The creation of a recombinant single-chain antibody against α/β-hydrolase of microsporidium <Emphasis Type="Italic">Paranosema locustae</Emphasis> and immunolocalization of the enzyme in an infected host cell

Microsporidia are a group of widespread fungi-related obligate intracellular parasites. Direct contact of most microsporidia with the cytoplasm of an infected host cell entails possible secretion of various proteins from the parasite that allows control physiological processes of the host. Earlier, by means of polyclonal antibodies against α/β-hydrolase of microsporidium Paranosema locustae, the secretion of large amounts of the enzyme into the cytoplasm of fat body cells of infected migratory locust Locusta migratoria was demonstrated. However, yeast fungi Pichia pastoris did not recognize this enzyme as a secretory one during its heterologous expression. In the present study, a library of recombinant single-chain antibodies (scFv fragments) against proteins of the infected fat body of locust was constructed. The use of the phage display technology enabled choosing a miniantibody that specifically recognized the studied enzyme. Immunoblotting and immunolabeling of frozen sections of locust fat body with the selected scFv fragment confirmed the fact of secretion of P. locustae α/β-hydrolase (as two forms of different size) into the infected host cell. Prospects of using the selected scFv fragment for further studies of the secretion mechanism of the parasite’s protein and its role in host–parasite interactions are discussed.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

The role of adipokinetic hormone in the control of vitellogenesis in locusts

Vitellogenesis in locusts is synchronized with the cyclic maturation of oocytes. Vitellogenesis by excised fat body of gravid females is differentially inhibited 80–90% when locust adipokinetic hormone I (AKH-I) is added to the incubation media. Hemolymph methanolic extracts completely inhibit fat body protein synthesis in vitro when the donor females are at the end of the ovarian cycle (6 mm stage), but not when taken from earlier stages. Hemolymph methanolic extracts from vitellogenic females at the 6 mm stage are separated by HPLC into three distinct inhibitors of protein synthesis, one of which is AKH-I. AKH-RIA of hemolymph during the first ovarian cycle reveals no AKH-I during active vitellogenesis, but a marked increase to about 5 ng per female at the end of egg maturation. A development of responsiveness to AKH-I is evident in female fat body as vitellogenesis proceeds. AKH-I is involved in the negative control of vitellogenesis.     

Protein synthesis and secretion by the locust fat body after ovariectomy

The synthesis and secretion of proteins, including vitellogenin, by the locust fat body were investigated in vivo and in vitro at various times after ovariectomy. The rate of overall protein synthesis and secretion by the fat body in vivo is significantly less in ovariectomized animals than in the controls two to three weeks after the operation, the difference in the rates of secretion of vitellin antibody-precipitable protein being even more pronounced and detectable earlier. The significantly greater amount of newly synthesized vitellogenin retained in the fat body of ovariectomized animals is insufficient to account for this difference. The secretion of newly synthesized protein by the fat bodies of ovariectomized locusts in vitro is significantly less than that of control fat bodies, the difference being particularly marked in the case of vitellogenin. The polysome populations of fat bodies of ovariectomized and control females are quantitatively similar and the amounts of total protein and vitellin antibody-precipitable protein synthesized by these polysomes in a cell-free system do not differ significantly.     

Effect of O-allyl-hydroxy amino acids on protein synthesis and secretion by cultured fat body and salivary glands in the fruit fly Ceratitis capitata

• 1.1. The amino acid analogs O-allyl-tyrosine, O-allyl-threonine, O-allyl-serine and O-allyl-homoserine, have been synthesized and their effect on protein synthesis and secretion in cultured C. capitata fat body and salivary glands have been studied.
• 2.2. The analogs had an inhibitory effect on protein synthesis in both tissues. Moreover, the analogs caused a slight increase in protein secretion in the fat body in comparison to the control.
• 3.3. The intracellular transit time of protein secretion in treated and non-treated fat body was found essentially the same (about 10 min).

     

Synthesis of adipokinetic hormone (AKH-I) in the locust brain

Methanol extracts of vitellogenic female locust brains contain two factors that inhibit protein synthesis in fat body tissue excised from such individuals. One of these factors (BI) elicits lipid mobilization when injected into adult male locusts. The retention times of BI on an RP-18 column and on an RP-4 column are identical to those of synthetic locust adipokinetic hormone (AKH-I) on each of these columns. Half maximal inhibition of protein synthesis in excised adult locust fat bodies is exerted by 0.05 brain extract equivalents of BI, which is equivalent to activity elicited by 1.5 pmol of AKH-I, as previously determined by AKH-radioimmunoassay. Enzymatic hydrolysis of the N-terminal pyroglutamate, followed by amino acid sequence analysis, indicates that the structure of BI is similar to that of the decapeptide AKH-I synthesized in the glandular lobe of the locust corpora cardiaca (CC). Incorporation of [5-³H]tryptophan into BI of locust brains incubated in vitro indicates that the AKH-I present in the brain is synthesized in situ and is not transported from the CC. Similar incorporation of radiolabel into AKH-I is obtained when excised CC are incubated in vitro.     

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca²⁺. The hormones stimulate both the influx and the efflux of Ca²⁺, the higher influx probably causing an increase in intracellular [Ca²⁺]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca²⁺ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.     

Juvenile hormone action on locust fat body

Production of vitellogenin (Vg) in fat body of adult female Locusta migratoria is abolished by removal or inactivation of the corpora allata and restored by administration of (RS)-methoprene. Juvenile hormone III injection alone has little effect, but when it is injected together with the JH esterase inhibitor OTFP, active Vg synthesis is induced. This supports the assumption that methoprene acts in place of the natural hormone in this system. When fat body from vitellogenic females is maintained in synthetic medium for 48 hr, the proportion of Vg in the secreted protein drops greatly, but when methoprene is present in the medium the proportion of Vg is sustained. When fat body from JH-withdrawn locusts, in which Vg synthesis has declined to zero, is cultured with methoprene, Vg synthesis is re-induced. These results show that the JH analog can act directly on locust fat body to bring about expression of the Vg genes. Experiments to optimize JH action on fat body in vitro are continuing.     

Activation of fat body glycogen phosphorylase in Locusta migratoria by corpus cardiacum extract and synthetic adipokinetic hormone

Between 10 and 20 per cent of the total glycogen phosphorylase in the fat body of mature Locusta migratoria of both sexes is in the active form. Injection of an aqueous corpus cardiacum (CC) extract results in a rapid activation: within 2 min the level of active phosphorylase is significantly increased and full activation is reached within 10 to 20 min. As little as 0.002 CC gland equivalents stimulate fat body glycogen phosphorylase significantly and maximum activation is obtained with 0.05 CC gland equivalents. From experiments with known quantities of injected synthetic adipokinetic hormone (SAKH), it appears that this hormone cannot account for all the activation. This is supported by results obtained when extracts of carefully isolated storage lobes are injected; at the dose used here these have no adipokinetic activity, but activate fat body phosphorylase. Furthermore, when locusts are ‘stressed’ by rotation, although no adipokinetic hormone is released, an activation of phosphorylase occurs. Starvation causes also an increase in the active form of the enzyme. The fat body receptor sites of the locust recognise also the crustacean red pigment concentrating hormone (RPCH), whose structure closely resembles that of the locust adipokinetic hormone, leading to activation of the phosphorylase. However, RPCH is about 2.5–5 times less potent than SAKH. Crude CC extracts of a stick insect (Carausius morosus), a cockroach (Periplaneta americana) and the tobacco hornworm (Manduca sexta) activate locust fat body phosphorylase, although this last extract has no effect on lipid elevation. On the other hand, CC extracts of the death's head hawk moth (Acherontia atropos) and purified crustacean hyperglycaemic hormone from a crayfish (Orconectes limosus) have no effect.     

The conserved transmembrane protein TMEM-39 coordinates with COPII to promote collagen secretion and regulate ER stress response

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.     

Feeding causes the appearance of a factor in the haemolymph that stimulates protein synthesis

Soon after a locust (Locusta migratoria) begins to feed, an increase in protein synthesis can be detected in the animal. Isolation of fat body shows that this tissue synthesizes protein at a faster rate in recently fed animals than it does in fasting insects. Fasting locusts injected with haemolymph from fed insects increased protein synthesis when compared with locusts injected with haemolymph from fasting locusts. The factor causing this increase was present in the haemolymph within 5 min of feeding. Feeding or direct contact with the food was not essential to increase protein synthesis. Exposure of fasting locusts to feeding insects was sufficient to elevate the rates of protein synthesis in the fasting animals.The increase inprotein synthesis was not a result of general excitation or an increase in the concentration of tryptophan or isoleucine in the haemolymph. Ecdysteroid titres were uniformly low during the first ten days of adult life. Gel filtration of the fed haemolymph revealed a low molecular weight fraction (about 600 daltons) which stimulated protein synthesis upon injection into fasting locusts.     

Factors influencing cyclic AMP and diacylglycerol levels in fat body of Locusta migratoria

Forskolin (FORSK), octopamine (OA) and adipokinetic hormone (AKH) stimulate the production of diacylglycerol (DG) in fat body of the locust, Locusta migratoria and the release of DG from fat body into hemolmph. The three effectors also increase the level of cAMP in fat body, but the cAMP content is not proportional to DG production. AKH stimulates the uptake of Ca²⁺ by fat body cells and requires the presence of extracellular Ca²⁺ to increase cAMP and DG levels in fat body. The production of DG seems to be an energy-dependent process. The uptake of DG by lipophorin (LP) from fat body is also energy-dependent but does not require extracellular Ca²⁺.     

Signal transduction for Schistocerca gregaria ion transport peptide is mediated via both cyclic AMP and cyclic GMP

The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl⁻, K⁺ and Na⁺ reabsorption, whereas secretion of H⁺ into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl⁻ uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.     

Colchicine-binding protein and the secretion of thyroid hormone

The role of microtubules in the thyrotropin- or adenosine 3'',5'' cyclic monophosphate (cyclic AMP)-stimulated accumulation of cytoplasmic colloid droplets and secretion of iodine from the mouse thyroid gland has been investigated by means of different classes of agents that affect the stability of microtubules. The onset of inhibition of secretion by colchicine, the uptake of colchicine-³H by thyroid lobes, and the binding of colchicine-³H to thyroidal soluble protein are shown to have similar time courses Colloid droplet accumulation is also inhibited and does not readily resume upon removal of colchicine from the medium. This appears to be due to the slow washout of the drug (t _½ ∼ hr). Thyroids contain a soluble colchicine-binding protein that resembles microtubule proteins of other tissues with respect to apparent K_m for colchicine, pH optimum, and stability characteristics Colchicine analogues inhibit iodine secretion and colchicine binding in a parallel manner and as a function of their antimitotic potencies. Microtubule-stabilizing agents such as hexylene glycol and D₂O also inhibit secretion. Thus, inhibition of thyroid secretion by antimitotic agents appears to be mediated by an effect on microtubules. The inhibitory locus of colchicine inhibition occurs after the generation of cyclic AMP, since stimulation of secretion by this nucleotide is blocked by colchicine, whereas thyroid-stimulating hormone—induced accumulation of cyclic AMP is not affected. Thus, the functioning microtubule appears to play a role in the induction of colloid endocytosis.     

Locust adipokinetic hormone stimulates lipid mobilization in Manduca sexta

Adipokinetic hormone, a decapeptide isolated from the locust, stimulates mobilization of diacylglycerols from the locust fat body and loading of the lipid transport protein, lipophorin. Injection of the synthetic locust adipokinetic hormone into a sphinx moth, Manduca sexta, causes lipid loading of lipophorin. The lipophorin decreases in density from 1.11 to 1.06 g/ml, and a soluble protein from the hemolymph (apolipophorin III) associates with the lipophorin particle. Administration of intermediate doses of hormone indicates that lipophorin is converted directly to the low density form; no appreciable amounts of intermediate density particles are formed.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Relationships between milks differentiated on native milk fat globule characteristics and fat,protein and calcium compositions

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.     

In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria

Both adipokinetic hormone and octopamine have a stimulating effect on lipid release from locust fat body in vitro, when incubated in diluted haemolymph. The presence of adipokinetic hormone results in the formation of the flight-specific haemolymph lipoprotein A⁺ accepting the increased amount of lipids released into the incubation medium. In contrast, interconversions of lipoproteins do not occur when octopamine is added to the incubation medium, which is in line with the expectations: the lipid-mobilizing effect of octopamine is a limited and short-term effect. When fat body tissue is incubated with isolated haemolymph protein fractions, the lipid-mobilizing effect of adipokinetic hormone only occurs when the incubation medium contains both lipoprotein, A_y and protein fraction C, resulting in the formation of lipoprotein A⁺. In similar control incubations with the hormone omitted, some lipoprotein A⁺ is also formed (concomitant with a slight amount of lipid released), though significantly less than in incubations with hormone. Besides a stimulating function on lipolytic processes in the fat body, adipokinetic hormone is suggested to influence haemolymph lipoprotein rearrangement. A possible counteracting function of another factor in the haemolymph is discussed.     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.