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The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.

Several factors, including body size, age, and temporal and spatial variation, which may influence the amount of cadmium (Cd) in the phantom midge larvae, Chaoborus spp., were examined in eight lakes, four from south-central Ontario (Dorset Region) and four from northwestern Ontario (Experimental Lakes Area). All three species of Chaoborus (C. punctipennis, C. trivittatus, C. flavicans) found in Chub Lake (south-central Ontario), were analyzed seasonally. One species, C. punctipennis, was collected and analyzed from all lakes. Smaller Chaoborus tended to have higher Cd concentrations (micrograms Cd/g dry weight), and lower Cd burdens (ng Cd/Animal) than larger Chaoborus. The Cd concentrations of Chaoborus in Chub Lake were lowest during the summer, which coincided with the fastest growth of the organisms. The Cd burdens differed between species, which was attributed to differences in their asynchronous life histories. Cadmium burdens and concentrations were significantly lower in the organisms from northwestern Ontario than those from south-central Ontario. Lower atmospheric deposition rates, lower Cd concentrations in the lake water, as well as life history differences, may explain these results.

Five compounds were isolated from the n-hexane extract and n-butanol of extract Eupolyphaga sinensis. These compounds were identified as octacosanol, beta-sitosterol, batyl alcohol, 2,4-pyrimidinedione and allantoin.

Burrowing mayflies (Hexagenia bilineata) were analyzed to assess longitudinal patterns in their cadmium and mercury content along the upper Mississippi River. Emergent mayflies (subimagoes and imagoes) were collected in 1988 at 34 sites (25 navigation pools), extending 1260 km from Little Falls, Minnesota, downstream to St. Louis, Missouri. Cadmium concentrations in composite samples of whole mayflies ranged from 7 to 219 ng/g dry weight in females and from less than 7 to 265 ng/g in males. Cadmium concentrations were highest (greater than or equal to 150 ng/g) in samples from Pools 2 and 3 (downstream from the Twin Cities metropolitan area), 15 (near the Quad Cities metropolitan area), and 27 (near the St. Louis metropolitan area). Cadmium concentrations in female mayflies decreased significantly with distance downstream from Pool 2 at river mile 825 to Pool 9 at river mile 648, paralleling spatial trends in the cadmium contamination of sediments in the reach downstream from the Twin Cities metropolitan area, reported in earlier studies; cadmium burdens in mayflies followed a similar spatial trend. Concentrations of mercury were much less variable, ranging from 44 to 102 ng/g dry weight in female mayflies and from 60 to 177 ng/g in males; concentrations and burdens were highest in mayflies collected at Pools 2, 20, 22, 25, and 27. Mercury concentrations in females decreased significantly with distance downstream from Pool 2 at river mile 825 to Pool 5A at river mile 728.5. Concentrations of mercury and cadmium in composite samples of female mayflies were not correlated, indicating dissimilar longitudinal patterns in concentrations of the two metals.(ABSTRACT TRUNCATED AT 250 WORDS)

The major proteins stored in the yolk of developing oocytes are thought to provide a nutritional store for utilization during embryogenesis. They seem to fall into two major families of proteins. The first are called vitellogenins and are found in frog, chicken, nematode, fish, and some insects such as the boll weevil. The other group are called yolk proteins and are found in dipteran insects such as fruitfly, housefly, fleshfly, and blue-bottles. Both groups are the major proteins found in the oocyte and are female-specific proteins endocytosed from the serum or hemolymph. The yolk protein group were found to have sequence similarity to the triacylglycerol lipases and lipoprotein lipases of vertebrates, including rat, pig, and human. The yolk proteins do not have lipase activity, but the sequences conserved between yolk proteins and lipases surround the active site where there are interactions with lipids. The likely reason for the presence of this domain in the yolk proteins is to bind a steroid hormone in a storage form conjugated to lipids. This permits the storage of the hormone in an inactive form until the yolk proteins are degraded, when it can be released from its conjugate to induce developmental decisions in embryogenesis. They may also transport lipids into the oocyte for use in embryogenesis. Whilst the vitellogenin family of proteins do not share this homology with the lipases they do have similarity to the human serum protein, apolipoprotein B, which also has a role in binding lipids. These findings are discussed in relation to the evolution and functions of lipases, apolipoproteins, vitellogenins, and yolk proteins. Experiments aimed at isolating genes encoding lipases in insects and at further elucidating the function of the yolk proteins are suggested.

Systematic elucidations of the chemical structures of glycosphingolipids from members of the phyla Arthropoda (class:insecta) of the Protostomia have shown several characteristic differences to those of the Deuterostomia, e.g., the Vertebrata. The ceramide constituents of the arthropod sphingolipids are C14:1- and C16:1-sphingoid and a majority of saturated C18-, C20-, C22- and C24-fatty acids. The GSL of the dipteran insects, L. caesar and C. vicina, consist of a series of homologous compounds of increasing sugar chain length. The carbohydrate moieties of these arthropod GSL are all derived from one unique neutral sugar core sequence, the arthro-series. The longest member known, an arthrononaosylceramide has the structure Gal beta,3GalNAc beta,3GlcNAc beta,3Gal beta,3GalNAc alpha,4GalNAc beta,4GlcNAc beta,3Man beta,4Glc beta Cer. Three pentahexosylceramides have been characterized that differ in their nonreducing terminus, i.e., IV4GalNAc alpha-Ap4Cer, IV3Gal alpha-Ap4Cer and IV3Gal beta-Ap4Cer. A large repertoire of structural variations of the arthro-series GSL is created by two types of derivatisations of the neutral carbohydrate core: addition of a zwitterionic 2-aminoethylphosphate group in phosphodiester linkage to the 6-position of the III-N-acetylglucosamine of the arthrotriaosylceramide core and/or substitution of a terminal galactose in 3-position by a glucuronic acid residue. The latter acidic arthro-series GSL were given the name arthrosides. Preliminary results have indicated that the blowfly GSL component distribution is strongly organ specific. GSL of various larval organs are distinguished by the length of their neutral core carbohydrate chain, as well as, the degree of PEtn- and GlcA-substitutions. Normal human sera contain antibodies that recognize epitopes on arthro-series GSL. Thereby, sites of the neutral carbohydrate chain, as well as, the zwitterionic and acidic epitopes are involved in the immune recognition. In a pathological condition, a M-protein of patients with autoallergic demyelinating peripheral neuropathy was found to react strongly with a glucuronic acid-containing molecular structure on arthrosides. Some indication was obtained for a possible functional role of glucuronic acid in dipteran-cell adhesion by using a mouse monoclonal antibody, mAb CAF-I. This antibody, directed against an arthroside epitope involving glucuronic acid, could induce an in vitro detachment of insect cells from one another or from their substratum. Obviously, present research on the GSL of insects is only at an early stage. Nevertheless, from the results available so far it was become increasingly apparent that a new chapter in our knowledge of GSL structures has been opened.(ABSTRACT TRUNCATED AT 400 WORDS)

Experiments are described for T-independent polyclonal activation of B cells in vitro by the immunoglobulin binding substance (IBS) from the granary weevil. The affinity-chromatographically purified IBS was used. IBS is a heat-, alkali- and acid-stable glucopeptide which is characterized by non-specific immunoglobulin binding to the Fab fragment. The purified IBS consists of three polymer homologues whose molecular weights are 12-14,000, 25-30,000 and more than 150,000 Da. IBS did not stimulate DNA synthesis by murine T cells, macrophages or plasma cells whereas it did stimulate that by mature B cells without any help from T cells or macrophages. IBS also stimulated both in vitro IgG production by spleen cells and in vitro sensitization of spleen cells by sheep red cells (SRBC). IBS was found to stimulate DNA synthesis by B cells mediated by binding to surface immunoglobulins of B cells. IBS is thought to be a useful amplifier for inducing human hybridomas and a valuable tool for examining mature B cells, both diagnostically and experimentally.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His7]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.

The presence of carotenoids in Catopus tritis infernus Szymanowski 1957 and Choleva lederiana gracilenta Szymanowski 1957 from a cave was studies, using columnar and thin-layer chromatography. Qualitative and quantitative differences in the carotenoid content of these insect species were found.

By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At least, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.

A neuropeptide with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana was purified by reversed-phase high performance liquid chromatography from the corpus cardiacum of the dragonfly, Libellula auripennis. After brief enzymatic digestion by 5-oxoprolyl-peptidase the primary structure of the peptide was determined by pulsed-liquid phase sequencing employing Edman degradation. As the peptide was not cleaved by carboxypeptidase the C-terminus was blocked, too. The peptide was assigned as a blocked uncharged octapeptide: Glu-Val-Asn-Phe-Thr-Pro-Ser-TrpNH2. The synthetic peptide was chromatographically indistinguishable from the natural compound and, upon injection in low quantities into dragonflies, elicited mainly haemolymph lipids. Therefore it is called dragonfly adipokinetic hormone (Lia-AKH). It is a new member of the large AKH/RPCH family of peptides. Because of its structural features and its origin from a very primitive insect order it is assumed to represent the putative ancestral peptide of this family. Synthesis was shown to occur in the corpus cardiacum by in vitro incorporation of tritium-labelled Trp into Lia-AKH.

A method was developed to determine in the same extract juvenile hormone and various types of ecdysteroids in precisely staged eggs and larvae of Trichoplusia ni. Ecdysteroids were tentatively identified on the basis of their retention time in ion suppression reversed-phase HPLC and their cross-reactivity with two relatively non-specific, complimentary antibodies, whereas juvenile hormone was identified using reversed-phase HPLC combined with Galleria bioassay. Freshly laid eggs contained low levels of immunoreactive ecdysteroids. Mid-polar ecdysteroids increased in the phase of segmentation (14-18 h) and 1st larval cuticle formation (36-44 h), when 20-hydroxyecdysone and 20,26-dihydroxyecdysone were found to be predominant. Only traces of ecdysone and 26-hydroxyecdysone were seen. Toward hatching ecdysteroids decreased and represented mainly compounds more polar than 20,26-dihydroxyecdysone. In larval development ecdysteroids were low at the beginning of the feeding phases, increased toward cessation of feeding, and reached highest levels 12-15 h before ecdysis. In feeding stages ecdysone and 20-hydroxyecdysone were predominant, whereas in molting stages they were seen together with 20,26-dihydroxyecdysone and 20-hydroxyecdysonoic acid. The juvenile hormone titer was very low in freshly laid eggs and was high (approximately 25 ng/g) in embryos at the stage of 1st larval cuticle formation and eye pigmentation. In eggs we tentatively identified juvenile hormones I and II, whereas in larval stages juvenile hormone II appeared to be the predominant or exclusive juvenile hormone. Its titer fluctuated rapidly and was high in early 1st-instar larvae and again before the molts into the 3rd, 4th, and 5th instar. Highest titers were reached concomitant with the peak in 20-hydroxyecdysone 12-15 h before ecdysis.