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Poster Full Abstracts - Physiology and Aging
Poster board number is above title. The first author is the presenter
329
(health) under optimal conditions.
740B
Quantifying main ecdysteroides throughout the
Drosophila
developmental cycle.
Oksana Lavrynenko, Maria Carvalho, Jonathan Rodenfels, Julio
Sampaio, Suzanne Eaton, Andrej Shevchenko. MPI CBG, Dresden, Germany.
Ecdysteroides are major regulators of developmental timing in
Drosophila
. Although it has been generally assumed that developmental transitions are
accompanied by puls releases of ecdysteroides [1], little is known on how concentrations of individual hormones are changing throughout the larvae
development. Current biological enzyme immunoassay (EIA) and radioimmunoassay (RIA) methods lack specificity towards individual hormones, are
laborious and relatively inaccurate. We developed LC-MS/MS method for simultaneous quantification of major ecdysteroides in Drosophila larvae:
ecdysone (E), 20-hydroxyecdysone (20H) and makisterone A (Maki A) with the detection limit of 3 pg and better than 7% RSD. We then acquired the full
time course of these hormones from the beginning of embryonic stage to adult’s emergence with the time intervals of four hours. We observed synchronized
pulses of 20H and Maki A in the middle embryonic period, the end of first and second instars directly followed by cuticle formation and molting. Maximal
content of 20H was 180 pg during wings and legs formation metamorphosis. The maximal release of ecdysone was observed 8 hours prior to 20H and Maki
A pulses. Overall, the highest E concentration was 800 pg/animal which is 5 to 10 times higher compared with 20H and Maki A. The total of all
ecdysteroides was reaching 1 ng per animal. We were able to quantify ecdysteroides in a single larvae and keep track on individual variability of the
hormone concentration throughout larvae population. Within white pupa hormone concentration could deviate for as much as 30%. While it has become
possible to quantify individual ecdysteroides in their active form, further efforts are directed towards comprehensive quantitative profiling of the full
ecdysteroidome, including multiple storage and metabolized forms. [1] Molecular Mechanisms of Developmental Timing in C. elegans and Drosophila, Carl
S. Thummel, developmental Cell, Vol. 1, 453-465, 2001.
741C
The Involvement of the Electron Transport Chain in the Isoflurane Response in Drosophila melanogaster.
Christopher R. Pope
1
, Gerald B. Call
2
. 1)
Department of Biomedical Sciences, College of Health Sciences, Midwestern University, Glendale, AZ; 2) Department of Pharmacology, Arizona College
of Osteopathic of Medicine, Midwestern University, Glendale, AZ.
Over 160 years has passed since the ground breaking public demonstration of the use of general anesthetics (GA) in surgery. However, the mechanism for
GA action is still unknown. Drosophila exhibits all four anesthetic endpoints: analgesia, amnesia, muscle relaxation and loss of consciousness. Because of
this and the numerous genetic tools available, Drosophila has been recognized as an excellent model organism for identifying the mechanism of GAs
through genetic approaches. The
bellwether
gene, which codes for the beta subunit of ATP Synthase, was one of a few genes identified in an initial screen of
Drosophila mutants for sensitivity or resistance to the commonly used GA, isoflurane. These results, along with other scientific evidence linking
mitochondrial function and GAs, led to this study on determining the involvement of the mitochondrial electron transport chain (ETC) in GA action.
Available mutants from the Bloomington stock center, along with RNAi stocks from different collections (Harvard TRiP, NIG and VDRC), for different
nuclear encoded ETC genes were used. RNAi lines were crossed with the ELAV-GAL4, UAS-Dcr2 stock to produce pan-neuronal silencing of the ETC
genes. Adult flies (100-150, with a minimum of four repetitions) are tested in an inebriometer with 1% isoflurane. The inebriometer is a device that can
quantitatively measure the response of a population of flies to GAs. The data from each mutant or RNAi line is then compared to appropriate controls to
characterize whether a gene confers sensitivity or resistance to isoflurane. Preliminary results from mutants or RNAi lines includes: complex I (
Pdsw
=67%
of wt,
CG6020
=46%), complex II (
SdhA
=196%,
SdhB
=166%), complex III (
RFeSP
=321%,
CG3560
49%), complex IV (CG11015=300%, CG11043=288%),
ATP Synthase (
CG5389
=302%,
Oscp
=285%). More comprehensive data will be presented at the meeting. Currently, this is the most comprehensive study to
determine the role of ETC genes in the isoflurane response.
742A
The Involvement of Ion Channels in the Response to Isoflurane.
Ryan Stopher-Mitchell
1
, Krista Pearman
2
, Erik Nelson
1
, Michael J. Murray
3
, Gerald B.
Call
2
. 1) Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ; 2) Department of Pharmacology, Arizona College of Osteopathic
Medicine, Midwestern University, Glendale, AZ; 3) Department of Anesthesiology, Mayo Clinic, Scottsdale, AZ.
For more than 160 years volatile anesthetics (VAs) have been used, yet the workings of VAs upon the central nervous system is unknown. Different and
separate mechanisms have been proposed for the effects of amnesia, analgesia, and immobility in addition to different target locations in the brain and spinal
cord. Recent evidence suggests that these effects occur through multiple targets as opposed to a single common mechanism. Drosophila melanogaster is a
useful species to study the effects of VAs: it passes through each stage of anesthesia at equivalent human dosages, its central nervous system is more
complex than other invertebrates, its genome has been completely mapped, it has a short life cycle, and it has an easily altered genome. An inebriometer was
used to quantitatively study the effects of the common VA, isoflurane, on Drosophila. Publically available Drosophila with UAS-RNAi constructs provides
an ideal resource for gene-specific silencing. Previous studies have indicated that ion channels, particularly sodium and nicotinic channels, may be potential
candidates for the VA mechanism. We identified 380 potential ion channels within the D. mel. genome. So far, 300 of these have been used in an RNAi
screen to determine potential candidates for future studies of the mechanism of VAs. RNAi silencing of genes such as
Nckx30C
, a K
+
dependent Na
+
/Ca
+
exchanger, showed sensitivity, while others like
CG7333
, a secondary active organic cation transporter, and
nAcRβ-64B
, a nicotinic acetylcholine receptor
showed resistance. Analysis of the data reveals many potential pathways for VA action and confirms the efficacy of using this method to further study
anesthetics in vivo. The full results of our screen will be presented at the meeting. This study is the first comprehensive analysis of ion channels in the
response to isoflurane.
743B
Arginine kinase function in adult tissues.
Glen E. Collier. Dept Biological Sci, Univ Tulsa, Tulsa, OK.
Arginine kinase is encoded by a single locus (
Argk
) in
Drosophila melanogaster
that produces six putative alternative transcripts. These all share a
common C-terminal catalytic domain, but differ in amino acid sequences at the N-terminus of the protein products. One product (PD) is extremely abundant
in muscle tissue, but is found in other tissues as well. The other protein products of this locus are less abundant and are more restricted in tissue distribution.