionizing radiation pressure. RF was 2.0; the resistance was lost after 6 mo of culturing without ionizing radiation pressure. With intermittent radiation doses of 0.5 and 1.0 Gy, no significant resistance could be induced. Cells intermittently exposed to 0.5, 1.0, and 1.5 Gy during 6 mo demonstrated increased sensitivity to CDDP, with 0.22 less than RF less than 0.43. Increased sensitivity was associated with proportionally increased formation of the platinum-DNA adducts. Differences in sensitivity for both ionizing radiation and CDDP were lost after 6 mo of culturing without radiation pressure; therefore, resistance toward ionizing radiation and, likewise, the increased sensitivity to CDDP, were judged to be unstable. In conclusion, data of the present study demonstrated that development of stable resistance to CDDP is associated with development of stable resistance to ionizing radiation in human ovarian cancer. Contrastingly, increased sensitivity to CDDP was found when resistance against irradiation was induced in the same cells.