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.

Hymenolepis diminuta: an investigation of juvenile hormone titre, degradation and supplementation in the intermediate host, Tenebrio molitor.

Metacestodes of Hymenolepis diminuta cause a perturbance of vitellogenesis in the intermediate host Tenebrio molitor. The reduction in host reproductive output associated with infection may be due to this pathophysiology. Many of these events are regulated by host juvenile hormone (JH). A comparison of the titre of JH and its rate of degradation in female control and parasitized 15-day-old insects has been made. Haemolymph from female beetles contained 1.27 pMol JH equivalents/100 microliters. No significant difference was associated with infection. Likewise, the activity of JH esterase in female haemolymph was not affected by infection. However, topical application of a JH analogue, methoprene, at the time of infection or 8 days post-infection reduced the significant accumulation of vitellogenin usually found in the haemolymph of females 12 days or more post-infection. These findings indicate that parasite-induced alteration of host vitellogenesis is not mediated via alteration in JH titres, although observations made after hormone supplementation suggest some form of interaction between the parasite and the host endocrine system.

Biochemistry of the evolution of Triatoma infestans: XI. Hemolymph lipophorin

Se aisló y purificó la lipoforina del T.infestans a partir de hemolinfa de machos adultos por ultracentrifugación en gradiente de densidad en dos etapas. Esta lipoproteína tiene una densidad de 1.10 g/ml y está constituida por 53% de proteínas y 47% de lípidos. El radio de Stokes (Rs) de la molécula, determinado por cromatografía de filtración en gel, es aproximadamente de 73A y e;l peso molecular (Mr) calculado por el método de Margolis, es de 743 000 daltons. La lipoforina contiene dos apoproteínas: apolipoforina I (apoLp-I) con Mr = 255 000 y apolipoforina II (apoLp-II) con Mr = 78 000, siendo ambas glicosiladas. La composición de aminoácidos de la apoLp-I y de la apoLp-II presenta un elevado contenido de aspartato, glutamato y leucina y muy pequeña cantidad de metionina y cisteína. Los lípidos de la lipoforina comprenden: diacilgliceroles (41.4% de los lípidos totales), fosfolípidos (31.5%), hidrocarburos (12.2%), ácidos grasos libres (6.1%), colesterol (4.7%) y triacilgliceroles (4.1%). La fosfatidiletanolamina es el fosfolípido predominante con cantidades menores de fosfatidilcolina. Al tratar la lipoforina con tripsina, la apoLp-I sufre ruptura proteolítica, mientras que la apoLp-II es resistente. La anisotropía de fluorescencia del difenilhexatrieno (DPH) incluido en la lipoforina señala una fuerte interacción lípido-apoproteína, la cual no se modifica después de una extensa proteólisis de la apoLp-I con tripsina. Durante la tripsinización de la lipoproteína no se detectó...

Biochemistry of the evolution of Triatoma infestans: XI. Hemolymph lipophorin

Se aisló y purificó la lipoforina del T.infestans a partir de hemolinfa de machos adultos por ultracentrifugación en gradiente de densidad en dos etapas. Esta lipoproteína tiene una densidad de 1.10 g/ml y está constituida por 53% de proteínas y 47% de lípidos. El radio de Stokes (Rs) de la molécula, determinado por cromatografía de filtración en gel, es aproximadamente de 73A y e;l peso molecular (Mr) calculado por el método de Margolis, es de 743 000 daltons. La lipoforina contiene dos apoproteínas: apolipoforina I (apoLp-I) con Mr = 255 000 y apolipoforina II (apoLp-II) con Mr = 78 000, siendo ambas glicosiladas. La composición de aminoácidos de la apoLp-I y de la apoLp-II presenta un elevado contenido de aspartato, glutamato y leucina y muy pequeña cantidad de metionina y cisteína. Los lípidos de la lipoforina comprenden: diacilgliceroles (41.4% de los lípidos totales), fosfolípidos (31.5%), hidrocarburos (12.2%), ácidos grasos libres (6.1%), colesterol (4.7%) y triacilgliceroles (4.1%). La fosfatidiletanolamina es el fosfolípido predominante con cantidades menores de fosfatidilcolina. Al tratar la lipoforina con tripsina, la apoLp-I sufre ruptura proteolítica, mientras que la apoLp-II es resistente. La anisotropía de fluorescencia del difenilhexatrieno (DPH) incluido en la lipoforina señala una fuerte interacción lípido-apoproteína, la cual no se modifica después de una extensa proteólisis de la apoLp-I con tripsina. Durante la tripsinización de la lipoproteína no se detectó... (AU)

Purification and characterization of lipopolysaccharide-binding protein from hemolymph of American cockroach Periplaneta americana.

A protein having affinity to lipopolysaccharide of Escherichia coli K12 was purified to homogeneity from the hemolymph of Periplaneta americana. This protein, with an average molecular mass of 450 kDa. was a homooligomer of a 28-kDa subunit protein. Comparative studies using lipopolysaccharide molecules of E. coli and Salmonella minnesota suggested that this protein recognizes and binds to a specific carbohydrate structure of E. coli lipopolysaccharide. Ca2+ was required for this protein to bind to lipopolysaccharide, but other divalent cations could not replace Ca2+.

Protein constituents of mosquito saliva: studies on Culex molestus.

The protein constituents of saliva, salivary gland extracts, haemolymph and whole body extract from the Culex molestus mosquito were compared by polyacrylamide gel electrophoresis. When developed by the sensitive silver staining technique, saliva and salivary gland extract were found to contain 15 comparable protein bands. Salivary gland extract contained additional bands, presumed to be structural proteins, and saliva contained some unique protein bands which were not present in the gland extract, possibly originating in the salivary stylet lining. Some differences were found in the protein components of salivary gland extracts prepared from mosquitoes of different ages. Salivary proteins were found to be poorly represented in both haemolymph and whole body extracts. Immunoblotting of salivary gland extract with antibody raised against pure saliva exhibited binding to at least nine protein bands indicating the potential for using salivary gland extracts in place of mosquito saliva for further studies.

Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta.

A lipoprotein receptor has been purified from the fat body of Manduca sexta larvae. The purification involves solubilization of membrane proteins in detergent, DEAE-, and hydroxyapatite chromatography, affinity chromatography on a concanavalin A column, and affinity chromatography on a lipoprotein-Sepharose column. An overall purification of 220-fold from the solubilized membranes was achieved. The receptor has an apparent molecular mass of 120 kDa. The receptor has an absolute requirement for Ca2+ and is inhibited by Suramin. The pH optimum of the receptor is 6.5, which is near the pH of the hemolymph. Binding data indicate a single high affinity binding site with a Kd = 4.1 +/- 0.19 x 10(-8) M as measured with the lipoprotein isolated from larval hemolymph. The major neutral lipid carried by insect lipoproteins is diacylglycerol, and it was shown that the affinity of the receptor for lipoprotein ligands correlates with their diacylglycerol content. It is proposed that the decrease in affinity of the receptor for lipoproteins depleted of diacylglycerol plays a key role in facilitating the transport of diacylglycerol from the midgut to the fat body during the larval feeding period. The insect receptor has some properties which are similar to those of vertebrate lipoprotein receptors, viz. molecular weight, requirement for Ca2+, and inhibition by Suramin. However, the insect receptor does not bind human low density lipoprotein.

A new method of detecting hormone-binding proteins electroblotted onto glass fiber filter: juvenile hormone-binding proteins from grasshopper hemolymph.

We have developed a new method to identify juvenile hormone (JH)-binding proteins blotted onto glass fiber filter (GFF) after electrophoretic separation. Insect JH regulates reproduction in the two-striped grasshopper, Melanoplus bivittatus. A number of proteins are involved in the delivery of JH from its site of synthesis to the nuclei of fat body cells where it acts to induce vitellogenesis. To identify JH binding proteins, hemolymph was separated by PAGE, electroblotted onto GFF, and incubated in [10-3H]JH-III. The amount of hormone bound by blotted proteins increased with the amount of protein on the filter, was competitively displaced by excess non-labeled hormone, and was affiliated with individual bands on fluorograms of proteins blotted after electrophoretic separation. GFF etched with trifluoroacetic acid was better than nitrocellulose, Zeta Probe, cellulose acetate or unetched GFF. Phosphate (pH 6.0-7.3) or Tris buffers (pH 7.3-8.0) worked equally well for the procedure. Unbound hormone was easily removed by short washes in buffer, and adequate binding for detection was achieved in a 15 min incubation. Preliminary data suggest that this technique may be used to detect receptors, carriers, and binding proteins of steroid hormones.

Immunomodulatory stimulation of an invertebrate circulatory lectin by its haptenic molecules of pathogenic origin.

2-keto-3-deoxy octonate and beta-glycerophosphate two important bacterial cell wall constituent molecules, haptenic in nature to an invertebrate (crab) circulatory lectin, carcinoscorpin, gave lectin induction after immunization of the crab, Carcinoscorpius rotunda-cauda. With the exception of erythrocyte, no other sialoglycoconjugate-containing substances (mucin, fetuin) sialodisaccharide O-[N-acetylneuraminyl-(2----6)2-acetamido-2-deoxy galactitol] and free sialic acid were effective in lectin induction. This induction of circulatory lectin failed to appear when, concanavalin A, epinephrine, cytochalasin B, methyl-alpha-D-mannoside and bovine serum albumin were used for immunization. But mechanical injury to crabs were extremely effective in such lectin induction by live crabs. LPS is lethal if used for immunization. The immunoregulatory induction of the circulatory lectin by pathogen-originated cell wall constituent molecules and mechanical injury may be considered as a humoral immune response.

Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera).

Honeybee (Apis mellifera) are frequently exposed to and likely to be infected by plant-associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387-2391]. We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino-terminal half is related to the apidaecins; similar proline motifs are also present in the amino-terminal quarter of the much longer fly diptericins. The newly identified peptide's broad spectrum, lower specific activities against Gram-negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin-resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name 'abaecin' for this new antibacterial response peptide.

Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori.

1. Cecropins, inducible antibacterial peptides, were purified by simple two step chromatography from immunized larval hemolymph of the silkworm, Bombyx mori. 2. The cecropins were further separated into four major and two minor forms by reverse-phase HPLC. 3. The four major cecropins were sequenced and divided into two types, A and B, whose structures were quite similar to cecropins A and B of Hyalophora cecropia. 4. Three of them contained an unusual amino acid, delta-hydroxylysine. 5. No remarkable difference in antibacterial activity against E. coli was detected among these cecropins.

Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta.

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.

Purification and characterization of trehalase inhibitor from hemolymph of the American cockroach, Periplaneta americana.

An endogenous proteinaceous inhibitor of trehalase (alpha,alpha-trehalose-1-glucohydrolase: EC 3.2.1.28) has been isolated and purified from the serum of resting adult American cockroaches, Periplaneta americana. Purification procedures involved decreasing ionic strength, gel filtration, and reversed phase high performance liquid chromatography. Homogeneity was confirmed by polyacrylamide gel electrophoresis and end group analysis. The purified protein inhibited trehalase activity in a dose-dependent manner and was estimated to have a molecular weight of 86,000 and to contain sugar chains. An automated gas-phase sequencer was used to determine the following sequence for the N-terminal amino acid residues: H-Ala-Ilu-Pro-Thr-Pro-His-Val-Tyr-Lys-Val-X-Val-Pro-Asp-Gly-Ala-Le u-Asn-Asp.

Comparison of insect hemoglobins (Erythrocruorins) from Chironomus thummi thummi and Chironomus thummi piger (Diptera). The primary structure of the monomeric hemoglobin CTP III.

The monomeric hemoglobin fractions of Chironomus thummi thummi (CTT) and Chironomus thummi piger (CTP) differ in the ratio of their components. The determination of the primary structure of the component CTP III was achieved by automatic Edman degradation of the native chain, the tryptic peptides and the C-terminal fragment, obtained by cleavage at the single tryptophan residue. It revealed two chains in the ratio 1:1 which share the ambiguity threonine/isoleucine in position 57 with CTT III. Whereas one chain is identical to the CTT III hemoglobin, the other differs in having isoleucine in position 105 and alanine in position 134. The CTP monomeric hemoglobin fraction comprises 8% of a component (CTP IV A) with a more negative charge than CTT IV but with an identical sequence up to position 44. This study reveals a very high polymorphism within Chironomus species and points out the need for more data at the gene level in order to provide better understanding of this striking phenomenon.

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus.

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].

Schistosoma mansoni antigens recognized in Biomphalaria glabrata hemolymph by monoclonal antibodies.

In order to identify and characterize Schistosoma mansoni antigens in Biomphalaria glabrata, we examined 19 murine monoclonal antibodies (Mabs) for specific binding to schistosome larvae. None of the murine Mabs induced by infection or by immunization with a crude cercarial antigen (CCA) served this purpose. Two Mabs out of 9 (KCSme22-3 and KCSme22-4) induced by soluble egg antigens reacted with CCA but not with normal snail (NSN) extract. We selected these 2 for studies on detection and characterization of schistosomal antigens in snails. When employed in an ELISA, they differentially detected schistosomal antigens in extracts and cell-free hemolymph (plasma) of infected snails. The selected Mabs bind to cercarial surface as demonstrated by the indirect fluorescent antibody technique (IFAT) with paraformaldehyde-fixed cercariae. The epitopes corresponding to the selected Mabs are periodate sensitive, suggesting the glycoprotein nature of the antigens recognized. Immunoblotting analysis employing the selected Mab revealed 1 antigen in CCA (Mr = 205 kDa) and 3 antigens in snail plasma (Mr = 220 kDa, 180 kDa, and 135 kDa). Schistosomal antigens were first detectable in the snails' plasma 2 weeks after snail infection, and their quantity increased afterwards.

Identification of snails infected with schistosomes by ELISA employing monoclonal antibodies: Schistosoma mansoni in laboratory snails (Biomphalaria glabrata) and in field snails (Biomphalaria pfeifferi) from Kenya.

An enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibodies was used for detecting Schistosoma mansoni antigens in hemolymph of laboratory snails (Biomphalaria glabrata) in Kenya. Infected laboratory snails shedding cercariae were differentially identified by ELISA from uninfected snails with 100% sensitivity and specificity. Prepatent infections were detected by ELISA from 2 weeks after exposure to miracidia. Thus, ELISA revealed infection 3 weeks before maximal patency was reached (5-6 weeks post-exposure). Infected field snails (B. pfeifferi) shedding cercariae were differentially identified by ELISA, with 100% sensitivity and specificity, from uninfected field snails and from snails naturally infected with other trematodes (echinostomes and strigeids). Prepatent infections with S. mansoni were readily identified by ELISA in field snails. A case is demonstrated where infection rate, as determined by shedding test alone, was 9.8%, whereas the combined figure of prepatent and patent infection rates was 22.9%

Purification and properties of the very high density lipoprotein from the hemolymph of adult Triatoma infestans.

The very high density lipoprotein (VHDL) of Triatoma infestans hemolymph from adult males has been isolated and purified by two-step density gradient ultracentrifugation. It appears to be homogeneous as judged by native polyacrylamide gel electrophoresis. The content of VHDL in hemolymph was estimated to be 8 mg protein/ml. The purified protein has a molecular weight (Mr) of 450,000, is composed of six subunits of Mr approximately equal to 77,000, and possesses a high content of aromatic amino acids. This protein is glycosylated and contains 3% of lipids by weight with a remarkable amount of free fatty acids (25% of total lipids). The T. infestans VHDL has a different lipid and amino acid composition from lipophorin. The lipid composition and the spectroscopic studies using cis-parinaric acid indicated a high fatty acid binding affinity. It has nine binding sites per mol of VHDL. Competence studies revealed that VHDL has its highest affinity for the binding of palmitic acid followed by stearic and arachidonic acids.

Developmental and diurnal changes in ecdysteroid biosynthesis by prothoracic glands of Rhodnius prolixus (Hemiptera) in vitro during the last larval instar.

The synthesis of ecdysteroids by prothoracic glands (PGs) of male last instar larvae of Rhodnius prolixus was measured in vitro by radioimmunoassay throughout the course of larval-adult development. Large and systematic changes in relative rates of synthesis occur during development. Two bursts of elevated synthetic activity were found. The first commences as soon as development is initiated by a blood meal and lasts approximately 1 day. The second commences 4 days later and increases progressively to a peak at Days 11-13 after feeding (up to 25 ng of 20-hydroxyecdysone eq. gland-1/4 hr-1). The onset of each of these bursts of activity coincides with apparent times of PG stimulation in vivo by release of the prothoracicotropic hormone from the brain. Both bursts result in increases in hemolymph ecdysteroid titer measured in the donor animals. PGs exhibit an abrupt attenuation of synthesis on Day 14, which is followed by a rapid decline in the hemolymph ecdysteroid titer. Clearly, ecdysteroid synthesis by PGs is a major factor regulating the hemolymph titer. Ecdysteroid synthesis by PGs exhibits diurnal changes in vitro. The amount of ecdysteroid synthesized by PGs from animals during the scotophase is two to five times higher than that from animals during the photophase. A corresponding rhythm is seen in the hemolymph ecdysteroid titer. The rhythm in the titer is known to be under circadian control. It is therefore suggested that ecdysteroid synthesis in PGs of Rhodnius is regulated by a circadian system, possibly located in the PGs themselves.

Effect of Schistosoma mansoni infection, starvation and molluscicides on acid phosphate, transaminases and total protein in tissues and hemolymph of Biomphalaria alexandrina.

The activities of GOT and GPT in the hemolymph of B. alexandrina were significantly decreased by S. mansoni infection. However, the total protein concentration and AcP activity were increased. Although the snail starvation decreased AcP activity in the ovotestis, it increased GOT activity in the other organs of the snails. On the other hand, the snail feeding after starvation increased significantly AcP activity in ovotestis. Natural and synthetic molluscicides inhibited the activities of GOT and GPT, however, they increased the total protein concentrations and AcP activities in the examined organs.