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[formula: see text] The 6,8-dioxabicyclo[3.2.1]octane skeleton is a common structural subunit in natural products. A conceptionally new strategy affording these structures is described for the syntheses of (+)-exo-brevicomin and rac-endo- and enantiomerically enriched (+)-endo-brevicomin, employing desymmetrization of trienes derived from diols with C2 and meso symmetry via ring-closing metathesis.

Background: We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase., Methods: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction., Results: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44)., Conclusions: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.

The 62 kDa protein firefly luciferase folds very rapidly upon translation on eukaryotic ribosomes. In contrast, the chaperone-mediated refolding of chemically denatured luciferase occurs with significantly slower kinetics. Here we investigate the structural basis for this difference in folding kinetics. We find that an N-terminal domain of luciferase (residues 1-190) folds co-translationally, followed by rapid formation of native protein upon release of the full-length polypeptide from the ribosome. In contrast sequential domain formation is not observed during in vitro refolding. Discrete unfolding steps, corresponding to domain unfolding, are however observed when the native protein is exposed to increasing concentrations of denaturant. Thus, the co-translational folding reaction bears more similarities to the unfolding reaction than to refolding from denaturant. We propose that co-translational domain formation avoids intramolecular misfolding and may be critical in the folding of multidomain proteins.

From whole body extracts of Photuris versicolor fireflies, the defensive betaine N-methylquinolinium 2-carboxylate (1) was isolated and characterized on the basis of spectroscopic data and comparison with a synthetic sample.

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.

Antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis are among the most active antifreeze proteins known. The Dendroides AFPs (DAFPs) consist of 6 or 7, 12- or 13-mer repeat units with a consensus sequence of -C-T-X3-S-X5-X6-C-X8-X9-A-X11-T-X13-. Nearly all of the Cys residues are in internal disulfide bridges between positions 1 and 7 within the repeats. The study presented here identified the secondary structure of the DAFPs using infrared and circular dichroism (CD) spectroscopies. The eight disulfide bridges impose significant constraints on potential secondary structural features (i.e., a number of three-residue gamma-turns) which may lead to unusual infrared and CD spectra that require special interpretation. At 25 degreesC the DAFPs contain approximately 46% beta-sheet, 39% turn, 2% helix, and 13% random structure. In the presence of ice there is a slight increase in helix and beta-sheet structures and a decrease in both turn and especially random structures. This change in the presence of ice may reflect a certain amount of flexibility in the DAFP structure. These structural changes may permit an improved lattice match between the DAFPs and ice, a requisite for the noncolligative freezing-point-depressing activity of the DAFPs., (Copyright 1998 Academic Press.)

The pupal defensive secretion of the 24-pointed ladybird beetle, Subcoccinella vigintiquatuorpunctata, consists of a mixture of macrocyclic polyamines, dominated by the three dimeric, 30-membered macrocycles 11-13, derived from the two building blocks 11-(2-hydoxyethylamino)-5-tetradecenoic acid (9) and 11-(2-hydoxyethylamino)-5,8-tetradecadienoic acid (10). Smaller amounts of the four possible cyclic trimers of 9 and 10 were also detected, corresponding to 45-membered macrocycles. Structural assignments were based on NMR-spectroscopic investigations and HPLC-MS analyses. In addition, the all-S absolute configuration of the S. vigintiquatuorpunctata macrocycles was determined by comparison of derivatives of the natural material with enantiomerically pure synthetic samples. Comparing this alkaloid mixture with that of the pupal defensive secretion in related ladybird beetle species indicates that the degree of oligomerization of the 2-hydroxyethylamino carboxylic acid building blocks can be carefully controlled by the insects.

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

The pupal defensive secretion of the coccinellid beetle Epilachna borealis is composed principally of a combinatorial library of macrocyclic polyamines. These compounds constitute a previously unrecognized family of natural products, characterized by extremely large-ring lactonic structures derived from a small set of (2-hydroxyethylamino)alkanoic acids. The combinatorial assembly of these simple building blocks generates a high degree of structural diversity, which is further increased by slow, spontaneous intramolecular rearrangement of the macrocycles.

A new hexacyclic alkaloid, chilocorine C (4), has been isolated from Chilocorus cacti and characterized on the basis of its IR, UV, MS, and NMR data. Although its structure is closely related to that of exochomine (1) (isolated from Exochomus quadripustulatus) and to chilocorine A (2) and B (3) (obtained previously from C. cacti), the presence of a hydroxymethyl substituent on the saturated tricyclic moiety represents an unexpected structural variation on the dimeric alkaloid theme.

The sunburst diving beetle, Thermonectus marmoratus, ejects a milky fluid from its prothoracic defensive glands when disturbed. Two major volatile components of this secretion are steroids; cybisterone (structure 7) constitutes about 20% of the volatiles, and a new steroid, mirasorvone, about 50%. Mirasorvone is assigned an 18-oxygenated pregnane structure (structure 9) on the basis of extensive spectroscopic data. Although no 18-oxygenated steroid has been described previously from an insect source, a closely related hormone with mineralocorticoid activity, 18-hydroxydeoxycorticosterone (structure 13), has been isolated from the adrenal glands of rats.

Two cases of periorbital dermatitis and one case of keratoconjunctivitis following contact with blister beetle are presented. In Tanzania and Kenya the commonest blister beetle is known as Nairobi Fly and is of the genus Paederus. Ocular symptoms are common, usually secondary to transfer by the fingers of the toxic chemical involved from elsewhere on the skin. Blister beetle keratoconjunctivitis has not previously been described in detail.

From the mixture of alkaloids obtained from adults of two species of ladybird beetles, Epilachna varivestis and Epilachna borealis, a novel bicyclic alkaloid, 5-(12'-aminotridecyl)pyrrolidinoöxazolidine [(5 alpha, 7 a beta)-hexahydro-alpha-methylpyrrolo[2,1-b]oxazole-5-dodecaneami ne] (2) was characterized on the basis of spectrometric and synthetic investigations. This new alkaloid is related structurally to a monocyclic congener 1-(2-hydroxyethyl)-2-(12'-aminotridecyl)pyrrolidine (1), previously characterized from E. varivestis. Two additional alkaloids (lower homologs of 1 and 2) from E. borealis were characterized as 5-(10'-aminoündecyl)pyrrolidinoöxazolidine [(5 alpha,-7 a beta)-hexahydro-alpha-methylpyrrolo[2,1-b]oxazole-5-decaneamine ] (7) and 1-(2-hydroxyethyl)-2-(10'-aminoündecyl)pyrrolidine (8), on the basis of their mass spectra.

Iranian firefly larvae, Lampyris turkestanicus collected from north of Iran and their luciferin were analyzed by HPLC, TLC, MS and spectroscopic Fourier transform ([FT]-IR, FT-NMR, UV and fluorescence) methods. The results showed that luciferin in L turkestanicus lanterns was the same as in the American firefly, Photinus pyralis and synthetic D-luciferin.

Ethyl 4-methyloctanoate, which has already been described in Oryctes monoceros, has been identified, using extracts of effluvia collected from males, as being a major component of the male pheromone of O. rhinoceros. Field trials have been carried out in North Sumatra, Indonesia. Ethyl 4-methyloctanoate synthesized in the laboratory and released at 10 mg/d resulted in the capture of 6.8 insects per week per trap, whereas ethyl chrysanthemate (40 mg/d), an allelochemical compound once used as an attractant, only led to the capture of 0.3 insects, and the control none at all. The insects captured with the pheromone were 81% females, the majority being sexually mature. Discovery of this compound opens up new prospects for O. rhinoceros control.

Two novel neuropeptides with neuropeptide F (NPF)-like immunoreactivity have been isolated from brain extracts of the Colorado potato beetle. Purification was achieved primarily by use of reverse phase chromatography including initial C-18 Sep-Pak cartridges and 4 subsequent analytical HPLC columns. Combined data from automated Edman degradation, immunochemical analysis, u.v. absorbance and mass spectrometry led to the elucidation of their full primary structures. The deduced sequences are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH2 (ARGPQLRLRFamide) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH2 (APSLRLRFamide). On the basis of their primary structure both peptides can be appended to the invertebrate group of neuropeptide Y (NPY)-like peptides, generally referred to as NPFs. We suggest these peptides to be designated Led-NPF-1 and Led-NPF-2.

Background: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions., Results: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft., Conclusions: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

The structure of firefly luciferase reveals a new protein fold which may be representative of a growing family of homologous enzymes.

A combination of the Horner-Emmons synthesis of alkyl 2,4-dienoates with their hydrogenation over complex L.Cr(CO)3 catalysts (L = 3CO or arene) provides a versatile, stereocontrolled and operationally simple approach to the (Z)-disubstituted, (Z)-trisubstituted, (E)-trisubstituted alkenes and skipped (Z,Z)-disubstituted diolefins with a homoallylic type of functionally. This protocol, sometimes supplemented by an enzymatic hydrolysis, was successfully applied to the synthesis of configurationally pure (gp > or = 98%) pheromones of the furniture carpet beetle, dry bean beetle, rusty grain beetle, square-necked grain beetle and a trail-following pheromone mimic for subterranean termites.

Five hydroxylated aliphatic molecules were identified as the pheromone produced by male West Indian Sugarcane Borer (WISB): 4-methyl-5-nonanol (1), 2-methyl-4-heptanol (2), 2-methyl-4-octanol (3), 5-nonanol (4) and 3-hydroxy-4-methyl-5-nonanone (5). Electroantennographic recordings revealed antennal responses to compounds 1, 2, 3 and 4. Significant EAGs were also recorded in response to pheromone compounds of weevils belonging to the same subfamily and structurally related to the WISB pheromone. The natural pheromone elicited aggregation behavior on WISB adults in laboratory bioassays.

It has previously been shown in field-trapping experiments and laboratory olfactometer bioassays that virgin females of Maladera matrida Argaman (Coleoptera, Scarabaeidae) and their volatiles, both in the presence of food (cut peanut leaves), are efficient attractants for M. matrida males and females. In this study GC-EAD experiments using male antennae and GC-MS experiments revealed that (Z,E)-alpha-farnesene is an active component of M. matrida female volatiles. The identification and quantitive electrophysiological responses (EAG) of synthetic (Z,E)-alpha-farnesene were obtained with male and female antennae. It was also shown that (Z,E)-alpha-farnesene is not a component of the plant volatiles that serve as synergistic components of the mixture of attractants or of the source of food for M. matrida.

Chiral amplification, a new strategy for determining the absolute configuration of difficulty available natural secondary alcohols or analogous amines, is described. Using this technique, the R configuration can be assigned to both components of the male sex pheromone emitted by the longhorn beetle, Anaglyptus subfasciatus. The application of this approach to fourteen alcohols and four amines illustrates its scope and limitations.

An active component of the sex pheromone system of the yellowish elongate chafer, Heptophylla picea was identified by GC-EAD. Mass spectral data and hydrogenation revealed that the active compound was a hexadecadien-4-olide. It was not possible to determine the double bond positions by direct DMDS derivatization of the pheromone, but partial hydrogenation (diimide) followed by DMDS derivatization showed that the double bonds were located in positions 7 and 15. FTIR (tracer) of the pheromone corroborated the lactone structure (1772 cm-1) and showed a band characteristic of a terminal double bond at 3073 cm-1, and one of a double bond in the cis-configuration at 3002 cm-1. Chiral resolution of the pheromone, after hydrogenation, demonstrated that the natural lactone had the (R)-stereochemistry. Synthetic (R,Z)-7,15-hexadecadien-4-olide, prepared from L-malic acid in 14 steps, was identical to the natural product in MS, IR, retention times and biological activity. This is the first fatty acid derivative compound found as a sex pheromone of a Melolonthinae species and as far as biosynthesis is concerned this is the most complex pheromone constituent of a scarab species.

Head-space volatiles obtained from male mountain pine beetles, Dendroctonus ponderosae, were analyzed by coupled GC-MS and chiral gas chromatography. 5-Ethyl-7-methyl-6,8-dioxabicyclo[3.2.1]octane (6) was found as a new naturally occurring isomer of brevicomin (1). In addition, several stereoisomers of 7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octan-2-ol (11) and 1-(5-methyl-6,8-dioxabicyclo[3.2.1]octyl)ethanol (12) could be identified. Relative and absolute configurations of the compounds were determined by unambiguous syntheses, which are described.

Haemolytic activity was identified in cell-free haemolymph from larval and imago stages of Leptinotarsa decemlineata. The haemolytically active fraction of the haemolymph was active against human, sheep, bull, toad and mouse erythrocytes. There was no haemolysis in the presence of 0.001 M EDTA and 0.5% glutathione. The titre of haemolytic activity did not increase after injury or vaccination of the larvae with Microccocus lysodeikticus. Haemolysin, a heat-labile protein was partially purified by ammonium sulphate precipitation, gel filtration, and ion-exchange separation. SDS PAGE, electrophoresis and immunoblotting showed that the active factor was a protein with a molecular weight of approximately 55 kD. It was not bactericidal for various micro-organisms but the antibacterial activity of the lysozyme increased in the presence of haemolysin only when M. lysodeikticus were used as target cells. Spherulocytes synthesized and released the haemolytic protein in vitro. The haemolytic activity increased in the presence of lipopolysaccharide from Escherichia coli and Ca++ ions. The physiological role of the haemolysin is as yet unknown.

In this paper, the proteins in 6 crude drugs (Prunus persica; P. armeniaca; Dolichos lablab; Strychnos nux-vomica; Mylabris phalerata; Whitmania pigra) and their processed products were analysed by polyacrylamide gel electrophoresis technique, and the effect of different processing methods on the quantity and kind of protein was explored. Protein electrophorograms of 20 samples are drawn.

We purified and characterized a sapecin homologue, named holotricin 1, from the hemolymph of immunized larvae of a coleopteran insect, Holotrichia diomphalia. We determined its complete amino acid sequence and three disulfide pairs. Holotricin 1 consisted of 43 amino acid residues and showed potent antibacterial activity against gram-positive bacteria, but antibacterial activity against gram-negative bacteria was not obvious.

Male-released sex pheromone constituents of the longhorn beetle Anaglyptus subfasciatus (Coleoptera: Cerambycidae) are identified by GC-MS and GC-Fourier transform infrared as a 7:1 molar mixture of 3-hydroxy-2-hexanone and 3-hydroxy-2-octanone. These two compounds undergo thermal isomerization during GC analyses to give the corresponding 2-hydroxy-3-alkanones. Comparison of GC retention times of the natural products with those of synthesized enantiomerically pure compounds revealed that both semiochemicals have (R)-stereochemistry. These absolute configurations were confirmed by comparisons of the (R)-methoxy(trifluoromethyl)phenylacetic acid esters of insect-derived and synthetic samples.

A novel myotropic heptapeptide was isolated from an extract of 54,000 heads of adult Leptinotarsa decemlineata by means of high performance liquid chromatography (HPLC), using the Locusta migratoria oviduct motility bioassay as monitoring system. The full primary structure was established as H-Ala-Tyr-Asn-Gly-Pro-Leu-Ala-NH2. This peptide, designated as Led-MNP-I, has a unique structure and does not belong to any known vertebrate or invertebrate peptide family. Two adjacent Led-MNP-I-immunoreactive perikarya were found in each optic lobe and in each half of all thoracic ganglia. Its absence from the pars intercerebralis and neurohemal organs suggests that Led-MNP-I is not a neurohormone but a neurotransmitter or neuromodulator. Treatment of isolated oviducts with varying concentrations of Led-MNP-I did not elicit significant changes in the level of cAMP concentration, suggesting that cAMP does not act as a second messenger for Led-MNP-I. Instead, Led-MNP-I induces an elevation of IP3. Treatment with Led-MNP-I did not stimulate cAMP production in the Colorado beetle brain, but this could be due to the very small number of receptive cells present. Both tissues contained a forskolin-sensitive adenylate cyclase enzyme.

Chaperonins are ubiquitous proteins found in all prokaryotic and eukaryotic cells. They are overproduced in several parasitic bacteria and are implicated in at least 2 types of endocytobiosis: in amoebae and in aphids. This work puts in evidence that a protein named symbionin, which shows an immunological homology with the E. coli protein GroEL, is present in the symbiotic relationship of 3 species of Sitophilus (S. oryzae, S. granarius, and S. zeamais). This protein is neither found in the naturally asymbiotic specie S. linearis nor in the aposymbiotic strain of S. oryzae obtained in the laboratory. This symbionin is stored in a great quantity within endocytobiotes and its amino acid composition seems to corroborate its chaperonin resemblance rather than its possible function as one of the insect storage proteins already described in the literature.

Elucidation of the secondary structure of the exchangeable apolipoproteins has been hindered by the difficulty in producing crystals suitable for X-ray spectrographic analyses. Consequently, in order to analyze potential structure-function relationships in the family of insect exchangeable apolipoproteins, apolipophorins-III (apoLps-III), two apoLps-III cDNA clones, one from the palo verde beetle (Derobrachus geminatus) and one from the house cricket (Acheta domesticus), have been isolated and sequenced. Multiple sequence alignments of the deduced protein sequences with two previously reported apolipophorins-III from Manduca sexta and Locusta migratoria reveal low sequence identity, suggesting that these proteins are very old and are highly divergent. Computer-assisted predictions of protein structure and subsequent analyses, using the known secondary structure of Locusta migratoria apolipophorin-III as a control, indicate that these insect proteins are composed of five amphipathic helices with characteristics similar to those of the helical domains of the mammalian exchangeable apolipoproteins. Thus, although insect and vertebrate exchangeable apolipoproteins share a common function in assisting lipid transport, precise amino acid identity is less important than the common structural feature of multiple amphipathic helices. Moreover, because these proteins occur widely among insect species, even in those where flight is limited or absent, we hypothesize that apolipophorin-III has a more generalized function in lipid metabolism than had been previously proposed.

In May 1993, at the end of the rainy season, outbreaks of Paederus sabaeus (Coleoptera, Staphylinidae) were recorded in Brazzaville (Congo), Kinshasa (Zaire), Franceville and Libreville (Gabon) and even in Bangui (CAR) at the North of the equator. A short review of previous outbreaks in Africa and on vesicant substances is given by the authors. These beetles are attracted to neon lights and they rest on the walls or on the skin of the occupants. When the insects are crushed on the bare skin their haemolymph liberate pederine and related vesicant components which provocate dermatitis. The insects disappeared spontaneously after three to four weeks.

Injection of Escherichia coli into larvae of the coleopteran Holotrichia diomphalia results in the appearance of antibacterial activity in the hemolymph. An antibacterial protein, named holotricin 2, was purified from larvae of this insect and characterized. A cDNA clone for holotricin 2 was isolated and its complete sequence was determined. This protein was found to inhibit the growth of Gram-negative bacteria and to consist of 72-amino acid residues with no cysteine residues. Its amino acid sequence is similar to that of coleoptericine, an antibacterial protein isolated from larvae of the coleopteran Zophobas atratus.

Clinical and epidemiological aspects of the contact dermatitis by Paederus are studied. This zoodermatosis is observed in many countries of the Ceará, State of Brasil, during the rainy season, specially at the months of april and may. The etiological agent of this disease is an insect of the generus Paederus. Two species were identified in the State: Paederus brasiliensis and Paederus columbinus.

During the month of May 1993, at the end of the rainy season, an outbreak of dermatitis occurred in Brazzaville (Congo). It was caused by Paederus sabaeus, a Staphylinid beetle which invaded the town during three weeks. The patients recovered spontaneously or after topical application of creams. No eye affection was recorded.

'Nairobi Eye' is a condition caused by a blister beetle, Paederus eximius, found in Northern Kenya. It has not previously been described as a hazard for troops exercising in this area. Four cases are described. Recommended management is to wash the contact area initially with soap and water, and to treat subsequent lesions with flamazine.

Experimental detection of deadly toxic cantharidin C10H12O4 by chemical analysis was performed in imagoes of two species of cryptotoxic Meloidae beetles--Lytta vesicatoria and Mylabris variabilis. Samples were processed by extraction and sublimation and analyzed by gas chromatography. Graphic documentation proved the presence of cantharidin. A part of microcristalline sublimate was studied microscopically and determinant features of crystalline cantharidin were identified. Moreover, cantharidin levels in single body parts of the beetle were investigated.

A peptide with the same retention time on gradient reversed-phase high-performance liquid chromatography was present in the corpora cardiaca of 5 scarabaeid beetles, subfamily Cetoniinae: the three fruit beetle species Pachnoda marginata, P. sinuata and P. aemulae and the two protea beetle species Trichostetha fascularis and T. albopicta. Crude corpora cardiaca material from P. sinuata had a small hypertrehalosaemic effect in American cockroaches and a very weak hyperlipaemic activity in migratory locusts. Injections into P. sinuata caused hypertrehalosaemia when a dose of 1.0 corpora cardiaca equivalents was injected. An identical neuropeptide was isolated, by RP-HPLC, and sequenced by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal 5-oxopyrrolidine-2-carboxylic acid residue, as well as by collision-induced decomposition tandem fast atom bombardment mass spectrometry. The peptide is a blocked octapeptide: Glu-Leu-Asn-Tyr-Ser-Pro-Asp-TrpNH2, previously designated Mem-CC. The synthetic peptide is able to elicit haemolymph carbohydrates in P. sinuata upon injection of low doses. Activity studies using synthetic analogues of this peptide revealed that Tyr4 may be important for receptor recognition/binding. The peptide is synthesized in intrinsic cells of the corpus cardiacum as shown by in vitro incorporation of [3H]Trp and [14C]Tyr in Mem-CC.

Proteins present at high concentrations in hemolymph of the larval weevil Diaprepes abbreviatus were previously shown to bind a synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10). One of the two native proteins previously identified (protein I) is now shown to separate into two distinct bands (proteins Ia and Ib) using native gradient pore-limiting electrophoresis. The high concentration of proteins Ia, Ib, and II in larval hemolymph, their disappearance from hemolymph upon pupation, and an apparent hexameric structure shown by chemical crosslinking identify them as hexameric storage proteins (hexamerins). At least one chromatographic form of Ib isolated by anion exchange HPLC is now shown to bind riboflavin (Rb). Binding was also demonstrated by quenching of Rb fluorescence by a partially isolated mixture of the storage proteins. Lipophorin did not quench Rb fluorescence. Rb was heat-extracted from whole hemolymph and identified by its fluorescence spectra and by reverse phase HPLC with fluorescence detection. The two subunits shared by the three holoproteins have been isolated by sequential density gradient ultracentrifugation, gel permeation HPLC, and reverse phase HPLC. All three holoproteins shared the alpha subunit (M(r) 75,000), while the beta subunit (M(r) 71,000) was lacking from one of the three. Repeated passage through an anion exchange column yielded two of the three proteins (Ib and II) in homogeneous form. Chemical crosslinking with dimethylsuberimidate indicated a hexameric structure for the holoproteins. All subunits and holoproteins stained as high mannose glycoproteins when probed with biotinylated concanavalin A on PVDF membranes. The alpha subunit was high in Met, His, and Thr, and the beta subunit was high in Lys. Both were high in Pro and had approximately 16% Phe+Tyr. Sequences of the 20 N-terminal amino acid residues of each subunit showed 45-60% homology between subunits. These coleopteran proteins also showed some sequential homology but no immunological cross-reactivity with storage proteins from the lepidopterans Galleria mellonella and Heliothis virescens.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.

From March 1988 through March 1990, at the Philadelphia Medical Examiner's Office toxicology laboratory, samples from 77 decomposed human bodies were tested for the presence of cocaine, employing gas chromatography/mass spectrometry (GC-MS). The material analyzed included decomposed soft tissue, bloody decomposition fluid, mummified tissue, maggots, and beetle feces. Twenty-two cases (28.6%) were positive for cocaine, many of these cases in states of advanced decomposition. These findings indicate the usefulness of testing decomposed tissue for cocaine in all cases where its presence is suspected. This is contrary to what might be expected, since cocaine is generally labile and rapidly broken down by both enzymatic and nonenzymatic mechanisms.

The structure of lipophorin, isolated from hemolymph of the Colorado potato beetle, was investigated by differential scanning calorimetry (DSC) and small-angle X-ray scattering. The DSC heating curves of intact lipophorin showed endothermic peaks that were similar to peaks obtained with the hydrocarbon fraction isolated from this lipophorin. The observed peaks correlated with the transition of the hydrocarbons from an ordered into a more disordered state. Changes in structure of the lipophorin particles with increasing temperature were also observed by small-angle X-ray scattering studies. The structural organization of lipophorin was further elucidated by simulation analysis, using a three-layered symmetrical sphere as a model. These studies revealed that lipophorin from the Colorado potato beetle is a sphere with a maximum diameter of 175 A. The sphere is composed of three radially symmetrical layers of different electron densities. The outer layer (37.5-39.5 A in thickness) is composed of phospholipid, apolipophorin I, and part of apolipophorin II. The middle layer (5-10 A) contains diacylglycerol, the rest of apolipophorin II, and probably beta-carotene. The core of the particle (40-45 A) only contains hydrocarbons. This structure differs from another model, previously proposed for cockroach and locust lipophorins [Katagiri, C., Sato, M., & Tanaka N. (1987) J. Biol. Chem. 262, 15857-15861], in the small size of the middle layer. The volume of the middle layer correlated well with the low diacylglycerol content of this lipophorin.

A conservative sequence motif was extracted from an alignment of the firefly luciferase family. AngR derived from a pathogenic bacterium and acetyl-CoA synthetases derived from two ascomycete fungi were identified as members of the firefly luciferase family by a homology search with the motif and other sequence comparison analyses. The motif sequence shares several characteristics with the phosphate-binding sites of phosphoproteins and nucleotide-binding proteins. A multiple alignment and an unrooted phylogenetic tree were constructed for the investigation of evolutionary relationships within the firefly luciferase family.