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H H Rees

Publications and source records attributed to H H Rees.

At least 37 records · Page 2Linked to original sources
PubMed1997

A novel neuropeptide-endocrine interaction controlling ecdysteroid production in ixodid ticks.

Ixodid (hard) ticks are blood-feeding arthropods that require a blood meal to complete each stage of development. However, the hormonal events coordinating aspects of feeding and development are only poorly understood. We have delineated a new neuropeptide-endocrine interaction in the adult tick, Amblyomma hebraeum, that stimulates the synthesis of the moulting hormones, the ecdysteroids. In adult female ticks, ecdysteroid synthesis could be demonstrated in integumental tissue incubated in vitro with a synganglial (central nervous system) extract, but not in its absence. Stimulation by the synganglial extract is both time- and dose-dependent, but is completely abolished by trypsin treatment, suggesting that the activity is due to a peptide/protein. Integumental tissue ecdysteroidogenesis is also stimulated by elevation of the cAMP concentration using forskolin and 3-isobutyl-l-methyl-xanthine, or by 8-bromo-cAMP. This suggests the involvement of at least a cAMP second messenger system in the neuropeptide-ecdysteroidogenesis axis, without precluding a role for other second messengers as well. Despite involving a quite different steroidogenic tissue, the foregoing system has some parallels with the known prothoracicotropic hormone (neuropeptide)-prothoracic gland endocrine axis of insects.

Animals↗
PubMed1997

Induction of enzymes involved in molting hormone (ecdysteroid) inactivation by ecdysteroids and an agonist, 1,2-dibenzoyl-1-tert-butylhydrazine (RH-5849).

Molting in insects is regulated by molting hormones (ecdysteroids). The major active hormone, 20-hydroxyecdysone, is formed by ecdysone 20-monooxygenase-catalyzed hydroxylation of ecdysone. During times of decreasing hormone titers, inactivation occurs by several routes including (i) 26-hydroxylation and further oxidation to the 26-oic acid, (ii) formation of various conjugates (e.g. phosphates), and (iii) in Lepidoptera in particular, ecdysone oxidase-catalyzed formation of 3-dehydroecdysteroid, which is reduced to 3-epiecdysteroid, followed by phosphotransferase-catalyzed formation of phosphate conjugates. Administration of the nonsteroidal ecdysteroid agonist RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), but not 20-hydroxyecdysone, to tobacco hornworm (Manduca sexta) resulted in induction of midgut cytosolic ecdysone oxidase and ecdysteroid phosphotransferase activities. In addition, both 20-hydroxyecdysone and RH-5849 caused induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes, whereas 20-hydroxylase was induced to a lesser extent by 20-hydroxyecdysone in mitochondria and by either RH-5849 or 20-hydroxyecdysone in microsomes. Commensurate with induction of the enzymes by ecdysteroid and RH-5849 is a requirement for RNA and protein synthesis, without precluding indirect mechanisms. These results indicate that molting hormone stimulates at least one universal route of its own inactivation by inducing ecdysteroid 26-hydroxylase activity and are discussed in relation to an analogous phenomenon observed for vitamin D inactivation in vertebrates.

Animals↗
PubMed1996

Purification and characterisation of haemolymph 3-dehydroecdysone 3 beta-reductase in relation to ecdysteroid biosynthesis in the cotton leafworm Spodoptera littoralis.

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (approximately 82%), with lesser amounts of ecdysone (approximately 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable. 3-Dehydroecdysone 3 beta-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and Km values for NADPH and NADH were determined to be 0.94 microM, and 22.8 microM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 microM). The results suggest that the 3-dehydroecdysone 3 beta-reductase has a high-affinity (low Km) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site. The 3 beta-reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Structure and significance of mandibular organ-inhibiting hormone in the crab, Cancer pagurus. Involvement in multihormonal regulation of growth and reproduction.

Current evidence indicates that methyl farnesoate is the crustacean equivalent of the juvenile hormones of insects. This putative hormone is produced by the mandibular organs and is negatively regulated by a neuropeptide produced and secreted by the X-organ-sinus gland complex of the eyestalk. To identify this neuropeptide, a bioassay was developed which measures the inhibition of methyl farnesoate synthesis by mandibular organs exposed to fractionated sinus gland extracts from the crab, Cancer pagurus. Two neuropeptides, named mandibular organ-inhibiting hormones (MOIH-1 and -2) repressed methyl farnesoate synthesis. MOIH-1 was fully sequenced by automated Edman degradation of endoproteinase-derived fragments and further characterized by mass spectrometry. This peptide consisted of 78 residues (Mr 9235.6), with unblocked termini and three intrachain disulfide bridges. MOIH-2 appeared to be almost identical to MOIH-1 with the exception of a Gln for Lys substitution at position 33. Comparison with previously sequenced crustacean neuropeptides shows that these MOIHs are members of the ever expanding crustacean hyperglycemic hormone family, with significant sequence similarity to molt-inhibiting hormones (MIHs). It is possible that these two structurally similar peptides (MIH, MOIH) may control mutually exclusive physiological phenomena (somatic and gonadal growth), suggesting a complex hormonal integration of these processes in crustaceans.

Amino Acid Sequence↗
PubMed1996

Effect of insect-growth-disrupting amines and amides on Schistosoma mansoni in vitro.

A series of nonsteroidal alkylamines and alkylamides, which apparently inhibit phytosterol dealkylation in insects and free-living nematodes, as well as steps in the ecdysteroid biosynthetic pathway of insects, are also known to affect growth and development of animal-parasitic nematodes. The effect of these compounds on the motility, pairing, and viability of the trematode, Schistosoma mansoni, maintained in vitro was examined by incorporating the potential inhibitors into the medium employed for parasite culture. The alkylamines were more active than their amide counterparts, killing all parasites within 18 hr when tested at 25 parts per million. At lower concentrations, reductions in schistosome pairing and motility were observed. The functional group of the amine compounds influenced the schistosomicidal activity, with N-ethylamines being more effective than N,N-dimethylamines of identical carbon chain-length. Of all the compounds tested, N-ethyldodecanamine appeared to be the most promising schistosomicidal agent, killing all parasites within 22 hr at 10 parts per million and preventing parasite pairing at 3 parts per million.

Amides↗
PubMed1995

Enzymes of ecdysteroid transformation and inactivation in the midgut of the cotton leafworm, Spodoptera littoralis: properties and developmental profiles.

In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.

Animals↗
PubMed1994

Induction of an inactivation pathway for ecdysteroids in larvae of the cotton leafworm, Spodoptera littoralis.

Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been head-ligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: Ecdysteroid-->26-hydroxyecdysteroid-->ecdysteroid 26-aldehyde-->ecdysteroid 26-oic acid.

Animals↗
PubMed1994

Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis.

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.

Adrenodoxin↗
PubMed1993

Ovarian and hemolymph ecdysteroid titers during vitellogenesis in Macrobrachium rosenbergii.

Changes in ovarian and hemolymph ecdysteroid concentration and composition during vitellogenesis have been investigated in the freshwater prawn Macrobrachium rosenbergii. Free ecdysteroids (20-hydroxyecdysone and ecdysone) in hemolymph increased in concentration during vitellogenesis from zero at stage 0 to 1.5 ng/ml at stage I to 7.3 ng/ml in mature, stage IV animals. 20-Hydroxyecdysonoic acid (1.2 ng/ml) was detected in the stage IV hemolymph. Ovarian-free ecdysteroid concentration, expressed as nanograms per gram of tissue, fell during vitellogenesis from 83.2 ng/g at stage 0, non-pigmented tissue to 14.2 ng/g at stage IV tissue, being minimal at stage I (6.3 ng/g). However, expression of ovarian free ecdysteroid content as nanograms per ovary revealed a rise from 7.7 ng/ovary at stage 0, nonpigmented tissue to 28.3 ng/ovary at stage IV, again being minimal at stage I (2.0 ng/ovary). 20-Hydroxyecdysonoic acid and ecdysonoic acid were identified at ovarian stages II-III and stage IV.

Animals↗
PubMed1993

Biosynthesis of isoprenoid compounds in Schistosoma mansoni.

The isoprenoid biosynthetic capacity of digenetic trematodes was investigated by following the fate of [14C]mevalonic acid incorporated by Schistosoma mansoni during maintenance in axenic culture. Isoprenoid compounds were analyzed by thin-layer chromatography and high-performance liquid chromatography. Of the nonsaponifiable lipid classes, radioactivity was recovered in the dolichols, ubiquinones and in the short-chain isoprenoid alcohols. The latter group of lipids included geraniol, farnesol, geranylgeraniol and a compound tentatively identified as 2,3-dihydrogeranylgeraniol. Radioactivity derived from [14C]mevalonate was also incorporated into the saponifiable lipids, with 2,3-dihydrogeranylgeranoic acid accompanied by much less geranylgeranoic acid being detected in the triacylglycerol-containing fraction. Similarly, geranylgeraniol and the dihydro derivative were also detected as esters, presumably with fatty acids. The possible significance of the results is discussed.

Alcohols↗
PubMed1993

The effect of invertebrate hormones and potential hormone inhibitors on the third larval moult of the filarial nematode, Dirofilaria immitis, in vitro.

The effects of the insect hormones, ecdysone and 20-hydroxyecdysone, certain non-steroidal ecdysteroid agonists (RH compounds) and the inhibitor, azadirachtin, on the timing of the 3rd-stage moult of Dirofilaria immitis were investigated. 20-Hydroxyecdysone and RH 5849 when used at a concentration of 10(-5) M, resulted in a premature timing of this moult. Azadiracthin, at a similar concentration, prevented moulting of most of the larvae to the 4th stage. The results are discussed in relation to the possibility of a hormonal role for ecdysteroids and neuropeptide-like compounds in the control of ecdysis in filarial nematodes, that maybe somewhat comparable to the system which is found in insects.

Aedes↗