Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta.

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.

Bioactive compounds from aquatic and terrestrial sources.

The world of nature provides a never-ending set of fascinating problems for the chemist. Many of the most intriguing problems, however, concern compounds available in only truly minute quantities. One solution is to focus on bioassay-guided separations. In so doing one can isolate compounds with novel structures or unsuspected activities from almost any phylum, including tunicates, sponges, insects, or even the much-studied terrestrial plants, as exemplified in several recent studies in our laboratory involving activities ranging from antiviral and antimicrobial activity to cytotoxicity and immunomodulation. Moreover, newer spectroscopic techniques, especially fast atom bombardment mass spectrometry and tandem mass spectrometry, enhance one's ability to study compounds present in minute quantities, including those of importance to the host organism, such as neuropeptides in insects or marine invertebrates.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

Isolation of high-molecular-weight DNA from insects.

A simple and rapid method for the isolation of high-molecular-weight DNA from insects is described. The method does not require CsCl ultracentrifugation or extensive dialysis. High-molecular-weight DNA was obtained within 24 h. Since the entire insect was used for DNA isolation, an initial nuclei-enriched fraction was required. Genomic DNA was extracted from lysed nuclei by organic phase separation (liquid/liquid extraction). This method has been successfully applied to the isolation and purification of DNA from eight different adult insects (Heliothis zea, Musca autumnalis, M. domestica, Blatta orientalis, Tenebrio molitor, Lymantria dispar, Ostrinia nubilalis, and Manduca sexta). The recovered DNA can be cleaved with restriction endonucleases, ligated efficiently using standard cloning vectors, and hybridized to synthetic oligonucleotides.

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata.

A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the Lom-AKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.

Effect of gelatin on recovery of hair fragments in filth analysis.

Prior literature has recommended the use of gelatin capsules or gelatin film for transferring spike elements to samples being examined for recovery studies. It was believed that gelatin had no effect on the recovery of filth spike elements. However, this study shows that hair recovery is lower when gelatin is present in direct trap-off procedures. Two types of gelatin capsules, gelatin film, and strips of filter paper were used to transfer spike filth elements. A comparison study employing an acid digestion and wet sieving procedure was also performed and showed that gelatin had little or no effect on the recovery of hairs using this type of procedure. An additional test was performed using gelatin added to water containing the same type of spiked filth elements. No hair fragments were recovered but all insect fragments were recovered. All recovery studies were performed using only water in the liquid phase of the trap flask extractions, with mineral oil or heptane as the flotation medium. No food product was used.

Biochemical identification of thymosin alpha-1: its phylogenetic distribution and evolutionary implications.

1. Thymosin alpha-1, like reactivity, was found in several different species (insects, crab, protozoan, fungus and bacteria) by radioimmunoassay and immune fluorescence and as an extracellular product from the bacterial genus Mycobacterium. 2. Biochemically, thymosin alpha-1 has been isolated from combined crab visceral and nervous tissue by reverse phase HPLC. 3. The identification of thymosin alpha-1 in lower life forms suggests a more generalized exocrine origin in unicellular organisms prior to the development of the immune system or exocrine differentiation.

Composition and distribution of retinal and 3-hydroxyretinal in the compound eye of the dragonfly.

Retinoids in the compound eyes of nymphs and adult dragonflies in 11 families of the 3 suborders were extracted by the oxime method, and analysed by high performance liquid chromatography. Almost all of the species examined contained both retinal and 3-hydroxyretinal in the compound eye. The ratio of 11-cis 3-hydroxyretinal to 11-cis retinal (3-OH ratio) was calculated as an index of the retinoid composition. The 3-OH ratios of the whole eye of nymphs in all the suborders and of adults of the suborder Zygoptera were very high, 2.2 at the minimum, but in Anisozygoptera and Anisoptera most of the ratios were distributed between 1 and 2.7. In the family Gomphidae, exceptionally low 3-OH ratios, less than 1, were observed in several species. The regional distributions of the retinals in the adult compound eyes were also examined. In the Zygopteran compound eye, both retinals were distributed evenly all over the eye, while in the compound eye of the other two suborders, the 3-OH ratios in the dorsal area of the eye were extremely low. In several species of Gomphidae and Libellulidae the ratios in the dorsal areas were zero. From the correspondence of these results and the compartment of the compound eye, it appeared that the large ommatidia in the dorsal area contained only retinal. This was confirmed when the large facet region in the dorsal part of the compound eye of an Anax was excised and examined, and only retinal was detected. However, the ventral area of the true dragonflies' compound eye which did not include the large ommatidia contained both retinals, and the 3-OH ratio was more than ten. The biological significance of using both retinals as chromophores of visual pigments in the dragonfly eye is discussed in relation to the structure of the ommatidia and to the vision of dragonflies.

Detection and microsequencing of juvenile hormone-binding proteins of an insect by the use of an iodinated juvenile hormone analog.

An [125I]iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high-affinity, (JH)-specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two-dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I-labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound gamma-emitter can be found by exposure of the dried gel or the electroblot to X-ray film. The radiolabeled protein spot can be excised from the Coomassie-stained glass fiber paper and subjected directly to gas-phase N-terminal amino acid sequencing. This non-destructive, non-denaturing technique may have wide applicability in identifying and sequencing ligand-specific binding proteins in complex mixtures.

Metasternal gland secretion of the locust tree borer, Megacyllene robiniae (Coleoptera: Cerambycidae).

1. The volatile components of metasternal gland extracts of male and female Megacyllene robiniae have been analyzed by gas chromatography-mass spectroscopy. 2. The major component was identified as 2-(1,3-hexadien-1-yl)-5-methyltetrahydrofuran, a new natural product. 3. 1-Phenylethanol is present only in male extracts. 4. Acetates of hexadecanol and octadecanol are also present.

Protein-structural heterogeneity in a non-allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy.

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180 degree rotation of the heme about the alpha, gamma-meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemoglobin, and a 'silent' amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8-methyl and 3-methyl and one of the alpha-vinyl proton resonances for the major heme rotational component and the 5-methyl and 1-methyl and one of the alpha-vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium-labeled heme. Decoupling experiments have been employed to assign vinyl beta protons in cis and trans position to the respective vinyl alpha protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8-methyl and 5-methyl proton resonances possibly reflecting titration of the propionate groups.

A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis.

A multiple homogenizer is described for preparing samples of small invertebrates or tissue in a flat-bottom immunoplate. Its efficiency was evaluated by immunoassay of carboxylesterase (E4), the enzyme conferring insecticide resistance in the peach potato aphid (Myzus persicae). This equipment was shown to release more enzyme, with less variability, than homogenizing individual aphids and its efficiency allows one person to analyze up to 3000 individual insects per day. It is also suitable for preparing samples for electrophoretic analysis. In the present study samples were loaded onto electrophoresis gels rapidly and accurately by using an eight-channel multipipette.

Purification of very high density lipoproteins by differential density gradient ultracentrifugation.

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.

Diversity in neurohaemal organs for homologous neurosecretory cells in different insect species as demonstrated by immunocytochemistry with an antiserum to molluscan cardioexcitatory peptide.

Neurosecretory cells were histochemically identified in the thoracic ganglia of the Colorado potato beetle and Drosophila melanogaster with an antiserum to the molluscan cardioexcitatory neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2). These cells and the FMRFamide-immunoreactive neurosecretory cells identified in a locust are most likely homologous. The neurosecretory axon terminals in the 3 species use different neurohaemal organs. Such a structural diversity in release sites of apparently homologous neurosecretory cells in different species may account for the large structural variability in neurohaemal organs in insects in general.