Ecdysten is the brand name for the
substance Ecdysterone which is extracted from the plant Leuzea (Rhaponticum
carthamoides). It is a powerful anabolic compound which has the ability
to enhance protein synthesis in muscle cells, but unlike other anabolic
steroids it shows no adverse side effects and does not affect natural
hormone levels. It works by increasing nitrogen preservation and protein
synthesis, thus stimulating muscle growth and lean weight. It causes an
increase in hemoglobin levels and enhances metabolism. This supplement
is very effective in combination with a high-protein diet and endurance
training to replace fat tissues with muscle mass. Ecdysterone also exercises
control on blood sugar levels, helping to maintain a healthy weight. Recent
research has found many other beneficial properties of the supplement,
some of which include increased libido, lowered cholesterol, antioxidant
and anti-inflammatory effects.
Dev Biol. 2005 Feb 1;278(1):163-74.
Ecdysteroid control of metamorphosis in the differentiating adult
leg structures of Drosophila melanogaster.
Mirth C.
Department of Biology, Box 351800, University of Washington, Seattle WA
98195, USA. mirthc@u.washington.edu
During insect metamorphosis, the steroid hormone 20-hydroxyecdysone (20E)
is responsible for coordinating the differentiation of adult structures.
Several structures of the Drosophila melanogaster adult leg, the six distalmost
joints, the bristles, and the pretarsal claws, were examined to investigate
how 20E controls their development in vitro. Joints, bristles, and claws
were dependent on 20E for differentiation between 20-22 and 24-26 h after
puparium formation (APF). After 26-28 h APF, differentiation became hormone
independent. Tissue-specific markers in 20E-free cultures showed that
the bristle and joint cells had not undergone any further morphogenetic
progression. In contrast, the pretarsi underwent partial differentiation.
The concentration of 20E required for differentiation was structure specific;
tarsal joints required higher concentrations of 20E (greater than 400
ng 20 E/ml) than pretarsal claws, bristles, and other joints (greater
than 40 ng 20E/ml). The 20E precursor ecdysone (E) was also able to induce
differentiation at concentrations over 700 ng E/ml, but did not show any
synergistic interactions with 20E. Lastly, leg structures had a finite
ability to respond to 20E; tarsal joints lost competence to respond after
32-34 h APF, while the remaining structures became incompetent after 44-46
h APF.
Yao Xue Xue Bao. 2004 Apr;39(4):241-4.
[Effect of ecdysterone on the expression of c-fos in the brain
of rats induced by microinjection beta-AP25-35 into the hippocampus]
[Article in Chinese]
Yang SF, Yang ZQ, Zhou QX, Wu Q, Huang XN, Shi JS.
Department of Pharmacology, Chongqing Medical University, Chongqing 400016,
China.
AIM: To observe the behavior in learning and memory and the expression
of c-fos gene from the brain of rats induced by beta-AP25-35, and the
intervention of ecdysterone, in order to explore the protective mechanism
of ecdysterone on the dysfunction of learning and memory of the rat induced
by beta-AP25-35. METHODS: Microinjection of beta-AP25-35 into hippocampus
induced learning and memory dysfunction of rats. The learning and memory
of rats were observed by Morris Water Maze. The expression of c-fos gene
in the brain was detected by immunohistochemistry. RESULTS: The results
of Morris Water Maze showed that after rats were microinjected beta-AP25-35
into hippocampus, the rats in model group took longer latency and searching
distance compared with the ones in control group (P < 0.01), and the
rats in treated group (ECR 4 mg x kg(-1), ECR 8 mg x kg(-1) and nimodipine
7.2 mg x kg(-1)) took shorter latency and searching distance, especially
the ECR 8 mg kg(-1) group (P < 0.01). At the same time, after the 5
days training, there was a higher expression of c-fos in hippocampus and
cortex from the rats in control group than that in model group (P <
0.01), but in the treated group, there was a relatively higher expression
of c-fos, especially the ECR 8 mg x kg(-1) group (P < 0.01). CONCLUSION:
Microinjection of beta-AP25-35 into the rat hippocampus resulted in dysfunction
of learning and memory. Ecdysterone was shown to improve the learning
and memory of the rats and increase the expression of c-fos. Increasing
the expression of c-fos is probably one of the most molecular mechanism
of its protection.
Neurochem Int. 2004 Oct;45(5):669-76.
G209A mutant alpha synuclein expression specifically enhances dopamine
induced oxidative damage.
Orth M, Tabrizi SJ, Tomlinson C, Messmer K, Korlipara LV, Schapira AH,
Cooper JM.
University Department of Clinical Neurosciences, Royal Free and University
College Medical School, Rowland Hill Street, London NW3 2PF, UK.
Alpha synuclein protein may play an important role in familial and sporadic
Parkinson's disease pathology. We have induced G209A mutant or wild-type
alpha-synuclein expression in stable HEK293 cell models to determine if
this influences markers of oxidative stress and damage under normal conditions
or in the presence of dopamine or paraquat. Induced wild-type or mutant
alpha-synuclein expression alone had no effect upon levels of oxidative
stress or damage, as measured by glutathione levels or aconitase activity.
Both wild-type and mutant alpha-synuclein expression decreased the oxidative
damage induced by paraquat, although the protection was less marked with
mutant alpha-synuclein expression. This suggests that alpha-synuclein
expression may either have anti-oxidant properties or may upregulate cellular
antioxidant levels, a function that was diminished by the G209A mutation.
However, mutant but not wild-type alpha-synuclein expression specifically
enhanced dopamine associated oxidative damage. Non-expressing cells treated
with reserpine to inhibit the vesicular monoamine compartmentalisation
produced similar results. However, consistent with the hypothesis that
mutant alpha-synuclein disrupts vesicular dopamine compartmentalization,
this effect was diminished in cells expressing mutant alpha-synuclein.
This may result in increased dopamine metabolism and cause selective oxidative
damage to dopaminergic cells.
Dev Biol. 2004 Aug 15;272(2):510-21.
Differential control of MHR3 promoter activity by isoforms of the ecdysone receptor and inhibitory effects of E75A and MHR3.
Hiruma K, Riddiford LM.
Department of Biology, University of Washington, Seattle, WA 98195-1800, USA.
MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis
and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone
response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1,
is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of
responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex
increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we
expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter
(pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50%. Moreover, when EcR-A was expressed
together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50%. Gel mobility
shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1
by about 50%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in
activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed
MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated
both by itself and by E75A.
Insect Mol Biol. 2004 Aug;13(4):337-47.
Transcription of a lepidopteran cytochrome P450 promoter is modulated by multiple elements in its 5' UTR and repressed by 20-hydroxyecdysone.
Petersen Brown R, Berenbaum MR, Schuler MA.
Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
The biochemical response to the phytochemical xanthotoxin encountered in the diet of black swallowtail larvae is
the induction of P450s capable of detoxifying this and other toxic furanocoumarins. As the xenobiotic response
element to xanthotoxin (XRE-xan) is necessary but not sufficient for transcription of the CYP6B1v3
gene in Sf9 cells, sequences upstream of it, such as a putative EcRE, and downstream of it, such as a putative
C/EBP binding site and Inr, have been tested for their roles in regulation. Mutation of the putative EcRE has
indicated that it affects basal transcription of this promoter but not repression by 20-hydroxyecdysone.
Mutation of the more proximal promoter sequence, including the C/EBP and Inr, have indicated that many core promoter elements
between the TATA box and translation start site modulate basal and xanthotoxin-inducible expression of this composite promoter.
Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10626-31. Epub 2004 Jun 30
Target of rapamycin-mediated amino acid signaling in mosquito anautogeny.
Hansen IA, Attardo GM, Park JH, Peng Q, Raikhel AS.
Department of Entomology and Program in Biochemistry and Molecular Biology, University of California, Riverside, CA 92506, USA.
Mosquitoes generate an enormous burden on human health worldwide. Disease-transmitting species use a reproductive strategy, termed
anautogeny, that requires a blood meal to initiate egg maturation. Whereas this strategy is important for
driving disease transmission, the molecular mechanisms underlying this phenomenon are still poorly understood. The production
of yolk protein precursors (YPPs), a central event in egg maturation, is called vitellogenesis. YPPs are synthesized in
the fat body, the insect analogue of the vertebrate liver. Mosquito vitellogenesis is regulated by the steroid hormone
20 hydroxyecdysone (20E). However, 20E alone is not capable of activating vitellogenesis in vivo. Here,
we report that amino acid signaling through the nutrient-sensitive target of rapamycin (TOR) pathway is essential for
the activation of YPP gene expression. An increase in extracellular amino acid levels, similar to the increase observed after
a blood meal, is critical for 20E stimulation of YPP gene expression. Treatment with the TOR kinase inhibitor rapamycin
significantly inhibits YPP expression. We used RNA interference to knockdown the expression of two key proteins of
the TOR signaling pathway, TOR, and tuberous sclerosis complex 2. Knockdown of TOR inhibited amino acid stimulation while knockdown of tuberous
sclerosis complex 2, a negative regulator of TOR signaling, resulted in enhanced YPP expression. Thus,
amino acid-based TOR signaling regulates the activation of egg development after a blood meal, an adaptation
to the unique life style of mosquitoes.
J Biol Chem. 2004 Jul 2;279(27):28000-8. Epub 2004 Apr 26.
The insect hemolymph protein HP19 mediates the nongenomic effect of ecdysteroids on acid phosphatase activity.
Arif A, Vasanthi P, Hansen IA, Scheller K, Dutta-Gupta A.
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
The activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in
vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the
ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph
of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and
non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer.
It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the
hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its
maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind
gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to
the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show
any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates
the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of
inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show
that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein.
Eur J Biochem. 2004 Jul;271(14):3017-27.
Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR, HR3, E75 gene expression in imaginal wing
cells of Plodia interpunctella lepidoptera.
Siaussat D, Bozzolan F, Queguiner I, Porcheron P, Debernard S.
Laboratoire de Physiologie Cellulaire des Invertebres, Universite Pierre et Marie Curie, Paris, France. dsiaussat@free.fr
The IAL-PID2 cells derived from imaginal wing discs of the last larval instar of Plodia interpunctella were responsive to
20-hydroxyecdysone (20E). These imaginal cells respond to 20E by proliferative arrest followed by a morphological differentiation.
These 20E-induced late responses were inhibited in presence of juvenile hormone (JH II). From
these imaginal wing cells, we have cloned a cDNA sequence encoding a P. interpunctella ecdysone
receptor-B1 isoform (PIEcR-B1). The amino acid sequence of PIEcR-B1 showed a high degree of identity with
EcR-B1 isoforms of Bombyx mori, Manduca sexta and Choristoneura fumiferana. The pattern of PIEcR-B1mRNA induction by
20E was characterized by a biphasic response with peaks at 2 h and 18 h. The presence of the protein synthesis
inhibitor anisomycin induced a slight reduction in level of PIEcR-B1 mRNA and prevented the subsequent declines
observed in 20E-treated cells. Therefore, PIEcR-B1 mRNA was directly induced by 20E and its downregulation depended
on protein synthesis. An exposure of imaginal wing cells to 20E in the presence of JH II caused
an increased expression of Plodia E75-B and HR3 transcription factors but inhibited the second increase of PIEcR-B1
mRNA. These findings showed that in vitro JH II was able to prevent the 20E-induced differentiation of imaginal
wing cells. This effect could result from a JH II action on the 20E-induced
genetic cascade through a modulation of EcR-B1, E75-B and HR3 expression.
J Exp Biol. 2004 Jun;207(Pt 14):2389-400.
Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta.
Schachtner J, Trosowski B, D'Hanis W, Stubner S, Homberg U.
Department of Biology, Animal Physiology, Philipps-University, 35032 Marburg, Germany. schachtj@staff.uni-marburg.de
During metamorphosis, the insect nervous system undergoes considerable remodeling: new neurons are integrated while larval neurons are remodeled or eliminated. To understand further the mechanisms involved in transforming larval to adult tissue we have mapped the metamorphic changes in a particularly well established brain area, the antennal lobe of the sphinx moth Manduca sexta, using an antiserum recognizing RFamide-related neuropeptides. Five types of RFamide-immunoreactive (ir) neurons could be distinguished in the antennal lobe, based on morphology and developmental appearance. Four cell types (types II-V, each consisting of one or two cells) showed RFamide immunostaining in the larva that persisted into metamorphosis. By contrast, the most prominent group (type I), a mixed population of local and projection neurons consisting of about 60 neurons in the adult antennal lobe, acquired immunostaining in a two-step process during metamorphosis. In a first step, from 5 to 7 days after pupal ecdysis, the number of labeled neurons reached about 25. In a second step, starting about 4 days later, the number of RFamide-ir neurons increased within 6 days to about 60. This two-step process parallels the rise and fall of the developmental hormone 20-hydroxyecdysone (20E) in the hemolymph. Artificially shifting the 20E peak to an earlier developmental time point resulted in the precocious appearance of RFamide immunostaining and led to premature formation of glomeruli. Prolonging high 20E concentrations to stages when the hormone titer starts to decline had no effect on the second increase of immunostained cell numbers. These results support the idea that the rise in 20E, which occurs after pupal ecdysis, plays a role in the first phase of RFamide expression and in glomeruli formation in the developing antennal lobes. The role of 20E in the second phase of RFamide expression is less clear, but increased cell numbers showing RFamide-ir do not appear to be a consequence of the declining levels in 20E that occur during adult development.
Anal Chem. 2004 May 15;76(10):2966-74.
Microflow NMR: concepts and capabilities.
Olson DL, Norcross JA, O'Neil-Johnson M, Molitor PF, Detlefsen DJ, Wilson AG, Peck TL.
Protasis/MRM Corporation, 101 Tomaras Avenue, Savoy, Illinois 61874, USA. dolson@microNMR.com
The principles and parameters to consider when choosing an NMR probe for analysis of a volume- or mass-limited sample
are identified and discussed. In particular, a capillary-based microflow probe is described which has a mass sensitivity comparable
to cryoprobes (observe volume approximately 40 microL), but with several distinct advantages. The microflow probe has a
flowcell volume of 5 microL and an observe volume of 1.5 microL and is equipped with proton and carbon observe channels,
deuterium lock, and z-gradient capability. The entire flow path is fused silica; inlet and outlet capillary inner diameters
are 50 microm to minimize sample dispersion, making it well-suited to volume-limited samples. An injected sample of 1 nmol
of sucrose (0.34 microg in 3 microL, 0.33 mM; MW = 342 g/mol) yields a 1D proton spectrum in 10 min on a spectrometer of
500 MHz or higher. In another example, 15 microg of sucrose (in 3 microL; 15 mM, 45 nmol) is injected and parked in the
probe to yield a heteronuclear multiple-quantum coherence (HMQC) spectrum in less than 15 h. The natural product muristerone
A (75 microg in 3 microL, 50 mM, 150 nmol; MW = 497 g/mol) was delivered to the flow cell, and a gradient correlation
spectroscopy spectrum was acquired in 7 min, a gradient HMQC in 4 h, and a gradient heteronuclear multiple-bond
correlation in 11 h. Four basic modes of sample injection into the probe vary in degree of user intervention, speed,
solvent consumption, and sample delivery efficiency. Manual, manual-assisted (employing a micropump), automated
(using an autosampler), and capillary HPLC modes of operation are described.
J Biol Chem. 2004 May 7;279(19):19634-42. Epub 2004 Feb 27.
Identification and characterization of a juvenile hormone (JH) response region in
the JH esterase gene from the spruce budworm, Choristoneura fumiferana.
Kethidi DR, Perera SC, Zheng S, Feng QL, Krell P, Retnakaran A, Palli SR.
Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY 40546, USA.
Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe)
from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is
suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the
regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified
a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E
suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to
the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE).
In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was
induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in
a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and
the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded
oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and
20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe
gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.
Exp Gerontol. 2004 May;39(5):767-73.
Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees.
Amdam GV, Simoes ZL, Hagen A, Norberg K, Schroder K, Mikkelsen Ø, Kirkwood TB, Omholt SW.
Department of Animal Science, Centre for Integrative Genetics, Agricultural University of Norway, P.O. Box 5025, 1432 Aas, Norway.
A striking example of plasticity in life span is seen in social insects such as ants and bees, where different castes may display distinct
ageing patterns. In particular, the honeybee offers an intriguing illustration of environmental control on ageing rate. Honeybee
workers display a temporal division of labour where young bees (or 'hive bees') perform tasks within the brood nest,
and older bees forage for nectar, pollen propolis and water. When bees switch from the hive bee to the forager stage, their
cellular defence machinery is down-regulated by a dramatic reduction in the number of functioning haemocytes (immunocytes).
This study documents that the yolk precursor vitellogenin is likely to be involved in a regulatory pathway that controls the
observed decline in somatic maintenance function of honeybee foragers. An association between the glyco-lipoprotein vitellogenin
and immune function has not previously been reported for any organism. Honeybee workers are functionally sterile, and via the expression
of juvenile hormone, a key gonotrophic hormone in adult insects, their vitellogenin levels are influenced by social
interactions with other bees. Our results therefore suggest that in terms of maintenance of the cellular immune system,
senescence of the honeybee worker is under social control.
Insect Biochem Mol Biol. 2004 May;34(5):451-8.
Functional and comparative analysis of two distinct ecdysteroid-responsive gene expression constructs in Drosophila S2 cells.
Poels J, Martinez A, Suner MM, De Loof A, Dunbar SJ, Vanden Broeck J.
Laboratory for Developmental Physiology, Genomics and Proteomics, Zoological Institute, Naamsestraat 59, B-3000 Leuven, Belgium.
Inducible expression systems have proven to be of major interest when analysing the function of specific genes or
when expressing cytotoxic proteins. In an effort to develop inducible switches allowing for flexible fine-tuning
of gene expression levels in insect cells, we have compared the induction capacities of two Drosophila
minimal promoters when linked to four consecutive ecdysone response elements. These minimal promoters, either containing a TATA-box
or a downstream promoter element, drove the expression of a luciferase reporter gene. Potent induction
capacities were observed with the insect moulting hormone, 20-hydroxyecdysone, and with ponasterone A, a
plant ecdysteroid. The developed inducible switches further expand the repertoire of molecular tools for functional expression of
proteins of interest in insect cells. In addition, the combination of an ecdysone switch with promoters that possess
different structural elements can provide novel insights into ecdysteroid-induced transcription in an insect cell line.
Insect Biochem Mol Biol. 2004 May;34(5):415-24.
Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.
do Nascimento AM, Cuvillier-Hot V, Barchuk AR, Simoes ZL, Hartfelder K.
Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo,
Avenida Bandeirantes 3.900, Ribeirao Preto, 14040-901 SP, Brazil. admendes@rge.fmrp.usp.br
Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy
to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin
(AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises
686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the
deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire
sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal
lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the
larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile
was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth
instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only
a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental
regulation of transferrin expression in an insect species.
Zoolog Sci. 2004 May;21(5):503-16.
Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions.
Sonobe H, Yamada R.
Department of Biology, Faculty of Science and Engineering, Konan University, Kobe, Japan. sonobe@konan-u.ac.jp
It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular
species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically
active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates)
and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In
de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by
ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase
(EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in
the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of
free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal
ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are
scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further
investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar
H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the
dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the
increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.
J Biol Chem. 2004 Apr 23;279(17):17019-26. Epub 2004 Jan 26.
Mitochondrial deoxyribonucleotides, pool sizes, synthesis, and regulation.
Rampazzo C, Ferraro P, Pontarin G, Fabris S, Reichard P, Bianchi V.
Department of Biology, University of Padova, I-35131 Padova, Italy.
We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a
recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million
cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc.
Natl. Acad. Sci. U. S. A. 100, 12159-12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine,
and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol,
whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of
the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for
mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly
in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by
phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that
the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of
mdN and measure the transfer of radioactivity from [(3)H]thymidine to mt thymidine phosphates. In cycling cells lacking
the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%,
and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN
modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase.
Regul Pept. 2004 Apr 15;118(1-2):25-31.
Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae).
Zhao JY, Xu WH, Kang L.
State Key Lab of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy
of Science, 19 Zhongguancun Road, Haidian District, Beijing 100080, China.
Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which
shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is
induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore,
Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of
Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required
for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal
stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the
combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae
were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a
competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH
gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be
activated by low temperature. Has-DTH might be
useful to elucidate the mechanism of diapause termination in pupal diapause species.
Insect Mol Biol. 2004 Apr;13(2):179-87.
Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone.
Siaussat D, Mottier V, Bozzolan F, Porcheron P, Debernard S.
Laboratoire de Physiologie Cellulaire des Invertebres, Universite Pierre et Marie Curie, Paris, France. David.Siaussat@snv.jussieu.fr
We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using
the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use
of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their
division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at
1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at
10(-6) m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed
from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced
by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These
findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.
Yao Xue Xue Bao. 2004 Apr;39(4):241-4.
Effect of ecdysterone on the expression of c-fos in the brain of rats induced by microinjection beta-AP25-35 into the hippocampus
Yang SF, Yang ZQ, Zhou QX, Wu Q, Huang XN, Shi JS.
Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China.
AIM: To observe the behavior in learning and memory and the expression of c-fos gene from the
brain of rats induced by beta-AP25-35, and the intervention of ecdysterone, in order to explore the protective
mechanism of ecdysterone on the dysfunction of learning and memory of the rat induced by beta-AP25-35.
METHODS: Microinjection of beta-AP25-35 into hippocampus induced learning and memory dysfunction of rats.
The learning and memory of rats were observed by Morris Water Maze. The expression of c-fos gene in the
brain was detected by immunohistochemistry. RESULTS: The results of Morris Water Maze showed that after rats
were microinjected beta-AP25-35 into hippocampus, the rats in model group took longer latency and searching distance
compared with the ones in control group (P < 0.01), and the rats in treated group
(ECR 4 mg x kg(-1), ECR 8 mg x kg(-1) and nimodipine 7.2 mg x kg(-1)) took shorter latency and
searching distance, especially the ECR 8 mg kg(-1) group (P < 0.01). At the same time, after the 5 days training,
there was a higher expression of c-fos in hippocampus and cortex from the rats in control group than that in model
group (P < 0.01), but in the treated group, there was a relatively higher expression of c-fos, especially the ECR
8 mg x kg(-1) group (P < 0.01). CONCLUSION: Microinjection of beta-AP25-35 into the rat hippocampus resulted in
dysfunction of learning and memory. Ecdysterone was shown to improve the learning and memory of the rats and
increase the expression of c-fos. Increasing the expression of c-fos is probably one of the most molecular
mechanism of its protection.
J Exp Biol. 2004 Mar;207(Pt 7):1151-61.
Overexpression of broad: a new insight into its role in the Drosophila prothoracic gland cells.
Zhou X, Zhou B, Truman JW, Riddiford LM.
Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA.
Insect molting is triggered by ecdysteroids, which are produced in the prothoracic glands (PG). The broad (br)
gene is one of the 'early genes' directly regulated by ecdysteroids. Ectopic expression of the BR-Z3 isoform in
early second instar Drosophila larvae (L2) before the rise of the ecdysteroid titer prevented molting to the
third instar, but the larvae subsequently formed L2 prepupae after prolonged feeding. When these larvae were
fed on diet containing 20-hydroxyecdysone (20E), they formed pharate third instar larvae. The critical weight
for normal L3 pupariation of w(1118) larvae was found to be 0.8 mg and that for L2 pupariation was 0.45 mg. We
also defined a threshold weight for metamorphosis of 0.3 mg, above which L2 larvae will metamorphose when
provided with 20E. BR-Z3 apparently works through the PG cells of the ring gland but not the putative neurosecretory
cells that drive ecdysone secretion, because ectopic expression of BR-Z3 specifically in the ring gland caused 53% of
the larvae to become permanent first instar larvae. Driving other BR isoforms in the ring gland prevented larval
molting or pupariation to varying degrees. These molting defects were rescued by feeding 20E. Overexpression of
each of the BR isoforms caused degeneration of the PG cells but on different time courses, indicating that
BR is a signal for the degeneration of the PG cells that normally occurs during the pupal-adult transition.
Phytochemistry. 2004 Mar;65(6):711-20.
Protective role of 20-hydroxyecdysone against lead stress in Chlorella vulgaris cultures.
Bajguz A, Godlewska-Zylkiewicz B.
Institute of Biology, University of Bialystok, Swierkowa 20 B, 15-950 Bialystok, Poland. abajguz@uwv.edu.pl
Treatment of cultured C. vulgaris cells with 10(-6)-10(-4) M lead decreased their growth and chemical composition
during the first 48 h of cultivation. However, at concentrations above 10(-4) M, lead is cytotoxic to Chlorella
vulgaris cells, resulting in cellular fragmentation and lysis. In contrast, at concentrations below 10(-6)
M lead had no influence on the growth and metabolism of C. vulgaris cells. 20-Hydroxyecdysone
(20E) (10(-10)-10(-8) M) increased growth and chemical composition of C. vulgaris cells over a
concentration range. Levels per cell of chlorophylls, protein, sugars are all increased by 20E treatment, when compared to
non-treated control cells. However, the cultures treated with 20E and lead show a lower stimulation than
the cultures treated with 20E alone. The effects of 20E mixed with lead on the growth and the level of
cellular lead, chlorophyll, sugar and protein in C. vulgaris are also reported. The decreased growth and composition of C.
vulgaris cells treated with lead was restored by the 20E. Application of 20E to C. vulgaris cultures reduced the impact of
lead stress on growth, prevented chlorophyll, sugar and protein loss and increased phytochelatins synthesis. Furthermore, 20E did
not restore toxic effect of lead on C. vulgaris cells. The combined treatment with lead and 20E appeared
to have a stimulatory effect on the above parameters during the 48 h of cultivation, as compared to the
control. 20E reduced the toxicity of lead and the growth recovered to the level of cells treated with 20E alone.
Concentration-dependent stimulation was observed with increasing concentration of 20E and decreasing concentration
of lead.
Arch Insect Biochem Physiol. 2004 Feb;55(2):68-78.
Morphological and molecular effects of 20-hydroxyecdysone and its agonist tebufenozide on CF-203, a midgut-derived cell
line from the spruce budworm, Choristoneura fumiferana.
Hu W, Cook BJ, Ampasala DR, Zheng S, Caputo G, Krell PJ, Retnakaran A, Arif BM, Feng Q.
Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, Sault Ste. Marie, Ontario, Canada.
The morphological and molecular responses of a midgut-derived cell line of the spruce budworm, Choristoneura fumiferana,
to 20-hydroxyecdysone (20E) and the nonsteroidal ecdysone agonist, tebufenozide (RH-5992), were investigated. The cells
responded to these compounds by clumping, generating filamentous extensions, increased mortality and expression of the
transcription factor, Choristoneura hormone receptor 3 (CHR3). This cell line can be used as a model system to study
the mode of action of ecdysone and its agonists. With subsequent passaging in ecdysteroid-containing medium, the
degree of clumping increased and the clumping could not be reversed by subculturing in ecdysteroid-free medium.
Cell numbers of the adapted cell lines in 20E and RH-5992 containing media were not significantly decreased,
compared to the control, but both cell lines accumulated less (14)C-labeled RH-5992 and lost the capability of
expressing CHR3 in response to these compounds. Taken together, the cell lines appeared to develop a mechanism to adapt
to the toxic effects of these compounds. Arch. Insect Biochem. Physiol. 55:68-78, 2004. Copyright 2004 Wiley-Liss, Inc.
J Insect Physiol. 2004 Feb-Mar;50(2-3):203-8.
Chilling stress effects on corpus allatum proliferation in the Hawaiian cockroach, Diploptera punctata: a role for ecdysteroids.
Pszczolkowski MA, Gelman DB.
Department of Entomology, Washington State University, 166 FSHN, Pullman, WA 99164, USA. pszcz@ksu.edu
Endocrine regulation of corpus allatum (CA) cell proliferation in response to chilling was studied in mated
females of the Hawaiian cockroach, Diploptera punctata. Chilling alone, when applied 24 h post-mating, suppressed
CA cell division, and elevated ecdysteroid levels in Diploptera's haemolymph. Application of 20-hydroxyecdysone (20E)
at 24 h post-mating similarly suppressed CA cell division, but had no effects at 48 h or 72 h post-mating.
Severance of the ventral nerve cord prior to chilling or to the application of 20E prevented suppression of
CA cell division, indicating that the effects of either chilling or 20E
application are mediated by the ventral nerve cord.
J Insect Physiol. 2004 Feb-Mar;50(2-3):123-33.
Commencement of pupal commitment in late penultimate instar and
its hormonal control in wing imaginal discs of the silkworm, Bombyx mori.
Koyama T, Obara Y, Iwami M, Sakurai S.
Division of Life Sciences, Graduate School of Science and Technology, Kanazawa University, Kakumamachi, Kanazawa 920-1192, Japan.
Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis.
Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule
slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E,
indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene
(JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs
at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing
discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a
pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E
effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that
begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to
begin the process and 20E is the factor that drives the discs to enter the reversible state.
Experientia. 1996 Jul 15;52(7):702-6.
Slama K, Koudela K, Tenora J, Mathova A.
Institute of Entomology, Czech Academy of Sciences, Praha, Czech Republic.
Insect hormones in vertebrates: anabolic effects of 20-hydroxyecdysone
in Japanese quail.
Ecdysteroids are hormones controlling cell proliferation, growth and
the developmental cycles of insects and other invertebrates. They are
occasionally present in various unrelated plants for no apparent reason;
no phytohormonal function has yet been identified. In certain cases, ecdysteroids
are accumulated to high levels in leaves, roots or seeds. Some ecdysteroid-containing
plants have been known as medicinal plants for centuries. One of them,
Leuzea carthamoides Iljin (Asteraceae), growing in Central Asia, contains
0.4% ecdysteroid in dry roots and 2% in seeds. A pharmacological preparation
from this plant, "Ecdisten', is already available as a commercial
preparation for its anabolic, tonic and other effects, for medical use
(review). It remained problematic, however, whether ecdysteroids were
truly responsible for these effects, because Leuzea contains a number
of other biologically active compounds in addition to ecdysteroids. We
extracted and purified ecdysteroids from the seeds of Leuzea. With 6 g
of 96% 20-hydroxyecdysone (20E), we made a large-scale feeding assay with
Japanese quail to find out whether ecdysteroid alone could duplicate the
anabolic effects of the seeds. We found that the 96% ecdysteroid increased
the mass of the developing quails in a dose-dependent manner, with the
rate of increase proportional to the ecdysteroid content in the seeds;
there was a 115% increase in living mass with 100 mg kg-1 of pure 20E
compared with 109.5% increase with 100-180 mg kg-1 20E equivalents in
the seeds. We conclude that the plethora of growth-promoting, vitamin-like
effects induced in vertebrates by Leuzea is mediated by ecdysteroids.
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