Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromatography, and amino-acid composition of factor II

The separation of two peptides with hypertrehalosaemic activity from the corpus cardiacum of the Indian stick insect, Carausius morosus, was achieved by reversed-phase high-performance liquid chromatography using a Nucleosil C-18 column with a trifluoroacetic acid/acetonitrile gradient. The eluant was monitored at 210 nm and the hypertrehalosaemic activity was detected in ligated stick insects using a bioassay. In addition, the isolated material was potent in causing hyperlipaemia in migratory locusts and also in raising blood carbohydrates in the American cockroach. The amino-acid composition of the major peptide, hypertrehalosaemic factor II, was determined after acid hydrolysis with HCl or methanesulfonic acid. The analyses suggest that factor II is a nonapeptide which contains the following amino-acid residues: Asp, 2 Thr, Glu, Pro, Gly, Leu, Phe and Trp. This composition is almost identical to that of locust adipokinetic hormone I, lacking only one Asp residue.     

C-mannosylation in the hypertrehalosaemic hormone from the stick insect Carausius morosus

The hypertrehalosaemic hormone from the stick insect Carausius morosus (Cam-HrTH) contains a hexose covalently bound to the ring of the tryptophan, which is in the eighth position in the molecule. We show by solution NMR spectroscopy that the tryptophan is modified at its C(delta1)(C2) by an alpha-mannopyranose. It is the first insect hormone to exhibit C-glycosylation whose exact nature has been determined experimentally. Chemical shift analysis reveals that the unmodified as well as the mannosylated Cam-HrTH are not completely random-coil in aqueous solution. Most prominently, C-mannosylation strongly influences the average orientation of the tryptophan ring in solution and stabilizes it in a position clearly different from that found in the unmodified peptide. NMR diffusion measurements indicate that mannosylation reduces the effective hydrodynamic radius. It induces a change of the average peptide conformation that also diminishes the propensity for aggregation of the peptide.     

Primary structures of hypertrehalosaemic neuropeptides isolated from the corpora cardiaca of the cockroaches Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica and Blatta orientalis and of the stick insect Extatosoma tiaratum assigned by tandem fast atom bombardment mass spectrometry

Hypertrehalosaemic peptides were isolated by reversed-phase high-performance liquid chromatography from corpora cardiaca of four species of cockroaches (Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica, and Blatta orientalis) and one stick insect species (Extatosoma tiaratum), and their primary sequences were assigned by collision-induced decomposition tandem fast atom bombardment mass spectrometry (FABMS/CID/MS). The members of the cockroach families Blaberidae (L. maderae and G. portentosa) and Blattellidae (B. germanica) contained an identical decapeptide (Glu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-ThrNH2), whereas the member of the cockroach family Blattidae (B. orientalis) had two octapeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-TrpNH2 and Glu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH2). The structure of the stick insect hypertrehalosaemic compound was assigned as a decapeptide (Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2). The respective synthetic peptides elevated blood carbohydrates in their respective acceptor species. The results are discussed in the light of family-specificity of members of the adipokinetic hormone/red pigment-concentrating hormone family.     

Ribosyl-diphthamide: Confirmation of structure by fast atom bombardment mass spectrometry

Diphtheria toxin inactivates protein synthesis elongation factor 2 by attaching ADP-ribose to an unusual post-translational amino acid derivative, diphthamide, in the factor. Previously, we prepared ribosyl-diphthamide from the ADP-ribosyl-factor and proposed on the basis of NMR spectral analysis that it is 1-α-d-ribofuranosyl-2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine [N. J. Oppenheimer, and J. W. Bodley, (1981) J. Biol. Chem.256, 8579–8581 and op. cit.]. Now, using fast atom bomardment mass spectrometry, the intact cation of ribosyl-diphthamide has been observed in the gas phase. The theoretical mass of the structure proposed for ribosyl-diphthamide uniquely agrees with the observed mass of the inact cation of the compound to within 2 ppm. Collisional activation decomposition mass spectral analysis provided additional structural confirmation. Thus, although the compound has not been synthesized, all available evidence appears uniquely consistent with the structure of ribosyl-diphthamide previously proposed.     

A model of leg coordination in the stick insect,Carausius morosus

A model of interleg coordination presented in a separate report is evaluated here by perturbing the step pattern in three ways. First, when the initial leg configuration is varied, the simulated leg movements assume a stable coordination from natural starting configurations in a natural way (Fig. 1a). They also rapidly re-establish the normal coordination when started from unnatural configurations (Fig. 1b-d). An explicit hierarchy of natural frequencies for the legs of the three thoracic segments is not required. Second, when the coordination is perturbed by assigning one or more legs a retraction velocity different from the rest, gliding coordination or various integer step ratios can be produced (Figs. 2–4). Third, when the swing of one leg is obstructed, characteristic changes in the stepping of other legs occur (Fig. 5). Overall differences between the step patterns of the model and those of the stick insect are related to the form of the coordinating mechanisms. Errors made by the model, such as overlapping swings by adjacent legs or discrepancies in step timing and step end-points, point out the limitations of a model restricted to kinematic parameters.     

A model of leg coordination in the stick insect,Carausius morosus

Mechanisms dependent upon leg position coordinate the alternate stepping of adjacent ipsilateral and contralateral legs in the stick insect. In this insect, swing duration and step amplitude are independent of walking speed. A simple geometrical model of the leg controller is used here to test different mechanisms for compatibility with these two invariant features. Leg position is the state variable of a relaxation oscillator and position thresholds determine the transitions between swing and stance. The coordination mechanisms alter these thresholds. The position-dependent mechanisms considered differ either in the form or the speed-dependence of the function relating the shift in the posterior threshold of the receiving leg to the position of the sending leg. The results identify parameter combinations leading to alternate stepping with symmetric or asymmetric phase distributions, to shifts in the posterior extreme position as a function of speed, to double stepping or to in-phase stepping. An optimal position-dependent excitatory mechanism is described. Finally the consequences of adding either inhibitory influences or time-dependent excitatory influences are analyzed.     

Sensory control of leg movement in the stick insect Carausius morosus

Hind legs with crossed receptor-apodemes of the femoral chordotonal organ when making a step during walking often do not release the ground after reaching the extreme posterior position. After putting a clamp on the trochanter (stimulation of the campaniform sensilla) the leg is no longer protracted during walking. However, during searching-movements the same leg is moved very far forwards. The anatomical situation of the campaniform sensilla on the trochanter and the sensory innervation of the trochanter is described. After removal of the hair-rows and continuously stimulating the hair-plate at the thorax-coxa-joint the extreme anterior and posterior positions of the leg in walking are displaced in the posterior direction. Front and middle legs operated in this way sometimes do not release the ground at the end of retraction. In searching-movements the same leg is moved in a normal way. If only one side of a decerebrated animal goes over a step, then on the other side a compensatory effect is observed. The main source of this compensatory information appears to be the BF1-hair-plates. If the animal has to drag a weight the extreme anterior and posterior positions of the middle and hind legs are displaced in the anterior direction. Crossing the receptor-apodeme of the femoral chordotonal organ, when it causes the leg to remain in the protraction phase, displaces the extreme posterior position of the ipsilateral leg in front of the operated one in the posterior direction. Influences of different sources on the extreme posterior position can superimpose. A model is presented which combines both a central programme and peripheral sensory influence. The word programme used here means that it does not only determine the motor output but also determines the reactions to particular afferences. The fact that the reaction to a stimulus depends on the internal state of the CNS is also represented by the model.Supported by Deutsche Forschungsgemeinschaft     

Primary structure of Paim I, an alpha-amylase inhibitor from Streptomyces corchorushii, determined by the combination of Edman degradation and fast atom bombardment mass spectrometry

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.     

Isolation, physiological characterization, release and sequence elucidation of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the stick insect, Sipyloidea sipylus

ABSTRACT. The corpora cardiaca of the stick insect, Sipyloidea sipylus Westwood, contain peptidic material which elevates blood lipids in migratory locusts, blood carbohydrates in American cockroaches, and activates glycogen phosphorylase in the fat body of the cockroach in a time- and dose-dependent manner. The active principle is found in appreciable amounts only in the corpora cardiaca; slight hyperlipaemia is caused by extracts made from corpora allata and abdominal ganglia, whereas brain, suboesophageal and thoracic ganglia are not active. The adipokinetic activity is already present in corpora cardiaca from second instar (first day) nymphs. The factor retains its adipokinetic activity after boiling for up to 1 h. Conspecific injections of extracts from corpora cardiaca of S.sipylus cause hypertrehalosaemia in ligated stick insects and activate glycogen phosphorylase in non-ligated S.sipylus. After incubation of corpora cardiaca in vitro in saline with high concentrations of potassium and calcium, one fraction with adipokinetic (in locusts) and hypertrehalosaemic (in stick insects) activity can be isolated from the medium by RP-HPLC. Fractionation of a methanolic extract of corpora cardiaca from S.sipylus by RP-HPLC shows that active compounds are confined to apparently three absorbance peaks. The material of the highest absorbance peak was purified to homogeneity by RP-HPLC, and its amino acid composition determined after acid hydrolysis with HCl and with methanesulfonic acid revealed the residues Asx, Thr₍₃₎, Glx, Pro, Gly, Leu, Phe and Trp. The primary structure of this hypertrehalosaemic factor is assigned as a blocked decapeptide, pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂, from its FAB spectrum and metastable scans of its FAB spectrum. The structure is confirmed by synthesis; the synthetic and natural peptide co-chromatograph, and the synthetic peptide elevates blood carbohydrates in ligated stick insects and activates fat body phosphorylase in non-ligated S.sipylus.     

Water and solute transport by the Malpighian tubules of the stick insect,Carausius morosus

Summary Structural features of the principal, urine-secreting cells (type 1 cells) of the Malpighian tubules of Carausius are de scribedquantitatively and discussed in relation to possible mechanisms of water and solute transport. Mitochondria are arranged in two bands of about equal volume near to the basal and apical surfaces, suggesting active processes occur at both surfaces. Basal infoldings and apical microvilli which greatly amplify the cell surface are probably primarily devices to increase the passive permeability of the tissue to solutes. They do not provide functionally significant standing-osmotic-gradients. The extensive endoplasmic reticulum is locally differentiated into several components and ramifies between the infoldings and along microvilli but probably is not an intracellular conduit for the majority of urinary constituents. Vesicles and stages in their formation or liberation are observed both basally and apically although they probably do not contribute significantly to transcellular transport. At present it remains a problem to satisfactorily account for observations that the urine of Carausius can be hypotonic.This investigation formed part of a dissertation for the degree of Ph. D. in the University of Newcastle upon Tyne. It is a pleasure to thank Prof. J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

Tryptophan 2,3-dioxygenase in the development of the stick insect,Carausius morosus Br

Summary A detailed kinematical analysis of oscillating fly wings using high speed cine films in three-table projection and model reconstructions show a variety of quick changes in beating and rotating movements. There are especially quick changes in the geometrical angle of attack during the upper and lower reversal phases and quick twisting movements and bending oscillations during some of the beating phases. A dozen possibilities for instationary aerodynamic effects are discussed, including quick oscillations in angle of attack, quick turning movements in the reversal points, circulation possibly induced by a quick supination during the beginning of upstroke (fast supination effect), oblique attack by the fluid, circulation possibly induced by a forced oscillation of the trailing edge during the beginning of downstroke (swinging edge effect), tangential transport of the boundary layer by undulatory movements, rolling movements during the turning phases, high-frequency small oscillations of the wing surface, and a quick oscillation during late upstroke. Weis-Fogh's clap and fling mechanism and flip mechanism could not be detected in Calliphorid flies.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft     

Hygro- and thermoreceptive triad in antennal sensillum of the stick insect,Carausius morosus

Summary The receptor cells in a poreless sensillum on the antenna ofCarausius morosus were examined electrophysiologically. Two of the units are antagonistic regarding humidity, one responding with an increase in impulse frequency to rising humidity (moistair unit) and the other to falling humidity (dry-air unit). Another reacts to falling temperature with a rise in impulse frequency (cold unit). In some cases responses from a fourth unit were also present. Its modality is uncertain. A method of marking the sensillum with a cactus needle for subsequent structural examination after recording is described.Supported by the Alexander von Humboldt Foundation and by the Fonds zur Förderung der wissenschaftlichen Forschung in ÖsterreichI wish to express my gratitude to Prof. Dr. F. Schaller and to Prof. Dr. H. Altner for their support of this study, to Dr. R. Loftus for very valuable discussion, and to Ms. C. Danner for her technical assistance.     

Fine structure of the abdominal neural fat-body sheath in the stick insect (Carausius morosus,Br.)

The neural fat-body sheath surrounding the abdominal ventral nerve cord of Carausius morosus has been examined by light and electron microscopy. A perineural chamber between the ventral nerve cord and the sheath is present in the ganglionic regions, but in the interconnective regions the sheath directly covers the neural lamella. The sheath is differentiated into secretory cells with abundant granular endoplasmic reticulum and many mitochondria and storage cells with lipid droplets and granules presumed to be glycogen. The whole sheath is pervaded with tracheoblast cells and associated tracheal and tracheolar tubules. Recent evidence suggests that this sheath may have little power of ionic regulation. The function of the sheath, deduced from the fine structure described here, appears to be to serve the nutritional needs of the central nervous system. Why a sheath of this type appears to be confined to herbivorous insects with unusual haemolymph cationic balance is still unexplained.     

Effects of circum-oesophageal lesion on the behaviour of the stick insect Carausius morosus

The co-ordination of the walking behaviour of decerebrate stick insects is examined and compared with normal behaviour. The walks are fully coordinated but undergo subtle changes in timing, have a longer average step period and show momentary pauses of 50 ms during the time course of protraction movements. In addition a new intersegmental reflex has been discovered. This tactile reflex is used to avoid errors in co-ordination that would be produced by posterior legs stepping onto the tarsi of the legs in front. The reflex has a latency of 100 ms and is easily observed in lesioned animals but is also active, although seldom seen, in slowly walking intact animals.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.