Novel members of the adipokinetic hormone family in beetles of the superfamily Scarabaeoidea.

Eight beetle species of the superfamily Scarabaeoidea were investigated with respect to peptides belonging to the adipokinetic hormone (AKH) family in their neurohemal organs, the corpora cardiaca (CC). The following beetle families are represented: Scarabaeidae, Lucanidae, and Geotrupidae. AKH peptides were identified through a heterospecific trehalose-mobilizing bioassay and by sequence analyses, using liquid chromatography coupled to positive electrospray mass spectrometry (LC-ESI-MS) and analysis of the tandem MS2 spectra obtained by collision-induced dissociation. All the beetle species have octapeptide AKHs; some have two AKHs, while others have only one. Novel AKH members were found in Euoniticellus intermedius and Circellium bacchus (family Scarabaeidae), as well as in Dorcus parallelipipedus (family Lucanidae). Two species of the family Geotrupidae and two species of the Scarabaeidae subfamily Cetoniinae contain one known AKH peptide, Melme-CC, while E. intermedius produces a novel peptide code named Euoin-AKH: pEINFTTGWamide. Two AKH peptides were each identified in CC of C. bacchus and D. parallelipipedus: the novel Cirba-AKH: pEFNFSAGWamide and the known peptide, Scade-CC-I in the former, and the novel Dorpa-AKH: pEVNYSPVW amide and the known peptide, Melme-CC in the latter. Kheper bonelli (subfamily Scarabaeinae) also has two AKHs, the known Scade-CC-I and Scade-CC-II. All the novel peptides were synthesized and the amino acid sequence assignments were unequivocally confirmed by co-elution of the synthetic peptides with their natural equivalent, and identical MS parameters of the two forms. The novel synthetic peptides are all active in inducing hypertrehalosemia in cockroaches.

Effects of chronic administration of adipokinetic and hypertrehalosemic hormone on animal behavior, BDNF, and CREB expression in the hippocampus and neurogenesis in mice.

Neurosecretory cells in corpus cardiacum of insects synthesize a set of hormones that are called adipokinetic, hypertrehalosaemic or hyperprolinaemic, depending on insect in question. This study investigated effects of chronic administration of Anax imperator adipokinetic hormone (Ani-AKH), Libellula auripennis adipokinetic hormone (Lia-AKH), and Phormia-Terra hypertrehalosaemic hormone (Pht-HrTH) on depression, anxiety, analgesy, locomotion in forced swimming (FST), elevated plus-maze (EPM), hot plate, and locomotor activity tests. Ani-AKH (1 and 2 mg/kg), Lia-AKH (1 and 2 mg/kg), and Pht-HrTH (1 and 2 mg/kg) had antidepressant effects in forced swimming test. Lia-AKH (2 mg/kg) and Pht-HrTH (1 and 2 mg/kg) had anxiolytic effects when given chronically in elevated plus-maze test. Ani-AKH (1 and 2 mg/kg) and Pht-HrTH (2 mg/kg) had antinociceptive effects in hot plate test in male balb-c mice. Ani-AKH (2 mg/kg), Lia-AKH (1 and 2 mg/kg), and Pht-HrTH had locomotion-enhancing effects in locomotor activity test in male balb-c mice. Drug treatment significantly increased brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) gene expression levels compared to control levels. Pht-HrTH and Ani-AKH groups had significantly increased numbers of BrdU-labeled cells, while neurodegeneration was lower in the Pht-HrTH group. Our study showed that AKH/RPCH family peptides may be used in treatment of psychiatric illness such as depression and anxiety, in treatment of pain and in diseases related to locomotion system. AKH/RPCH family peptides increase neurotrophic factors in brain and have potential proliferative and neuroprotective effects in hippocampal neurogenesis and neurodegeneration.

Removal of N-terminal blocking groups from proteins.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.

Biological activity and identification of neuropeptides in the neurosecretory complexes of the cabbage pest insect, Mamestra brassicae (Noctuidae; Lepidoptera).

The need for more environmentally sound strategies of plant protection has become a driving force in physiological entomology to combat insect pests more efficiently. Since neuropeptides regulate key biological processes, these "special agents" or their synthetic analogues, mimetics, agonists or antagonists may be useful tools. We examined brain-suboesophageal ganglia and corpora cardiaca-corpora allata complexes of the cabbage moth, Mamestra brassicae, in order to obtain clues about possible peptide candidates which may be appropriate for the biological control of this pest. With the aid of bioassays, reversed phase high performance liquid chromatography, and mass spectrometry, five neuropeptides were unequivocally identified and the presence of a further three were inferred solely by comparing mass spectra with known peptides. Only one neuropeptide with adipokinetic capability was identified in M. brassicae. Data from the established homologous bioassay indicated that the cabbage moths rely on a lipid-based metabolism which is aided by an adipokinetic hormone (viz. Manse-AKH) that had previously been isolated in many different lepidopterans. Other groups of neuropeptides identified in this study are: FLRFamides, corazonin, allatostatin and pheromonotropic peptide.

Isolation of an anti-angiogenic substance from Agaricus blazei Murill: its antitumor and antimetastatic actions.

We previously found that ergosterol isolated from Agaricus blazei inhibited tumor growth through the inhibition of tumor-induced neovascularization. In the present study, we isolated further anti-angiogenic substances (A-1 and A-2) from this fungus using an assay system of angiogenesis induced by Matrigel supplemented with vascular endothelial growth factor, and A-1 was identified as sodium pyroglutamate. Next, we examined the antitumor and antimetastatic actions of A-1 using Lewis lung carcinoma (LLC)-bearing mice. A-1 (30, 100 and 300 mg/kg) inhibited tumor growth and metastasis to the lung. The reduction of the numbers of splenic lymphocytes, CD4+ and CD8+ T cells in LLC-bearing mice was inhibited by the oral administration of A-1 (30, 100 and 300 mg/kg). Further, A-1 increased the number of apoptotic cells of tumors and the numbers of CD8+ T and natural killer cells invading the tumors, and inhibited the increase of von Willebrand factor expression (a measure of angiogenesis) in the tumors. These results suggest that the antitumor and antimetastatic actions of A-1 (sodium pyroglutamate) may be associated with inhibition of the reduction of immune response caused by the tumor growth and tumor-induced neovascularization. This is the first report showing that sodium pyroglutamate isolated from A. blazei as an anti-angiogenic substance has potent antitumor and antimetastatic actions, as well as immune-modulatory activity, in tumor-bearing mice.

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay.

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H](+) ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP(2b) and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP(2b) were confirmed by PSD analysis. Fourteen [M+H](+) ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP(2b), M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.

Studies on insulin-like substances in Korean red ginseng.

Korean red ginseng powder was found to contain adenosine and an acidic substance which inhibited epinephrine-induced lipolysis and stimulated insulin-mediated lipogenesis from glucose (3, 4). In the present experiment, the chemical structure of this acidic substance is determined to be pyro-glutamic acid. Pyroglutamic acid exhibits selective modulations toward the opposite metabolic pathways in rat adipocytes; it inhibits the lipolysis but rather stimulates the lipogenesis. Based on these results, we suggest to call these substances (adenosine and pyro-glutamic acid) "selective modulators".

Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus.

Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus. The longest and most predominant polypeptide was 138 amino acids in length, which is five residues shorter than the sequence predicted from the cDNA. The presence of multiple-carboxyl-terminal forms indicated possible proteolytic processing during induction or protein purification. Limited proteolytic digestion of full-length recombinant IFN-gamma with endoproteinase Lys-C and trypsin revealed that the carboxyl-terminal 15 residues could be released under conditions in which the core portion of the polypeptide chain remained intact. Thus, the heterogeneity of natural IFN-gamma may be explained by partial proteolytic degradation of the molecule and differences in the degree of glycosylation as previously reported [Rinderknecht, E., O'Conner, B. H. & Rodriguez, H. (1984) J. Biol. Chem. 259, 6790-6797].

Pyroglutamyl N-termini of thermal polyamino acids.

It has been established indirectly that the N-termini of the thermal polyamino acids are pyroglutamic acid. This was determined by trifluoroacetic acid hydrolysis of the lactam ring followed by Dansyl labelling. The polyamino acids contained Ala, Gly, Glu, Leu, Phe, and Pro. In the experiments described here, the presence of pyroglutamic acid at the N-terminus of a polyamino acid was determined directly by the use of pyrrolidone carboxylyl peptidase. The enzyme catalyzes the removal of pyroglutamyl residues at the N-terminus of polypeptide chains. The polyamino acids used in these studies contained glutamic acid, aspartic acid, alanine, glycine, isoleucine, proline and valine. Alkaline hydrolysis was also used to determine indirectly that the N-termini of these polyamino acids are pyroglutamic acid. Another interesting finding was that many of the amino acids in the polymerization mixture were found to occur penultimate to the N-terminal amino acid. This is interpreted to mean that the diffusible fraction contains many polyamino acids.

Reversed-phase high-performance liquid chromatography used to monitor enzymatic cleavage of pyrrolidone carboxylic acid from regulatory peptides.

Sequence determination of peptides blocked by amino-terminal pyrrolidone carboxylic acid (PCA) has in the past been hampered by the lack of a reliable and efficient method for removing this residue. We report here a rapid, efficient enzymatic method for removal of PCA. Reversed-phase high-performance liquid chromatography is used to monitor the reaction and to separate unblocked peptide from the reaction product. The method allows direct sequence analysis of newly purified PCA blocked peptides, eliminating the need for complicated enzymatic digestions and chromatography to determine the amino-terminal residue. Only a few micrograms of peptide are required instead of the several milligrams needed in the past.