Jan Kochansky

Locustatachykinin III and IV : two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin III and IV were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structures of Lom-TK III and IV were established as amidated decapeptides: Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2 (Lom-TK III) and Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2 (Lom-TK IV). The locustatachykinins were synthesized and shown to have chromatographic and biological properties identical with those of the native materials. They stimulate visceral muscle contractions of the oviduct and the foregut of Locusta migratoria and of the hindgut of Leucophaea maderae. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence similarity is greater with the fish and amphibian tachykinins (up to 40%) than with the mammalian tachykinins. In addition, the intestinal and oviducal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. Both chemical and biological similarities of vertebrate and insect tachykinins substantiates the evidence for a long evolutionary history of the tachykinin peptide family.

Analysis of sterol esters by capillary gas chromatography-electron impact and chemical lonization-mass spectrometry

Synthetic mixtures of C40 to C47 sterol esters in groups of 7 esters were effectively separated and analyzed by capillary gas chromatography-mass spectrometry. Ammonia chemical ionization of all 20 sterol esters analyzed at a source block temperature of 120 C yielded (M+NH4)+ and (M+H-RCO2H)+ ions of high abundance or as base peak, thereby indirectly indicating the molecular weights of the ester and the sterol and acid moieties. Ammonia CI spectra of all esters containing a Δ5-sterol moiety exhibited in addition to the above 2 ions an M+NH4-RCO2 H fragment. At a source block temperature of 150 C, M+H-RCO2 H fragment was the base peak for all esters, and there was little or no indication of an (M+NH4)+ adduct ion. Protonated molecules were not observed for any esters analyzed by methane or isobutane CI. Molecular ions of 3–14% intensity were obtained for only 3 of the esters analyzed by electron impact; they contained a Δ7-bond in the sterol nucleus, and the acid moiety was either saturated normal or branched chain or contained a single double bond. The base peak was a function of both the acid and sterol moieties of the sterol ester. The esters containing both saturated straight chain acid and saturated sterol moieties exhibited a base peak at m/z 215. The Δ5-sterol esters with saturated branched or straight chain acid moieties exhibited base peaks at M-RCO2 H. Other ions also were of diagnostic value.

The glycosylceramides of the nematodeCaenorhabditis elegans contain an unusual, branched-chain sphingoid base

Caenorhabditis elegans was cultured in semi-defined medium containing yeast extract, soy peptone, glucose, hemoglobin, Tween 80, and sitosterol. Monoglycosylceramides were chromatographically purified from nematode extracts. Their structures were elucidated with mass spectrometry, nuclear magnetic resonance spectroscopy, and analysis of methanolysis products of the parent cerebrosides. The glycosylceramides were unusual in that the only long-chain sphingoid base detected was aniso-branched compound with a C-4 double bond (i.e., 15-methyl-2-aminohexadec-4-en-1,3-diol). Glucose was the only sugar moiety detected. The fatty acids consisted of a series of primarily straint-chain, saturated, 2-hydroxylated C20–C26 acids; someiso-branched analogs also occurred. The sphingomyelins ofC. elegans were also hydrolyzed, and the sameiso-branched C17 compound was the only sphingoid base detected. This is the first structural analysis of a nematode glycosphingolipid and the first report of an organism in which the long-chain sphingoid bases are entirelyiso-branched.

Isolation, identification and synthesis of locustamyotropin III and IV, two additional neuropeptides of Locusta migratoria: Members of the locustamyotropin peptide family

Two neuropeptides, locustamyotropin III (Lom-MT III) and locustamyotropin IV (Lom-MT IV) were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion complexes of the migratory locust, Locusta migratoria. They were purified by reversed phase and normal phase high performance liquid chromatography and detected by a Leucophaea hindgut motility bioassay. Amino acid composition and sequence analysis combined with enzymatic digestion established the primary structure of Lom-MT III as Arg-Gln-Gln-Pro-Phe-Val-Pro-Arg-Leu and of Lom-MT IV as Arg-Leu-His-Gln-Asn-Gly-Met-Pro-Phe-Ser-Pro-Arg-Leu. Both candidate peptide sequences were synthesized and both chromatographic and biological properties of the synthetic peptides in the amide from were the same as those of the native peptides thus confirming the structural analysis. Both peptides have a Phe-X-Pro-Arg-Leu-NH2 carboxyl terminus and hence, are the third and fourth members of the locustamyotropin peptide family. This carboxyl terminal sequence is also found in other insect peptides and has a stimulating effect on pheromone biosynthesis and cuticular melanisation (PBAN/MRCH in Bombyx mori and Heliothis zea) and on myogenic hindgut contractions (leucopyrokinin in Leucophaea maderae). Like the other locustamyotropins, Lom-MT III and IV stimulate contractions of the oviduct in the locust.

Major hydrocarbons of the post-pharyngeal glands of mated queens of the red imported fire antSolenopsis invicta

Thin layer and column chromatographic analyses showed that hydrocarbons were the major lipoidal components of post-pharyngeal glands of mated queens of the red imported fire ant,Solenopsis invicta. Gas liquid chromatographic analyses on an OV-17 column showed four major hydrocarbons which have been identified and confirmed by synthesis and comparative mass spectral analyses as 13-methylheptacosane, 13,15-dimethylheptacosane, 3-methylheptacosane, and 3,9-dimethylheptacosane. When microgram quantitites of the natural alkanes on filter paper were placed in colonies of ants, the ants clustered on the paper about the sample and proceeded to masticate the paper in the area containing the alkanes.

Identification and characterization of an ecdysiotropic peptide from brain extracts of the gypsy moth, Lymantria dispar

A peptide (Lymantria TE) was isolated from brains of the gypsy moth, Lymantria dispar, which stimulates synthesis of ecdysteroid in the testes of larval and pupal insects. This ecdysiotropic peptide was purified and its structure determined to be NH2-IIe-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH using protein sequence analysis and electrospray mass spectrometry. The peptide was biphasic in activity, with maximal activity in the pupal testes at 10−13 M and 10−9 M, with a minimum at 10−10 M, and with maxima at 10−15 M and 10−10 M and minimum at 10−13 M for larval testes. Arch. Insect Biochem. Physiol. 34:175–189, 1997.

Transduction of the signal initiated by the neuropeptide, testis ecdysiotropin, in testes of the gypsy moth, Lymantria dispar

Abstract Testes of Lymantria dispar synthesize immunologically detectable ecdysteroid in vitro . Early last instar larval testes can be induced to produce ecdysteroid de novo by means of the brain peptide, testis ecdysiotropin (TE), although testes excised in mid pupal stage synthesize ecdysteroid without TE stimulus in vitro ; they can be boosted to higher levels of synthesis with TE. By use of agents H-7 and H-8, inhibitors of phosphokinases C (PKC) and cAMP-dependent protein kinase (PKA), respectively, we have confirmed previous data indicating that diacyl glycerol and its effector, PKC, are second messengers for the TE message. In larval testes, but not pupal testes, cAMP and PKA also regulate ecdysteroid synthesis. Data obtained from incubation of testes with analogs of GTP (GTPγS) and GDP (GDPβS) and the ribosylating agents, pertussis and cholera toxins, indicate that TE is transduced via inhibiting G protein (G i ) in larval testes, although both G i and stimulating G protein (G s ) influence ecdysteroid synthesis in pupal testes. Synthesis must be further modulated by feedback loops operating with G s and G i controlled factors in testes exposed to TE. Ecdysteroid synthesis appears to be constitutive in pupal testes in the absence of exogenous TE.

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea

The tritium-labeled bis-norleucine analog ofHelicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph fromManduca sexta orH. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 μM the degradation proceeded at a very slow but physiologically plausible rate (2–10 fmol/min/μl hemolymph). The primary [3H]NLPBAN degradation reaction inM. sexta hemolymph was not inhibited by 20 μM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph ofM. sexta andH. zea contains peptidase(s) capable of inactivating circulating PBAN.

Immunocytochemical localization of testis ecdysiotropin in the pupa of the gypsy moth,Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Antiserum against testis ecdysiotropin isolated from the gypsy moth,Lymantria dispar, reacted with neurons in the protocerebrum, optic and antennal lobes, subesophageal, thoracic and abdominal ganglia, as well as in nerve tracts extending through the optic lobes, tritocerebrum, and interganglionic connectives of the pupal stage of these insects. Testis ecdysiotropin is a peptide required by immature moths to initiate production of testes ecdysteroid, which is necessary for the development of the male reproductive system and initiation of spermatogenesis.Antiserum against testis ecdysiotropin also detected an accumulation of testis ecdysiotripic-like material between the inner and outer testis sheaths of pupae. The localization of this peptide in the imaginal disks of the last larval stage, cells and nerve fibers in the optic and antennal lobes of the pupa of both sexes, as well as in the testes during development of the adult reproductive system indicates that testis ecdysiotropin has a much larger impact on adult metamorphosis than development of the reproductive system and initiation of gametogenesis. Although this peptide may have a modulatory role in the central nervous system (CNS), it may also initiate a cascade of activity required for the development of the adult nervous system, in addition to its role in reproduction.

Structure–function analysis of Lymantria testis ecdysiotropin: A search for the active core

A structure-function study was performed on the synthetic 21 residue neuropeptide, Lymantria testis ecdysiotropin (LTE), originally isolated from brains of Lymantria dispar pupae. The peptide induces ecdysteroid synthesis by testis sheaths of various lepidopteran species. LTE, as well as synthetic LTE 1-11, 11-21, and 11-15, stimulated synthesis in larval and pupal testes of Lymantria dispar at concentrations of 10(-9) to 10(-15) M; LTE 16-21 was weakly active, and an elongated LEU-LTE was inhibitory to synthesis at all but extremely low concentrations (10(-15) M). Since the sequence and polarity of residues in LTE 1-11, 11-15, and 11-21 are quite different, several parts of the molecule must activate receptors which initiate the cascade, resulting in ecdysiogenesis in Lepidopteran testes.