bluuebunny/crossref_metadata_2025_split huggingface dataset:
Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol
Authors: Thompson Malcolm J. , Svoboda James A. , Lozano Ruben , Wilzer Kenneth R. 
Published: February 2005

AbstractThe levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1‐h old contain about 2.5 and 35 μ/g of the 2‐ and 26‐phosphates of 26‐hydroxyecdysone, respectively, and 1 μg/g of 26‐hydroxyecdysone. During embryogenesis of 26‐hydroxyedcdysone 26‐phosphate is hydrolyzed to 26‐hydroxyecdysone, which reaches a peak titer in 1–18‐h‐old eggs; the level of 26‐hydroxyecdysone 2‐phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26‐hydroxyecdysone 26‐phosphate decreases there is a progression of other ecdysteroids. C‐20 hydroxylation first appears in 24–40‐h‐old eggs and reaches peak activity in 48–64‐h‐old eggs, where 20‐hydroxyecdysone and 20, 26‐dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h‐old. C‐3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3‐epi‐26‐hydroxyecdysone and 3‐epi‐20, 26‐dihydroxyecdysone are the major free ecdysteroids in 96‐h‐old eggs. A new ecdysteroid conjugate, 26‐hydroxyecdysone 22‐glucoside, first appears at 24–40h and becomes the major conjugate in 72–80‐h‐old eggs; it represents an apparent end‐product as its peak titer is reached and maintained throughout the final embryonic stages. 20‐Hydroxyecdysonoic acid occurs in 48–64‐h‐old eggs, and along with 3‐epi‐20‐hydroxyecdysonoic and ecdysonoic acids in 72–88‐h‐old eggs. 20‐Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3‐epi‐20‐hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1‐h‐old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26‐hydroxyecdysone 26‐phosphate serves as source for 26‐hydroxyecdysone and the numerous metabolites; that 26‐hydroxyecdysone and 20,26‐dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26‐phosphate in the developing eggs of m. sexta is presented.
Ecdysteroids in Carausius eggs during embryonic development
Authors: Fournier Bernard , Radallah Driss 
Published: February 2005

AbstractPeaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin‐layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high‐polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co‐elution during HPLC with reference compounds of 20,26‐dihydroxyecdysone, 20‐hydroxyecdysone, ecdysone, and 2‐deoxy‐20‐hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20‐hydroxyecdysone and 2‐deoxy‐20‐hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI8 and VII6 whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20‐hydroxyecdysone, and both compounds underwent 26‐hydroxylation and/or conjugation to polar or apolar metabolites.
Sex pheromone biosynthesis in the leafroller moth Planotortix excessana by Δ10 desaturation
Authors: Foster S. P. , Roelofs W. L. 
Published: February 2005

AbstractWith the use of deuterium‐labeled saturated fatty acids coupled with gas chromatography‐mass spectrometric analysis, biosynthesis of the sex pheromone component (Z)‐8‐tetradecenyl acetate in the greenheaded leafroller moth Planotortrix excessana was shown to proceed via Δ10 desaturation of palmitate. The resultant (Z)‐10‐hexadecenoate is two carbon chain‐shortened to the precursor (Z)‐8‐tetradecenoate. The minor component (Z)‐10‐tetradecenyl acetate is biosynthesized by Δ10 desaturation of myristate. This is the first confirmation of Δ10 desaturation in an eukaryotic system.
Effects of ionophores on vitellogenin uptake by Hyalophora oocytes
Authors: Stynen Dirk , Woodruff Richard I. , Telfer William H. 
Published: February 2005

AbstractOocytes of Hyalophora cecropia that were incubated in vitro with [35S]vitellogenin incorporated label within 10 min into an intermediate‐density compartment identified by sucrose density gradient centrifugation. During a subsequent 20‐min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m‐cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K+ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H+‐K+ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.
Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta
Authors: Ryan Robert O. , Cole Kenneth D. , Kawooya John K. , Wells Michael A. , Law John H. 
Published: February 2005

AbstractThe identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [35S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of Mr ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ∼8.6). Electrophoresis under denaturing conditions reveals a subunit Mr ≈ 50,000 while the native protein has an apparent Mr ∼ 85,000 by gel permeation chromatography. Anti‐PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.
Experimental modifications of an insect vitellin affect its structure and its uptake by oocytes
Authors: Gochoco Caroline H. , Kunkel Joseph G. , Nordin John H. 
Published: February 2005

AbstractThe 18S and 33S vitellins (Vts) of Blattella germanica were subjected to periodate oxidation and digestions with α‐mannosidase, endo‐β‐Nacetylglucosaminidase H (endo‐H), and trypsin to study their effects on Vt structure and function. Periodate oxidation caused 33S Vt to dissociate to a form that cosedimented with 18S Vt upon glycerol gradient centrifugation but had no effect on the sedimentation of 18S Vt. This result implicates the oligosaccharides in stabilization of the 33S structure. Incubation of 18S and 33S Vts with α‐mannosidase and endo‐H revealed that the oligosaccharides of both Vts are largely shielded from attack by both glycosidases. However, the carbohydrate of 18S Vt was 3 to 5 times more susceptible to both enzymes, suggesting that the 18S to 33S transition results in decreased accessibility of the oligosaccharides to both glycosidases. Short‐term exposure of 18S and 33S Vts to trypsin resulted in limited hydrolysis; the Mr 102,000 subunit of each form was cleaved with an Mr79,000 peptide as a major product. However, the sedimentation properties of the Vts and their relative susceptibilities to α‐mannosidase were unchanged; therefore while the Mr102,000 subunit of the Vt is vulnerable to trypsin, it retains its higher‐order structure after limited digestion. Endocytosis of radiolabelled 18S Vt by oocytes in vivo decreased about 15‐fold after its modification by periodate and sixfold after treatment with α‐mannosidase. Limited trypsin digestion also severely diminished its uptake. All injected radioactivity of unmodified 18S and 33S Vts could be recovered from either the hemolymph or ovaries of recipient females. However, modified Vts were taken up from the hemolymph primarily by cells of the pericardium and the fat body, suggesting that these organs participate in a clearance mechanism that recognizes “damaged” Vt molecules.
Correlation of yolk phosphatase expression with the programmed proteolysis of vitellin in Blattella germanica during embryonic development
Authors: Purcell John P. , Quinn Theresa M. , Kunkel Joseph G. , Nordin John H. 
Published: February 2005

AbstractProteolytic processing of the vitellin in Blattella germanica eggs occurs 4 days postovulation and is correlated with both the onset of its utilization and the major portion of the embryo's growth. Yolk phosphatase is also expressed coincident with this event, and some aspects of its activation have been investigated. The enzyme is absent from the ooplasm (yolk) during the first 2 days following ovulation but increases approximately 20‐fold in specific activity between days 3 and 4, when assayed at pH 3.9 or 4.8 and 9‐fold at pH 6.5. No activation is observed for yolk‐bound α‐mannosidase, its specific activity remains elevated through the first 6 days following ovulation. This suggests that expression of the phosphatase is regulated independently of that of α‐mannosidase in the yolk. Yolk with active phosphatase can dephosphorylate native vitellin, delipidated vitellin, and phosphocasein. Sucrose density gradient centrifugation of yolk obtained from eggs 4 days postovulation, revealed that phosphatase activity cosediments with material which reacts with antivitellin antibodies, while α‐mannosidase and β‐N‐acetyl glucosaminidase are found near the top of the gradient. Oothecae derived from crossing certain translocational heterozygote males and wild‐type females contain some eggs with severely depressed levels of yolk phosphatase in which embryos do not grow. Vitellin in these eggs fails to undergo proteolytic processing as late as day 5 postovulation and retains the subunit composition of freshly ovulated vitellin.
Adipokinetic response of a flightless grasshopper (Barytettix psolus): Functional components, defective response
Authors: Ziegler Rolf , Ryan Robert O. , Arbas Edmund A. , Law John H. 
Published: February 2005

AbstractThe mature flightless grasshopper Barytettix psolus shows a very small adipokinetic response when injected with extracts of its own corpora cardiaca, although the fat body contains enough lipid for a strong response. When these extracts were injected into Melanoplus differentialis, a grasshopper capable of flight, or the moth Manduca sexta, much stronger adipokinetic responses were observed. Upon analysis of B. psolus extracts by HPLC, two components with adipokinetic activity were obtained. The major component appears to be identical to locust adipokinetic hormone (AKH) I. Extracts of B. psolus corpora cardiaca also activated fat body glycogen phosphorylase in B. psolus. This activation, however, did not result in an increase in hemolymph sugar, probably because of low levels of glycogen in the fat body.B. psolus hemolymph contains a high‐density lipophorin (HDLp) consisting of the apolipophorins (apoLp) I and II and lipid. Both apoproteins are glycosylated. The hemolymph also contains apoLp‐III, although this apoprotein apparently does not associate with HDLp to form a low‐density lipophorin (LDLp) following AKH or corpora cardiaca extract injections. When B. psolus lipophorin and AKH were injected into Schistocerca americana, lipophorin took up lipids and combined with apoLp‐III, forming LDLp. ApoLp‐III from B. psolus injected into S. americana can also form LDLp, demonstrating that the components are functional. A lipid transfer particle isolated from M. sexta and injected into B. psolus does not improve the adipokinetic response. Thus, it appears that the adipokinetic response of B. psolus is not deficient because of the lack of AKH or functional lipophorins, but may be caused by the lack of a full response to AKH by fat body or the deficiency in hemolymph of some as yet unknown factor.
Midgut amylase, lysozyme, aminopeptidase, and trehalase from larvae and adults of Musca domestica
Authors: Terra Walter R. , Espinoza‐Fuentes F. P. , Ferreira Clélia 
Published: February 2005

AbstractBased on polyacrylamide gel electrophoresis, density‐gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X‐100): membrane‐bound aminopeptidase (pH optimum 8.5; Km 0.21 mM L‐leucine p‐nitroanilide; Mr 322,000), amylase (pH optimum 6.5; Km 0.14% starch; Mr 66,000), lysozyme (pH optimum 3.5; Km 0.3 mg/ml; Mr 24,000); and membrane‐bound trehalase (pH optimum 5.0; Km 1.09 mM trehalose; Mr 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density‐gradient ultracentrifugation, and were used to raise antibodies in a rabbit.Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.
Polypeptide composition and biosynthesis of the yolk proteins in the firebrat, Thermobia domestica (insecta, thysanura)
Authors: Rousset André , Bitsch Colette , Bitsch Jacques 
Published: February 2005

AbstractIon‐exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (Mr 166,000–212,000) and small (around Mr 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse‐chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at Mr 210,000 and 212,000, is rapidly processed to Mr 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.
Endogenous and induced monooxygenase activity in gypsy moth larvae feeding on natural and artificial diets
Authors: Sheppard Carol A. , Friedman Stanley 
Published: February 2005

AbstractNADPH oxidase activity was measured in third to sixth instar gypsy moth larvae fed oak or pine foliage. Activity levels ranged from 400 to 1,900 pmol NADPH oxidized/min/mg microsomal protein, but enzyme activity was not correlated with host plant ingested. Similarly, activity levels in larvae fed diets containing inducers, such as the terpenoid α‐pinene or pentamethylbenzene, ranged from 700 to 1,500 pmol NADPH oxidized/min/mg protein, levels that were comparable to those measured for larvae fed control diets. O‐demethylase activity in older instar gypsy moth larvae fed pine averaged 109 pmol p‐nitrophenol/min/mg protein, and activity levels in those fed diet containing α‐pinene ranged from 22 to 55 pmol/min/mg protein. Although statistically significant, these induced O‐demethylase levels are well below those observed for Heliothis zea larvae. Our findings indicate that monooxygenases play a minor, if any, role in the ability of later instar gypsy moth larvae to develop successfully on pine foliage.
Metabolism of ecdysteroid by testes of the tobacco budworm, Heliothis virescens
Authors: Loeb Marcia J. , Woods Charles W. 
Published: February 2005

AbstractTestes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [3H]ecdysone and [3H]cholesterol. [3H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse‐phase high‐pressure liquid chromatography (RPHPLC); four of them were verified by normal‐phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26‐dihydroxyecdysone, 3‐epi‐20‐hydroxyecdysone, and 20‐hydroxyecdysone) and low‐polarity ecdysteroids, including 2‐deoxyecdysone, were detected after incubation with [3H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low‐polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [3H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high‐ and low‐polarity endpoints, 88% of the [3H]20‐hydroxyecdysone peak apparently remained unmetabolized. 20‐Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [3H]Cholesterol was not metabolized to any appreciable extent.
Complete amino acid sequence of Bombyx egg‐specific protein deduced from cDNA clone
Authors: Inagaki Shyuichiro , Yamashita Okitsugu 
Published: February 2005

AbstractComplementary (c)DNA coding for an insect yolk protein, the egg‐specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg‐specific protein consists of 559 amino acid residues. The NH2‐terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn‐X‐Thr, a potential N‐linked glycosylation site, is found at positions 191 to 193. A serine‐rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys132‐Asn133 and Arg228‐Asp229 are probably the sites cleaved by the egg‐specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.
Mediation of ecdysone synthesis in Manduca sexta by a hemolymph enzyme
Authors: Sakurai Sho , Warren James T. , Gilbert Lawrence I. 
Published: February 2005

AbstractThe prothoracic glands of Manduca sexta synthesize dehydroecdysone, which is rapidly converted to ecdysone through the mediation of a hemolymph enzyme, a 3 β‐forming‐3‐ketosteroid reductase. The hemolymph protein fraction (HPF) containing this enzyme was obtained from diapausing and non‐diapausing pupae, isolated abdomens, surgically manipulated pupae, etc., and in all cases had the capacity to affect the conversion of dehydroecdysone to ecdysone. The enzyme is heat labile, is inactivated by trypsin, and has a molecular weight of between 20,000 and 30,000. The data indicate that the conversion of dehydroecdysone to ecdysone exhibits linear kinetics and may be dependent on both the enzyme concentration and the concentration of NADPH at the beginning of the reaction but may be limited by the absolute amount of reducing equivalents after 10 min, under the experimental conditions utilized. The capacity of the enzyme to reduce dehydroecdysone was titered in the hemolymph during the last larval instar and during prepupal and pupal life with maximum capacity exhibited at the beginning of the instar, on day 8 of larval life and at day 1 of pupal life. Even at its lowest point at day 5, 1 ml of hemolymph was able to convert 77 pmol (∼35 ng) dehydroecdysone to ecdysone in 1 min. These results require a new interpretation of the control of molting in the Lepidoptera.
Properties, synthesis, and accumulation of storage proteins in Pieris rapae L.
Authors: Kim Hak Ryul , Seo Sook J. , Mayer Richard T. 
Published: February 2005

AbstractTwo kinds of storage proteins (SP‐1, SP‐2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP‐1 and SP‐2 dropped after pupation as the proteins were being deposited in fat bodies. SP‐2 is present in a larger amount than SP‐1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body.SP‐1 has a molecular weight of 500,000 and consists of one type of subunit (Mr 77,000), while SP‐2 has a molecular weight of 460,000 and is composed of two types of subunits (Mr 80,000 and 69,000). The pl values of SP‐1 and SP‐2 were determined to be 6.97 and 7.06, respectively.Fat body cells from 1‐day‐old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage.Morphological changes in the fat body tissues were observed during the larval‐pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.