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1.
Malcolm J. Thompson James A. Svoboda Ruben Lozano Kenneth R. Wilzer 《Archives of insect biochemistry and physiology》1988,7(3):157-172
The 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. 相似文献
2.
Malcolm J. Thompson Mark F. Feldlaufer Ruben Lozano Huw H. Rees William R. Lusby James A. Svoboda Kenneth R. Wilzer 《Archives of insect biochemistry and physiology》1987,4(1):1-15
Following injection into female Manduca sexta pupae, [14C]cholesterol is converted to a radiolabeled C21 nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C21-steroid conjugates). 相似文献
3.
The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult. 相似文献
4.
5.
Malcolm J. Thompson James A. Svoboda Huw H. Rees Kenneth R. Wilzer 《Archives of insect biochemistry and physiology》1987,4(3):183-190
Maturing eggs (48 to 64 h old) of the tobacco hornworm, Manduca sexta, contain at least three ecdysteroid conjugates, two of which have been previously identified as 26-hydroxyecdysone 26-phosphate (the major conjugate) and 26-hydroxyecdysone 22-glucoside. In this study we have isolated and identified the third conjugate as 26-hydroxyecdysone 2-phosphate by XAD-2 chromatography, C18 SEP-PAK separation, ion suppression reversed-phase high-performance liquid chromatography, nuclear magnetic resonance, and fast atom bombardment mass spectrometry. This compound is the second most abundant conjugate of ovaries from 4-day-old adult females. The possible role for this ecdysteroid conjugate is discussed. 相似文献
6.
Malcolm J. Thompson Gunter F. Weirich Huw H. Rees James A. Svoboda Mark F. Feldlaufer Kenneth R. Wilzer 《Archives of insect biochemistry and physiology》1985,2(3):227-236
The major ecdysteroid conjugate present in eggs (48–64 h old) of the tobacco hornworm has been purified by XAD-2 chromatography, C18 SEP-PAK separations, and ion suppression reversed-phase high-performance liquid chromatography. Enzymatic hydrolysis of the conjugate with acid phosphatase from human seminal fluid gave 26-hydroxyecdysone. The conjugate was identified as 26-hydroxyecdysone 26-phosphate by nuclear magnetic resonance and fast atom bombardment mass spectrometry. This compound is also the major conjugate of newly laid eggs (0–1 h old) of the tobacco hornworm. The role for ecdysteroid conjugates is discussed. 相似文献
7.
J. M. Gibson R. E. Isaac L. N. Dinan H. H. Rees 《Archives of insect biochemistry and physiology》1984,1(4):385-407
The metabolism of [3H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria. 相似文献
8.
After injection into male and female fifth-instar larvae of Manduca sexta, [14C]cholesterol was converted to C21 steroids, [14C]pregn-5-ene-3 beta,20-diols. These metabolites were isolated from 8-day-old pupae and were identified by TLC, HPLC, and GC-MS as the C-20 isomers of pregnene-3 beta,20-diol. They also were isolated from male and female meconium fluid (of 16-day-old pupae) following injection of [14C]cholesterol into 14-day-old pupae. 相似文献
9.
Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g). 相似文献
10.
Gunter F. Weirich Virginia P. Williams Mark F. Feldlaufer 《Archives of insect biochemistry and physiology》1996,31(3):305-312
The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The Km5 (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10−5 and 3.67 × 10−7 M, respectively. The Vmax was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher Vmax, at physiological ecdysone concentrations (10−7 − 10−8 M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America. 相似文献
11.
STUART E. REYNOLDS AUDREY M. BROWN RAKESH K. SETH LYNN M. RIDDIFORD KIYOSHI HIRUMA 《Physiological Entomology》2009,34(1):30-38
Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle. 相似文献
12.
Piotr Mikoajczyk Herbert Oberlander Donald L. Silhacek Isaac Ishaaya Eli Shaaya 《Archives of insect biochemistry and physiology》1994,25(3):245-258
The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [14C]N-acetyl-D-glucosamine ([14C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [14C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [14C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America. 相似文献
13.
M J Thompson J A Svoboda W R Lusby H H Rees J E Oliver G F Weirich K R Wilzer 《The Journal of biological chemistry》1985,260(29):15410-15412
Following injection into Manduca sexta (L.) female pupae (day 16), [14C]cholesterol was converted to a C21 steroid conjugate, 5-[14C]pregnen-3 beta,20 beta-diol glucoside. The conjugate was isolated from ovaries and eggs and contained three glucose units at least one of which is attached to C-20. The distribution of the other two glucose units remains to be determined. Other than the dealkylation of C-24 alkane or alkene substituents, side-chain cleavage of sterols is uncommon to insects. Here we report the first definitive proof of the biosynthesis of a C21 steroid conjugate from cholesterol in an insect species. The capability of M. sexta to so readily convert cholesterol to a C21 steroid suggests a physiological role for 5-pregnen-3 beta,20 beta-diol in this species. 相似文献
14.
15.
M J Garson V Partali S Liaaen-Jensen I L Stoilov 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,91(2):293-300
1. We present quantitative evidence from incorporation of [1-14C] acetate that the enzymes to synthesise isoprenoids are present in the marine sponge Amphimedon sp. and that efficient carotenoid synthesis takes place. 2. The de novo synthesis of b,b-carotene and (3R,3'R)-zeaxanthin may occur in a chlorophyll a-producing microalgal symbiont with subsequent aromatisation to (3R)-isoagelaxanthin by the sponge itself. 3. Amphimedon sp. contains nuclear-modified sterols derived by modification of conventional dietary sterols. 相似文献
16.
Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from omega 3-PUFAs have been found to be below detection limits of available assays. Because of the interest in omega3-PUFA dietary supplementation, we developed specific methods to measure nPF4 alpha-VI and iPF3 alpha-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF4 alpha-VI was below the detection limit of the assay, we conclusively identified iPF3 alpha-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were approximately 300 pg/mg creatinine. Our failure to detect nPF4 alpha-VI may have been due to its rapid metabolism by beta-oxidation to iPF3 alpha-VI, which we showed to occur in rat liver homogenates. In contrast, iPF3 alpha-VI is highly resistant to beta-oxidation in vitro. Thus iPF3 alpha-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) beta-oxidation of nPF4 alpha-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA. 相似文献
17.
The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins was investigated. Tissue supernatants were incubated with [4-14C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle. 相似文献
18.
J. M. Gibson M. S. I. Majumder A. H. W. Mendis H. H. Rees 《Archives of insect biochemistry and physiology》1983,1(1):105-120
The absence of phytosterol dealkylation in the cotton stainer bug, Dysdercus fasciatus, has been established and the major ecdysteroid in the fifth-stage larvae identified. The demonstration that the free and esterified sterols in D fasciatus consisted of 95–96% sitosterol and 4–5% campesterol, a similar composition to the cottonseed diet, together with the lack of conversion of [14C]sitosterol into cholesterol, establishes that phytosterol dealkylation does not occur in this insect species. The ecdysteroid titer determined by radioimmunoassay in the fifth instar of D fasciatus shows a distinct peak at day 6, the instar lasting for 7 days. Makisterone A was purified by HPLC from insects at a time of high ecdysteroid titer and identified as a major component by both fast atom bombardment and electron impact mass spectrometry. Gas-liquid chromatography/mass spectrometry (selected ion monitoring) confirmed the occurrence of makisterone A and revealed the presence of two unidentified compounds. One of these occurs in a similar amount to makisterone A and may be 26-hydroxymakisterone A, whereas only a minute amount of the other compound, which may be 20-deoxymakisterone A, was present; further identification of the latter compounds is necessary. C27 ecdysteroids (eg, ecdysone and 20-hydroxyecdysone) and C29 ecdysteroids (eg, podecdysone A) were undetectable. The specificity of the enzymes of ecdysteroid biosynthesis is discussed. 相似文献
19.
Conversion of [U-14C]glucose into carbon dioxide, glycogen, cholesterol and fatty acids in liver slices from embryonic and growing chicks 总被引:3,自引:7,他引:3
Alan G. Goodridge 《The Biochemical journal》1968,108(4):655-661
1. Rates of appearance and disappearance of total ketone bodies were determined in normal, starved and alloxan-diabetic rats by measuring specific radioactivities and concentrations of blood acetoacetate and 3-hydroxybutyrate at different times after injection of 3-hydroxy[(14)C]butyrate. 2. The mean rates of appearance were 1.7, 4.2 and 10.9mumoles/min./100g. body wt. respectively for normal, starved and alloxan-diabetic rats. The rates of disappearance were of the same order of magnitude as the rates of appearance. 3. There was a direct correlation between the rates of appearance and disappearance and the blood concentrations of the ketone bodies. 4. The results indicate that in the rat increased ketone-body production is paralleled by increased ketone-body utilization and that the raised ketone-body concentration in the blood in starvation and alloxan-diabetes is due to a slight imbalance between the rates of production and utilization. 5. The findings are discussed in relation to the concept that ketone bodies can serve as fuels of respiration when the supply of carbohydrate is limited. 相似文献
20.
Reverse-phase high-performance liquid chromatography was used to analyse 14C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [14C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [14C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [14C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC
High-performance liquid chromatography
- IAA
Indole-3-acetic acid 相似文献