ACCEPTED ABSTRACTS
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O R A L |
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Brewer, Gregory J. |
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AGING NEURON MITOCHONDRIAL FUNCTION AND REJUVENATION WITH ESTROGEN OR
MITOSIS
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G.
Brewer, J. Nash, T. Jones, J. Reichensperger, M. Parihar
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PO
Box 19626, Springfield, IL 62794-9626
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The mitochondrial
oxyradical theory of aging is gaining increased support from studies of
brain, heart and muscle tissue. Is this aging caused by systemic
hormones or intrinsic to aging mitochondria in neurons, for example.
The intrinsic hypothesis is supported by our findings with isolated neurons
of increased susceptibility with age to glutamate and A-beta toxicity, two
endogenous toxins important to age-related neurodegenerative disease
(Brewer, 1998). For neuron-specific function, we have established
culture techniques that allow us to compare neurons isolated from embryonic,
middle-age (12 month) and old (24 month) rat hippocampus (Brewer, 1997).
For use with these cultured neurons, we monitored cytochrome oxidase
activity (COX), cardiolipin specific staining of mitochondria with
nonyl-acrydine orange (NAO), as well as labeling with mitotracker-red and
immunostain for cytochrome C, fluorescent NADH, glutathione levels and
respiration. Cytochrome C immunostain and mitotracker red stained equal
numbers of mitochondria per cell for middle-age and old neurons, compared to
higher numbers per embryonic neuron. In contrast to mitochondrial
numbers, COX activity/cell declined sharply with age of the neurons: levels
of old neurons were reduced by 30% of those of middle-age neurons.
Similarly, NAO stain for mitochondria was 40% lower for old neurons compared
to middle-age neurons. By addition of FGF2 and low density passage,
old neurons divide in culture (Brewer, 1999). COX activity in these
dividing old neurons was equivalent to that of middle-age neurons.
Age-related reductions in glutathione levels were exacerbated by exposure to
glutamate. NADH levels and respiration in old neurons were normal, but
showed dramatic deficits in response to glutamate. In addition to
restoring youthful calcium dynamics, treatment of old neurons with
17-beta-estradiol restored NAO staining levels/cell to those of middle-age
neurons and provided full neuroprotection from glutamate and A-beta
toxicity. These results demonstrate that mitochondrial function is
impaired in old neurons, which correlates with increased susceptibility to
toxic stress, but function can be restored by treatment with FGF2 or
estradiol.
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Carter, Christy S. |
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ACE inhibition intervention: Implications for improving age-related
declines in physical performance and longevity.
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CS
Carter, M Cesari, M Pahor
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Wake Forest Univeristy School of Medicine, J. Paul Sticht Center on Aging, 1
Medical Center Blvd. Winston-Salem NC, 27157
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Age-related disregulation
of the renin-angiotensin system (RAS) leads to variety of pathologies
including end organ damage to kidney, heart, blood vessels, and more
recently has been suggested to exacerbate declining cognitive and physical
performance. Reversing or attenuating these effects could have a significant
impact on maintaining independence and extending longevity in the elderly.
In fact, both pharmacological and genetic studies have demonstrated that
lower circulating levels of angiotensin converting enzyme (ACE) results in
enhanced responsiveness to exercise, increased muscle strength, and lower
adiposity. The relationship between low physical performance in healthy
older adults, shifting body composition (increases in fat and decreases in
fat-free mass) and longevity has been documented and more recently this same
assessment has been made in aging rodents. Therefore, interventions
that retard declining performance such as ACE inhibition (ACEi) may also
have validity for studies of longevity. We have conducted studies to
assess the effects of ACEi on body composition and physical performance in
aged rats. Animals were randomized to daily injections of 40 or 80 mg/kg of
enalapril or saline at baseline (24 months of age) and were followed for 6
months. ACEi attenuated declining physical performance as measured by
both grip strength and inclined plane tasks, and was most likely explained
by a significant reduction in total fat mass, as no differences in lean mass
were observed between groups (as measured using DXA). A possible
interpretation is that beyond its powerful hemodynamic effects, ACEi
regulates many aspects of metabolic functioning, decreases oxidative stress
in many tissues, and may act ubiquitously to reduce age and disease related
chronic inflammatory states. Therefore, our understanding of these
results could be enhanced by a more thorough and mechanistic
characterization of the effects of long-term ACEi on lifespan and declining
performance.
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Chang, Sandy |
- ESSENTIAL ROLE OF LIMITING TELOMERES IN THE PATHOGENESIS OF
WERNER SYNDROME
AbstractAuthors: S. Chang*, A. Multani, N. Cabrera, M. Naylor, P. Laud, D.
Lombard, S. Pathak, R. de Pinho
MD Anderson Cancer Center, Houston, TX and Dana Farber
Cancer Center, Boston, MA
Mutational inactivation of the Wrn helicase gene causes Werner Syndrome (WS), an
autosomal recessive disease characterized by premature aging, elevated genomic
instability and increased cancer incidence. The capacity of enforced telomerase
expression to rescue premature senescence of cultured human WS cells and the
lack of a phenotype in Wrn deficient mice with long telomeres have implicated
telomere attrition in WS pathogenesis. Here, we show that the varied and complex
in vivo and cellular phenotypes of WS are precipitated upon exhaustion of
telomere reserves in mice. In mice doubly null for Wrn and the telomerase RNA
component (Terc), telomere dysfunction elicits a classical WS premature aging
syndrome typified by premature death, hair graying, alopecia, osteoporosis, type
II diabetes, and cataracts. This model also exhibited accelerated replicative
senescence and accumulation of DNA-damage foci in cultured cells as well as
increased chromosomal instability and cancer, particularly non-epithelial
malignancies typical of WS. This faithful mouse model of WS establishes that the
complex and pleiotropic in vivo sequelae of Wrn deficiency are provoked largely
by critical telomere shortening.
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Heilbronn, Leonie K. |
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THE EFFECTS OF 6-MONTHS OF CALORIE RESTRICTION ON BIOMARKERS OF AGING IN
NON-OBESE HUMANS.
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Leonie K Heilbronn*, Eric Ravussin and the Pennington CALERIE group.
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Pennington Biomdeical Research Center, 6400 Perkins Rd, Baton Rouge, LA
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Caloric restriction (CR)
extends life span and retards age-related diseases in rats, mice, flies,
worms and yeast. Several biomarkers of aging (fasting glucose, insulin,
DHEAS and core temperature) have been identified in human and monkey studies
of longevity. Whether prolonged CR improves biomarkers of aging in humans is
unknown. Forty-eight healthy, nonsmoking, male (25-50y) and female (25-45y),
overweight participants (25 < BMI < 30) were randomized into one of four
groups; group 1 = 25% CR, group 2 = 12.5% CR + 12.5% increase in physical
activity, group 3 = low calorie diet until 15% weight reduction, group 4 =
Control. Subjects were fed by the metabolic kitchen from Weeks 1-12 whilst
they were taught a calorie counting system to allow them to prepare their
own meals (Weeks 12-22). Subjects returned to infeeding from Weeks 22-24.
Fasting glucose, insulin, DHEAS and 24h core temperature were assessed at
baseline, weeks 12 and 24. At the time of writing, only 30 subjects had
completed the study and so CR groups (Groups 1-3) were combined for
analysis. Average weight loss was 10% in the CR groups. Weight was not
changed significantly in the controls. Fasting insulin was reduced 24% in CR
groups as compared to a 5% increase in the controls (p<0.05). Glucose was
reduced 1% in CR groups as compared to a 4% increase in controls (p<0.03).
DHEAS and daytime core temperature were not significantly changed after 6
months. However, nighttime temperature (recorded from 10pm to 6am) was
reduced by 0.2ºC in CR groups as compared to a 0.3ºC increase in controls
(p=0.06). This study suggests that 6-mo. of calorie restriction in non-obese
humans was sufficient to observe improvements in 3 out of 4 known biomarkers
of aging. Whether these changes are sustained over longer periods of time is
unknown.
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Kitani, Kenichi |
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THE PRESENCE OF AN OPTIMAL DOSE RANEGE MAY EXPLAIN DISCREPANCIES IN THE
EFFECTS OF (-)DEPRENYL (D) ON SURVIVALS OF ANIMALS IN PAST REPORTS
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K.Kitani*1, S.Kanai2, K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4
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Tokyo Metropolitan Institute of Gerontology
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THE PRESENCE OF AN OPTIMAL
DOSE RANGE MAY EXPLAIN DISCREPANCIES IN THE EFFECTS OF (-)DEPRENYL (D) ON
SURVIVALS OF ANIMALS IN PAST REPORTS
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K.Kitani*1, S.Kanai2,
K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4
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*1National Institute for
Longevity Sciences, 36-3, Gengo, Moriokacho, Obu-shi; 2Tokyo Metropolitan
Institute of Gerontology, Tokyo, Japan; 3Univ.Rosario,Rosario, Argentina;
4Univ.Toronto, Scarborough,Canada
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To
explain discrepancies in the effect of D on life spans of animals in past
reports, we examined the effect of D at a dose of 0.25 mg/kg/inj s.c. 3x a
week starting from 18 months of age on life spans of F344Du/Crj rats of both
sexes. Average life spans (days) were significantly increased in
both male (895.2±109.7,mean ±S.D., n=30; 967.8±88.6, n=30, control vs. D
treated,+8.1%, P<0.01) and female (924.7±132.2, n=38; 987.1±133.4,
n=39,+6.7%, P<0.05) rats that received D injections compared to control
animals given saline. Increases in life expectancy from 24 months of
age were 44% in males and 32% in females. We previously reported that
a dose of 0.5mg/kg/inj significantly increased the life span of male F344
rats (1). However, a dose of 1.0 mg/kg/inj somewhat shortened the life span
of animals, although not statistically significantly (2). Together,our data
indicate that a proper dose range of D can significantly increase the life
spans of rats of both sexes, however, a greater dose becomes less effective
and may adversely affect the life span of animals. The presence of
this effective dose range of D may explain discrepancies in the effect of D
on life spans of animals previously reported. Furthermore, the fact
that the effective dose range observed for the life prolonging effect of D
parallels that for increasing activities of SOD and catalase strengthens our
contention that these two effects are causally related. Our new
observation that the same dosage of D (0.25mg/kg) increases life spans of
both male and female rats further supports our contention, since we
previously reported that the optimally effective dose of D to increase
antioxidant enzyme activities becomes closer for male and female rats as
they get older, while at young ages, the optimal dose is 10 times greater in
male rats (3). Ref. 1) Life Sci. 52:281-288, 1993. 2) Life Sci.
67:577-585, 2000.3)Life Sci. 52:1925-1934,1993
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Ladiges, Warren C. |
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AGE-ASSOCIATED CELLULAR STRESS IN HSP-40 MOLECULAR CHAPERONE MUTANT MICE
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S
Knoblaugh, J Morton, G Moore-Sanders, A MacAuley, W Ladiges*
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Department of Comparative Medicine, University of Washington, Seattle,
WA 98195
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Cellular stress associated with a malfunctioning endoplasmic reticulum (ER)
is causally related to the inability of molecular chaperones to maintain
structural integrity of proteins. Proteins which are structurally damaged
under conditions of oxidative stress or aggregation during the aging process
place an increased burden on the processing capacity of the ER. The result
is an exaggerated and extended ER stress response, which triggers
transcriptional and translational pathways of cell death and decreased
protein synthesis. ER dysfunction has been implicated in a number of
age-related diseases including Parkinsons, Alzheimers, cardiovascular
conditions, and diabetes. The HSP-40 molecular chaperone family member,
p58ipk, functions in the later stages of the ER stress response as an off
switch to prevent excessive cell death and promote protein synthesis.
Mutant mice deficient in p58ipk have evidence of an exaggerated ER stress
response in several cell types including pancreatic beta cells, hepatocytes,
and plasma cells. Gene expression, Western immunoblots and
immunohistochemistry in cells from p58ipk mutant mice confirm the
upregulation of ER stress response genes and an enhanced cell death pattern
reflective of an age-dependent onset of diabetes and immune dysfunction, and
an average life span of 18 months. We conclude that ER resident
proteins, including HSP-40/p58ipk, are targets for investigating specific
aging and age-related disease questions. The p58ipk mutant mouse model will
be of interest in intervention studies to determine capabilities of
decreased disease and increased life span mediated by a functional ER.
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Nikolich-Zugich, Janko |
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PHENOTYPIC, FUNCTIONAL AND GENETIC PROFILES OF THE AGING T- LYMPHOCYTES
IN PRIMATES AND THEIR ALTERATION BY CALORIC RESTRICTION
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Janko Nikolich-Zugich, Jessica Warner, Bree Fisher, Dragana Nikolich-Zugich
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and Ilhem Messaoudi
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Vaccine and Gene Therapy Institute and the Oregon National Primate Research
Center, Oregon Health & Science University, Beaverton, OR, USA
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Numerous T-cell functions
are diminished or dysregulated in old age, including T-cell population
dysbalance (altered na ve:memory and CD4/CD8 T-cell ratios), diminished
T-cell responsiveness to a variety of signals and altered cytokine networks.
Yet, despite intense research, numerous gaps in the understanding of T-cell
senescence still exist. Moreover, much of our current knowledge on T-cell
biology and aging comes from inbred, specific pathogen-free rodents, and it
is unclear which observations translate to human immune aging.
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We begun to develop a
primate model of human immune senescence using Rhesus macaques (RM). We
evaluated T-cell surface phenotype, in vivo and in vitro turnover, the
complexity of T-cell receptor (TCR) repertoire and correlated them to
functional studies of isolated cell subsets and gene expression profile
analysis. We also evaluated the ability of an experimental manipulation -
caloric restriction, the only intervention that consistently and
reproducibly increases longevity and quality of life in a variety of animal
models - to impact upon these parameters.
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We showed that sharp
differences exist in the phenotypic and functional T-cell aging between the
CD8 and CD4 T-cell subsets in: (i) cell cycle programs (as assessed by both
in vitro proliferation and in vivo turnover measurement); (ii) CD28
regulation upon cell cycle entry; (iii) accumulation of immediate effector
cells amongst the CD28- cells, believed to be close to or at replicative
senescence. These results underscore poor reliability of CD28 as a marker
for senescence, and further suggest that some of the T-cell aging
phenomenology in RM can be ascribed to accentuation over time of the
inherent differences in activation programs in CD8 and CD4 T-cells. We also
found age-related differences in T-cell turnover, and modest, but
consistent, gene expression changes in certain resting T-cell subsets.
Caloric restriction was able to modulate most, if not all, of these
parameters, resulting in young-like characteristics of examined
T-cells. We conclude that the results so far are consistent with resource
conservation induced by caloric restriction.
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Shaikh, Aasef G. |
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cAMP
forms a neurochemical
correlate of tinnitus
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A, Shaikh; P, Finlayson
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Department of Otolaryngology, Wayne State
University, Detroit, MI 48201
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Cochlear damage that commonly occurs in
elderly is either due to ototoxicity of anticancer drugs (cisplatin) or a
process of aging. Such hearing loss is often accompanied by tinnitus. It was
suggested that increased spontaneous neural activity (SA) may be the
underlying mechanism for tinnitus. Neurochemical consequences of cochlear
ablation (CA) have also been widely explored. A significant synaptic
plasticity has been reported following cochlear insults.
Yet a direct relationship of increased SA with plastic neurochemical
alterations remains to be recognized. In central auditory neurons, the
signals emerging after CA are transduced by range of mechanisms including
extracellular signal-regulated kinase (ERK) pathway. One of the functional
roles of this pathway is to enhance the inhibition of phosphodisterase E 4
(PDE4), thereby elevating intracellular cAMP concentrations ([cAMP]i). We
sought to determine if increasing [cAMP]i affects SA, and if so what
possible mechanisms may be involved and the possible
physiological/pathological ramifications. We also investigated such
elevations in SA could mimic the neural code for the pure-tone. Forskolin
(an agent that systematically increases [cAMP]i) specifically increases
tone-evoked responses and SA of the SOC neurons in dose-dependent manner.
Interestingly, the increased SA following application of 50 µM
forskolin mimics the neural code that is normally generated by the tonal
stimulus. We also report that effects of forskolin are predominantly due
to PKA independent pathway that involves hyperpolarization activated inward
conductances. These results provide for the first time, direct evidence that
systematic increase in the [cAMP]i, in the auditory brainstem neurons, by
application of forskolin elevates the SA, which mimics the neural code for
the pure-tone. Here we suggest, increased levels of [cAMP]i that could
follow cochlear insults, forms the basis of increased SA and therefore could
be the “neurochemical correlate” of tinnitus.
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Sun, Liou Y. |
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HIPPOCAMPAL GH AND IGF-1 EXPRESSION IN GH-DEFICIENT MICE
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Liou
Y. Sun*, Khalid Al-Regaiey, Michal M. Masternak, Jian Wang and Andrzej
Bartke
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Geriatrics Research, Department of Medicine and Department of Physiology
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Southern Illinois
University, Springfield, IL 62794, USA
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Liou Y. Sun*, Khalid Al-Regaiey,
Michal M. Masternak, Jian Wang and Andrzej Bartke
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Geriatrics Research,
Department of Medicine and Department of Physiology
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Southern Illinois
University, Springfield, IL 62794, USA
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Beneficial effects of GH and IGF-1 on the development and function of the
central nervous system are well documented. In spite of primary deficiency
of GH and secondary IGF-1 deficiency, Ames dwarf mice live considerably
longer than normal animals, exhibit apparently normal cognitive functions
and maintain them into advanced age. In an attempt to reconcile these
findings, we have examined local expression of GH and IGF-1 in the
hippocampus, brain region involved in learning and memory, of these mice.
RNA and protein was extracted from the hippocampus of dwarf and normal mice
and assayed for the GH and IGF-1 transcripts and their encoded proteins.
With the real-time RT-PCR analyses, hippocampus of Ames dwarf mice was found
to express normal levels of GH and IGF-1 messenger RNA indicating ectopic GH
expression and the integrity of transcription capability in dwarf mice. The
identities of the PCR products were confirmed by sequencing. Hippocampal
levels of GH and IGF-1 were evaluated by western blotting using the
antibodies specific for the respective proteins. Both GH and IGF-1 protein
levels are significantly increased in the hippocampus of Ames dwarf compared
with normal mice suggesting a compensatory mechanism of peripheral hormonal
deficiency. In contrast, IGF-1 expression in the liver of Ames dwarf mice is
profoundly suppressed, consistent with congenital GH deficiency and lack of
detectable GH and IGF-1 in peripheral circulation. In addition, Increased
phosphorylation of Akt and CREB were also detected in the hippocampus of
Ames dwarf mice. Our results suggest that increase in hippocampal GH and
IGF-1 protein expression and subsequent activation of PI3K/Akt-CREB signal
transduction cascade might contribute to the maintenance of cognitive
function and is likely to be responsible for the integrity of neuronal
structure, and maintenance of youthful levels of cognitive function in these
long-lived mice during aging.
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Wilcock, Donna M. |
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PASSIVE AMYLOID IMMUNOTHERAPY CLEARS AMYLOID AND TRANSIENTLY ACTIVATES
MICROGLIA IN A TRANSGENIC MOUSE MODEL OF AMYLOID DEPOSITION
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Donna M. Wilcock1, Amyn Rojiani2, Arnon Rosenthal3, Jennifer Alamed1, David
Wilson1, Nedda Wilson1, Melissa J. Freeman1, Marcia N. Gordon1, Dave
Morgan1.
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AbstractLocation: 1,2:
Alzheimer’s Research Laboratory, 1Department of Pharmacology, 2Departments
of Interdisciplinary Oncology and Pathology, University of South Florida,
12901 Bruce B Downs Blvd, Tampa, FL 33612, USA.
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3: Rinat Neuroscience Corp.
3155 Porter Drive, Palo Alto, California, 94304, USA.
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The role of microglia in
the removal of amyloid deposits following systemically administered anti-Aß
antibodies remains unclear. In the current study we injected Tg2576 APP
transgenic mice weekly with anti-Aß antibody for a period of one, two or
three months such that all mice were 22 months at the end of the study. In
mice immunized for three months we found an improvement in alternation
performance in the Y maze. Histologically, we were able to detect mouse IgG
bound to congophilic amyloid deposits in those mice treated with anti-Aß
antibody but not in those treated with control antibody. We found that
Fcgamma receptor expression on microglia was increased following one month
of treatment while CD45 was increased following two months of treatment.
Associated with these microglial changes was a reduction in both diffuse and
compact amyloid deposits following two months of treatment.
Interestingly, the microglia markers were reduced to control levels
following three months of treatment while amyloid levels remained reduced.
Serum Aß levels and anti Aß antibody levels were elevated to similar levels
at all three survival times in mice given anti-Aß injections rather than
control antibody injections. These data show that antibody is able to enter
the brain and bind to the amyloid deposits, likely opsonizing the Aß and
resulting in Fcgamma receptor mediated phagocytosis. Together with our
earlier work, our data argue that all proposed mechanisms of anti-Aß
antibody mediated amyloid removal can be simultaneously active.
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P O S T E R |
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Al-Regaiey, Khalid |
43. EFFECTS OF LONG-TERM CALORIC
RESTRICTION ON WILD-TYPE AND GROWTH HORMONE RECEPTOR KNOCKOUT MICE
Khalid Al-Regaiey,
Michal M. Masternak, Michael Bonkowski, Liou Sun, Andrzej Bartke
Southern Illinois University School of Medicine, Springfield, IL 62794
Reduced IGF-1/insulin signaling and caloric restriction (CR) are known to extend
life span and delay age related diseases. To address the interaction of these
two interventions, we subjected normal (N) and growth hormone receptor knockout
(KO) mice to CR starting at weaning for 20 months. Molecules involved in
glucose homeostasis were investigated. CR resulted in decreased plasma
glucose levels in both phenotypes (N-CR and KO-CR). Circulating IGF-1 was
reduced in N-CR and was undetectable in KO animals. Insulin was also
reduced in N-CR to levels comparable to those in KO animals. Corticosterone and
adiponectin levels were higher in KO than in N animals while leptin was reduced
by CR in both phenotypes. We also analyzed hepatic gene and protein expressions
of molecules involved in insulin signaling using real-time PCR and western
blotting, respectively. Diet and phenotype did not affect Akt gene and total
protein expression while KO mice exhibited reduced Akt activation with no diet
effect. Phosphorylation of protein kinase C γ/ζ was not affected by
either diet or phenotype. KO mice exhibited increased CREB activation. Gene
expression and protein levels of Foxo1 and PGC-1 were increased in KO mice with
little diet effects. Genes that are known to be regulated by CREB, Foxo1, and/or
PGC-1including pepck, g6pase, igfbp-1, and MnSOD had increased expression in KO
mice. Genes encoding proteins that are involved in lipolysis, including
hormone-sensitive lipase (HSL) and lipo-protein lipase (LPL) were upregulated in
KO mice. We conclude that CR and growth hormone resistance probably affect
longevity of mice by different mechanisms and that gluconeogenesis pathway as
well as fatty acids mobilization are more active in KO mice. These data also
suggest that liver of KO mice is more protected against oxidative stress as
indicated by increased Foxo proteins and MnSOD mRNA.
Supported by NIA.
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Anderson, Rozalyn M. |
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44. AGING RETARDATION BY CALORIE RESTRICTION: ROLE OF REGULATORS OF ENERGY
METABOLISM.
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R.
Anderson (1), J.Barger (1), T. Pugh (1), S. Park (2), T. Prolla (2) and R.
Weindruch (1).
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(1)Department of Medicine, University of Wisconsin and GRECC, VA Hospital,
Madison WI 53705 USA. (2) Department of Genetics and Medical Genetics,
University of Wisconsin, Madison WI 53706 USA.
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University of Wisconsin
Madison, GRECC, VA Hospital, Madison WI 53705, USA.
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Caloric restriction (CR) extends lifespan in a broad spectrum of organisms
and retards the progression of a wide range of age-associated biological
changes; however, the underlying mechanisms are unclear. In mice,
there is an inverse linear relationship between calorie intake and lifespan
extension suggesting that factors central to energy metabolism may underlie
aging retardation by CR. One hypothesis is that CR triggers an active
response involving a reprogramming of energy metabolism that reduces the
rate of aging and the development of several age-related diseases.
Transcription profiling of tissues from control and CR animals points to
mitochondrial function as a central feature in this process. We have
used a combination of techniques to identify factors that may be involved in
the mechanism of lifespan extension by CR. We then investigated how
these potential effectors of CR are regulated and examined their
relationship to elements of known longevity pathways, including members of
the FOXO and Sirtuin families.
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Armbrecht, Harvey J. |
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45. REGULATION OF THE RENAL VITAMIN D 1ALPHA-HYDROXYLASE CYTOCHROME
P450(CYP27B1) BY DIETARY PHOSPHORUS CHANGES WITH AGE
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H.J. Armbrecht*, M.A. Boltz
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Geriatric Center, St. Louis VA Medical Center, St. Louis, MO 63125
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Young rats adapt to a low phosphorus diet by increasing plasma levels of
1,25-dihydroxyvitamin D (1,25D), the biologically active metabolite of
vitamin D, which then increases intestinal phosphate absorption.
Previous studies have shown that the capacity of rats to adapt to low
dietary phosphorus declines with age. The purpose of this study was to
determine whether this decreased adaptation was due to decreased expression
of the renal 1alpha-hydroxylase (1-OHase), which makes 1,25D. The
1-OHase consists of ferredoxin reductase, ferredoxin, and a terminal
cytochrome P450 – CYP27B1. Young (2 months) and adult (12 months) F344
rats were placed on a low phosphorus (0.1%) or high phosphorus (1.0%) diet
for 2 weeks. Plasma 1,25D was markedly increased by the low phosphorus
diet in young animals but not in adults. To determine whether this
difference was due to decreased 1-OHase expression, mRNA levels of CYP27B1
were measured by ribonuclease protection assay. In young animals, the
low phosphorus diet increased renal CYP27B1 mRNA levels 8-fold compared to
the high phosphorus diet, but there was no significant effect in adults.
The effect of low dietary phosphorus on plasma calcium, phosphate, and
parathyroid hormone levels was similar in both age groups. These
results suggest that the decreased CYP27B1 expression in the adult in
response to dietary phosphorus depravation is not due to changes in the
major regulators of 1-OHase activity.
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Ball, Sheldon S. |
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46. SENEX: INTEGRATING GERIATRICS AND MOLECULAR GERONTOLOGY
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S
Ball, A Nhalil and V Mah
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Department of Veterans Affairs, Greater Los Angeles Healthcare, 11301
Wilshire Boulevard, GRECC 11G, Los Angeles CA 90073
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Senex is a computer application 15 years in development integrating
geriatrics and molecular gerontology (from bench to bedside). It includes
information (and tools for interpretation of data) that span internal
medicine, molecular pathology (biochemistry, molecular biology, pathology),
laboratory medicine, radiology, pharmacology, anatomy, and statistics.
Clinical and molecular information is needed from laboratory bench to
patient bedside, during contact between physician and patient, educator and
student, scientist and technician. The information needs to be
structured so that specific and real-time retrieval is achievable. The
information needs to be presented in a manner that facilitates a conceptual
understanding of the details presented. Additionally, an information
system should display a certain degree of intelligence, including
flexibility in accepting input from the user, the capacity to reason with
structured information, and the esthetic display of context-specific
information. Implementation of such an information system should allow use
to become better physicians, scientists, educators, students and/or informed
citizens. Senex functions independently as a stand-alone application (i.e.
not dependent upon internet access). Thus it is fast, reliable, and mobile.
Senex also provides direct links into the world-wide-web, to molecular and
clinical databases and to the original literature when access to the
internet is available. Senex is a large application with: 1514
organisms, 2417 anatomic structures, 222 cells, 131 cellular compartments,
13,138 molecules, 9607 proteins, 628 genes, 795 motifs, 67 molecular
pathways, 1580 diseases, 540 clinical laboratory tests, 18,680
database links (6140 Swiss Prot, 6280 OMIM, 870 Prosite, 2000 PIR,
3390 Locus Link). Senex allows a user to add proprietary information
on top of the core Senex knowledge base, thus customizing the application
for a user’s own information needs. Senex runs on Macintosh and
Windows computers. The Macintosh version has a microarray data analysis and
data mining module. Reference: www.senex-medical-software.com
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Bergamini, Ettore |
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47. THE AGE-RELATED ACCUMULATION OF DOLICHOL IN TISSUES SATISFIES ALL
CRITERIA TO BE QUALIFIED A BIOMARKER OF AGING
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E. Bergamini, G. Cavallini, A. Donati, Z. Gori, A. Manfrini, I. Parentini
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-
Centro di Ricerca di Biologia e Patologia dell’Invecchiamento
dell’Universita’ di Pisa, Dipartimento di Patologia sperimentale, Via Roma
55, 56126 PISA (Italy); Tel 0039-050-2218584 Fax 0039-050-2218581
e-mail ebergami@med.unipi.it
-
-
It was stated that the
identification of specific biomarkers of aging would be an important
milestone in gerontological research, and criteria for defining biomarkers
were suggested (Mooradian, 1990). In this communication evidence on male and
female Sprague Dawley and Lewis rats fed ad-libitum or on regimens of food
restriction and on human tissues are reviewed, showing that accumulation of
dolichol in tissues of older animals meet these criteria: levels of dolichol
exhibit a quantitative correlation with age in all tissues; the biological
parameter is not altered by several age-dependent diseases in the same
direction as that of aging; data with transplanted organs show that the
age-related accumulation is not secondary to metabolic changes of aging;
factors that modulate the aging rate like caloric restriction and physical
exercise appropriately alter the accumulation of dolichol; this putative
biomarker of aging appears to be applicable to different tissues across
mammalian species including human; biomarker is applicable to the
hypothalamic digoxin-mediated model for trisomy 21 (Kurup & Kurup, 2003).
Reliable changes in tissue dolichol levels are seen in relatively short
intervals of time compared to life-time, and levels can be tested on a small
amount of tissue without causing death of the animal, which are desirable
features of a biomarker of aging. The age-associated alteration in dolichol
levels is likely to reflect an age-dependent derangement of free radical
metabolism in membranes. Useful applications are shown, which include a
study of the effect of the donor-recipient age-mismatch on the biological
age of the graft tissues, and the detection of gender-related differences in
aging.
-
Research was supported in
part by a grant of MIUR (Ministero Istruzione, Universita’ e Ricerca)
-
Mooradian, A.D. (1990) J
Gerontol 45(8) B183-186.
-
Kurup R.K., Kurup, P.A.
(2004) Pediatr Pathol Mol Med 22(5) 411-422.
|
|
Bowen,
Richard |
107. LIVING AND DYING FOR SEX: A THEORY OF AGING
BASED ON THE MODULATION OF CELL CYCLE SIGNALING OF REPRODUCTIVE HORMONES
R. Bowen, C. Atwood*
Voyager Pharmaceutical Corporation, Raleigh, North Carolina, USA
* Department of Pathology, Case Western Reserve University, Cleveland, Ohio and
School of Medicine, Section of Geriatrics and Gerontology, Department of
Medicine, University of Wisconsin, Madison, Wisconsin, USA
We put forth a new theory based on a novel definition of aging - any change in
an organism over time. This definition includes not only the changes associated
with the loss of function (i.e. senescence), but also the changes associated
with the gain of function (growth and development). Using this definition, the
rate of aging would be synonymous with the rate of change. The rate of
change/aging is most rapid during the fetal period when organisms develop from a
single cell at conception to a multicellular organism at birth. Therefore,
“fetal aging” would be determined by factors regulating the rate of mitogenesis,
differentiation, and cell death. We suggest these factors also are responsible
for regulating aging throughout life. Thus, whatever controls mitogenesis,
differentiation and cell death must also control aging. Since life-extending
modalities consistently affect reproduction, and reproductive hormones are known
to regulate mitogenesis and differentiation, we propose that aging is primarily
regulated by the hormones that control reproduction (hence, the
Reproductive-Cell Cycle Theory of Aging). In mammals, reproduction is controlled
by the hypothalamic-pituitary-gonadal (HPG) axis hormones. Longevity inducing
interventions, including caloric restriction, decrease fertility by suppressing
HPG axis hormones and HPG hormones are known to affect signaling through the
well-documented longevity regulating GH/IGF-1/PI3K/Akt/Forkhead pathway. This is
exemplified by genetic alterations in C. elegans where homologues of the HPG
axis pathways, as well as the daf-2 and daf-9 pathways, all converge on daf-16,
the homologue of human Forkhead that functions in the regulation of cell cycle
events. In summary, we propose that the hormones that regulate reproduction act
in an antagonistic pleiotrophic manner to control aging via cell cycle
signaling; promoting growth and development early in life in order to achieve
reproduction, but later in life, in a futile attempt to maintain reproduction,
become dysregulated
|
|
Chacon, M.A. |
108. UP-REGULATION OF FREE
FATTY ACIDS - A POTENTIAL MASTER SWITCH OF CALORIC RESTRICTION PROTECTION
P. Wills1, H.
Brown-Borg2,and M.A. Chacon1(P)
1Irazu
Biodiscovery, LLC, Baltimore, MD 21224; 2Department of Pharmacology,
Physiology and Therapeutics, University of North Dakota School of Medicine and
Health Sciences, Grand Forks, ND 58203.
Seventy years of scientific
investigation has demonstrated that caloric restriction (CR) without
malnutrition is the only experimental intervention that can extend life span,
and decrease the incidence and delay the onset of pathologies associated with
aging. Nevertheless, the precise mechanism by which caloric restriction induces
its life-extending effect remains poorly understood. One consequence of caloric
restriction observed in experimental rats, which has not been previously
investigated, is an elevation in serum levels of free fatty acids (FFA). In our
studies, we found that calorie restricted rats, mice, and dwarf mice all have
elevated free fatty acids. In addition to their well-characterized role as an
energy substrate, recent scientific studies have demonstrated that these
molecules possess signaling properties. Therefore, we tested the hypothesis
that free fatty acids may induce (mimic) some of the protective effects seen in
calorie restricted animals. Using an in vitro, cell-based ROS assay, we
demonstrated that supplementation of serum from ad lib animals with free fatty
acids to levels comparable to those found in the serum of caloric restricted
animals conferred protection from the cytotoxic effects of H2O2. We contend
that the up-regulation of FFA and their CoA derivatives, through their effects
on ion channels, oxygen utilization, and gene expression, offer a plausible
mechanism for the protective phenotype associated with CR.
|
|
Chai,
Weihang |
48. SENESCENT NORMAL HUMAN FIBROBLASTS MAINTAIN
THEIR 3' TELOMERIC OVERHANG LENGTH
W. Chai, J.W. Shay, and W.E. Wright
Department of Cell Biology, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9039
Replicative senescence involves progressive telomere shortening but the exact
molecular mechanisms triggering the growth arrest are poorly understood.
Recently, using a telomere-oligonucleotide ligation assay (T-OLA), it was
reported1 that the 3’ G-rich overhang was eroded at senescence, leading to the
hypothesis that loss of the 3’ G-rich overhang is the molecular signal that
triggers senescence. To better understand the molecular mechanisms triggering
replicative senescence, we have developed a quantitative assay to measure the
length of the G-rich 3’ telomeric overhangs from cultured cells. By using this
assay and the conventional non-denaturing hybridization assay for measuring
G-rich overhangs, we show that normal human fibroblasts maintain their 3’ G-rich
overhangs at senescence. Furthermore, cells maintain their G-rich overhangs when
they bypass senescence after the inactivation of p53 and retinoblastoma proteins
by human papillomavirus type 16 (HPV) oncoproteins E6 and E7 (E6/E7).
Interestingly, cells expressing just HPV E7 exhibit longer overhangs when they
growth arrest. We also found that expression of simian virus 40 large T-antigen
induces some telomeric overhang shortening. These results demonstrate that a
significant reduction in overhang length is not the molecular signal that
triggers senescence.
Reference:
1. Stewart, S.A., I. Ben-Porath, V.J. Carey, B.F. O'Connor, W.C. Hahn, and R.A.
Weinberg. 2003. Erosion of the telomeric single-strand overhang at replicative
senescence. Nat Genet 33: 492-6.
|
|
Chow, Vivian W.H. |
-
49. APOPTOTIC DNA FRAGMENTATION IN THE BRAINS OF YOUNG AND AGED eNOS-, iNOS-
and nNOS-KNOCKOUT MICE: MEASUREMENT BY NEW ULTRASENSITIVE CE-LIF TECHNIQUE
-
-
VW
Chow*, RR Fiscus, SB Chew
-
-
The
Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T.
Hong Kong.
-
-
Previously, we showed that
elevation of cGMP levels in PC12 cells exposed to atrial and brain
natriuretic peptides (ANP & BNP) inhibits onset of apoptosis and promotes
survival [1]. We further showed that 24h-preincubation of NG108-15 cells
with ANP protects against pro-apoptotic effects of nitric oxide (NO) donor
(high concentrations) [2]. At lower physiological concentrations, NO, via
cGMP elevations, inhibits apoptosis and improves neural survival [3].
Depending on the source of NO, whether from nNOS, iNOS or eNOS, and
depending on age, NO may promote or protect against apoptosis. The present
study determines if aging-induced-apoptosis in brain is affected by genetic
deletion of nNOS, iNOS and eNOS using nNOS-, iNOS-, eNOS-knockout mice.
Young (2-4-month-old) and aged (12-22-month-old) nNOS-/-, iNOS-/-, eNOS-/-
knockout and control mice (C57BL/6J & B6129SF2/J) were used to determine
levels of apoptosis in half-brain and different brain regions by a new
ultrasensitive technique using capillary electrophoresis with laser-induced
fluorescent detector (CE-LIF) to accurately quantify apoptotic-DNA
fragmentation. Using half-brain, amounts of apoptotic-DNA-fragments were
11-fold higher in aged than in young B6129SF2/J (nNOS-/-control) mice
(p<0.0001). Apoptotic-DNA-fragments in young nNOS-/- were 23-fold higher
than in young B6129SF2/J (p<0.0001). Apoptotic levels in aged B6129SF2/J
were 2.5-fold higher than in aged nNOS-/- (p<0.001). These data suggest that
nNOS contributes to aging-dependent-increase in brain apoptosis of
B6129SF2/J, but provide neuroprotective effect in young adults.
Neuroprotective effects of nNOS were especially large in young hippocampus.
Apoptotic-DNA-fragments in young iNOS-/- were 30-fold higher than in young
C57 controls (p<0.001). Aged iNOS-/- showed 6.5-fold lower level of
apoptosis compared to young adult (p<0.001). iNOS had anti-apoptotic effect
in young C57 but not aged C57. eNOS showed significant neuroprotective
effect in medulla oblongata of both young and aged C57. All NOS isoforms
provided neuroprotective function in young adult brain, but this
neuroprotection appeared change during aging.
-
1. Fiscus
RR, Tu AW, Chew SB. Neuroreport 2001;12:185-189.
-
2. Cheng
Chew SB, Leung PY, Fiscus RR. Histochem Cell Biol. 2003;120:163-171.
-
3. Fiscus
RR. Neurosignals 2002;11:175-190.
-
Support: Competitive
Earmarked Grant from RGC of Hong Kong to RRF. (#CUHK 4169/02M)
-
|
|
Citron, Bruce |
- 50. GENE EXPRESSION CHANGES IN NEURODEGENERATIVE MICE AND CULTURED
CELLS
- B.A. Citron*1,2, A. Surguchov3,4, I. Surgucheva3,4, and B. W.
Festoff3,4,5
1Lab of Molecular Biology, VA Medical Center, Bay Pines, FL, 2Dept. of
Biochemistry & Molecular Biology, USF College of Medicine, Tampa, FL,
3Neurobiology Research Lab, VA Medical Center, Kansas City, MO, Depts. of
4Neurology and 5Pharmacology, University of Kansas Medical School, Kansas City,
KS
A variety of genes have been implicated in late onset neurodegenerative
disorders. Several reports linked synucleins to motor neuron diseases including
ALS. Present in aggregates, α-synuclein is important for the long term survival
of neurons. The role of γ-synuclein is less clear but recent studies indicate
that it regulates other neurodegenerative factors. Synucleins modulate certain
proteases, however their own proteolytic degradation may be essential in the
process of formation of inclusion bodies. We have been studying cultured neurons
and wobbler mice, which undergo an age dependent loss of motor neurons in the
cervical region of the spinal cord and model sporadic ALS. We have measured
activation of transcription factors (with electrophoretic mobility shift assays)
in wobbler spinal cords and cultured neurons exposed to insults. Specifically,
we found that activated transcription factors SP1 and NF-kB, but not AP1 are
increased in wobbler mice and in motor neurons exposed to apoptosis inducing insults (thrombin or mechanical injury). We
have also examined mRNAs levels with Affymetrix gene arrays. Genes responsible
for CNS maintenance (e.g. myelin-associated oligodendrocytic basic protein) are
downregulated in the wobbler spinal cord. Ubiquitin C-terminal hydrolase is also
expressed at lower levels in the wobbler cord and this could affect the
ubiquitination of proteins that aggregate in the degenerating CNS. α-synuclein
is upregulated in the wobbler compared to the wild-type littermate spinal cord.
Directed elevation of α-synuclein, γ-synuclein or both, in transfected neurons
resulted in a marked dysregulation of gene expression in several functional
categories. For example, of the genes that are significantly expressed in these
cells, more genes involved in cell adhesion are upregulated compared to genes in
several other categories. The over and underrepresented functional classes in
gene groups upregulated and downregulated by α- and/or γ-synuclein introduction have been determined to obtain
a picture of synuclein regulatory influences.
|
|
Cohen, Haim Y. |
- 51.
The SIRT1 NAD+-dependent
deacetylase regulates Bax-mediated apoptosis
Haim Y. Cohen*, Christine Miller, Kevin Bitterman and David A. Sinclair.
Department of Pathology, Harvard Medical School. Boston MA USA 02115.
Decreased apoptosis and increased cell survival is observed in organisms whose
lifespan has been extended by calorie restriction or genetic manipulation. A
crucial but poorly understood step in the stress-induced apoptotic pathway is
activation of the pro-apoptotic factor Bax. In yeast, lifespan extension by
calorie restriction requires Sir2, a stress-responsive, NAD+-dependent
deacetylase. Here we show that the human Sir2 homolog, SIRT1, controls cell
survival in response to stress by inhibiting Bax-mediated apoptosis. SIRT1
deacetylates specific lysines in the Ku70 C-terminus, which increases the
Ku70-Bax interaction and prevents Bax from relocalizing to mitochondria. We show
that this pathway can be manipulated using small molecules and discuss the
implications of this finding to the general decline in physiological function
with age.
|
|
Coumoul, Xavier |
- 52. Modulation of IGF members expression in Brca1-knockout Mice
X Coumoul*, L Cao and C Deng
National Institute of Health, National Institute of
Diabetes and Digestive and Kidney Diseases, 9000 Rockville Pike, Bethesda MD
20892, USA
Breast cancer affects 200.000 women in the United States which results in 40.000
deaths every year. Heterozygous carriers of Breast Cancer Associated Protein 1
(Brca1) mutations are at great risk to develop breast or ovarian cancers through
their lifetime. Our mice models of Brca1 deletions have shown that indeed lack
of this gene plays a role in mammary tumorigenesis but also interestingly in
aging process. Here, we shown that deletion of Brca1 modulates expression of
several Insulin-like Growth Factor (IGF) members in mice liver. IGF members
expression has previously been linked to tumorigenesis and aging process.
Interestinlgy, IGF1 serum levels but also mRNA liver levels are increased as
shown by microarrays and RT-PCR analysis. Other members like IGF1-Receptor,
Insulin Receptor Substrate 1 or IGF-Binding Protein 1 and 2 also appears to be
modulated. We also use a human cell culture system UBR60, allowing modification
of Brca1 expression, to shown that those results are reproducible with a
different model. Those results allow us to hypothesize that modulation of IGF
members expression by Brca1 could be one of the mechanism that links lack of
this protein to aging and tumorigenesis process.
|
|
de Grey, Aubrey D. |
-
53.
THE UNFORTUNATE
INFLUENCE OF THE WEATHER ON THE RATE OF AGING
-
-
A. de Grey
-
-
Department of Genetics,
University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
-
-
Much research interest, and
recently even commercial interest, has been predicated on the assumption
that reasonably closely-related species -- humans and mice, for example --
should in principle respond to aging-retarding interventions with an
increase in life expectancy roughly proportional to their control lifespan
(life expectancy without the intervention). Here it is argued that the
best-studied life-extending manipulations of mice are examples of a category
that is highly unlikely to follow this rule, and more likely to exhibit only
a similar absolute increase in lifespan from one species to the next,
independent of the species' control lifespan. That category -- deprivation
of dietary calories or of the organism's ability to metabolise or sense them
-- is widely recognised to extend lifespan as an evolutionary adaptation to
transient starvation in the wild, a situation which alters the organism's
optimal partitioning of resources between maintenance and reproduction. What
has been generally overlooked is that the extent of the evolutionary
pressure to maintain adaptability to a given duration of starvation varies
with the frequency of that duration, something which is -- certainly for
terrestrial animals, and less directly for others -- determined principally
by the weather. The pattern of starvation that the weather imposes is
suggested here to be of a sort that will tend to cause all terrestrial
animals, even those as far apart phylogenetically as nematodes and mice, to
possess the ability to live a similar maximum absolute (rather than
proportional) amount longer when food is short than when it is plentiful.
This generalisation seems to be strikingly in line with available data,
leading to the biomedically and commercially sobering conclusion that human
interventions which manipulate caloric intake or its sensing are unlikely
ever to confer more than two or three years' increase in lifespan at the
most.
-
|
|
Ding, Shi-Ying |
-
54. CRP LEVELS ARE STRONGLY ASSOCIATED WITH AGING
-
-
SY.
Ding, X.T. Tigno, and B.C. Hansen
-
-
Obesity, Diabetes, and Aging Animal Resource Center, Department of
Physiology, School of Medicine, University of Maryland at Baltimore, 10 S.
Pine St, Baltimore MD 21201
-
-
Epidemiological studies
have consistently shown that the plasma level of C-Reactive Protein (CRP), a
marker of inflammation, is a strong, independent predictor of future
cardiovascular events, both in patients with a history of coronary heart
disease (CHD) and in apparently healthy subjects. Inflammatory markers have
also lately been implicated in aging and in type 2 diabetes. The plasma
concentrations of CRP were determined by ELISA in a cohort of 33 diabetic
and non-diabetic rhesus monkeys: 13 young normal (Y) with a mean age of
9.1±2.9 yr, 11 old normal (O, 25.9±1.5 yr), 8 old type 2 diabetic monkeys
(DM, 26.2±2.3 yr) and 4 calorie-restricted (for more than 20 years) monkeys
(CR, 30.1± 2.8 yr). Results show that CRP levels were positively correlated
to age (r=0.46, p<0.01) and body weight (r=0.33, p<0.02). The mean levels of
CRP were found to be highest among the O (246±16µg/L), a value significantly
greater than that among the Y (184±14µg/L). However, mean CRP values did not
differ significantly between O, DM (221.4±18µg/L) and CR (214±26µg/L), all
of whom were elderly. The data suggests that the proportion of the variation
in CRP that can be accounted for by variation in age is 0.21, and that age
is a stronger predictor of CRP than weight (R2= 0.18) or metabolic status,
as reflected by circulating levels of CRP in the non-human primate.
|
|
Dolle, Martijn |
55. AGE-RELATED GENOME INSTABILITY IN DNA
REPAIR-DEFICIENT MICE
M. Dollé1, R. Busuttil2, L. Niedernhofer3, G. van der Horst3, J. Hoeijmakers3,
S. Wijnhoven1, P. Lohman1, H. van Steeg1, J. Vijg2
1National Institute of Public Health and the Environment, Department of
Toxicology, Pathology and Genetics, Bilthoven, the Netherlands; 2University of
Texas Health Science Center, Department of Physiology, San Antonio, TX, USA;
3Erasmus University, Department of Cell Biology and Genetics, Rotterdam, the
Netherlands
Genomic instability has been implicated as a major cause of both cancer and
aging. Previously we have shown that mutations accumulate with age in an
organ-specific manner, using a transgenic mouse model with a chromosomally
integrated lacZ mutational reporter gene. To examine the correlation between age
and mutation load, we are investigating genomic instability in aging DNA
repair-deficient mice. Here we present our preliminary findings on different
mouse mutants with deficiencies in nucleotide excision repair (NER) and DNA
interstrand crosslink (ICL) repair. NER removes a broad range of lesions, such
as UV-induced damage, other bulky adducts and some forms of oxidative damage.
Life span studies and mutant frequency determinations in liver and kidney were
performed on male mice with defects in global and/or transcription-coupled
repair (Xpa, Csb or Xpd(ttd)). We observed a reduced life span for Xpa and
Xpd(ttd), as compared to Csb and wild type control animals. Conform to our
initial studies with just wild type mice, all NER-deficient models showed an
age-related increase of mutant frequencies in liver and kidney. However, only
the Xpa-mice showed elevated mutant frequencies over control animals in both
organs. In addition, we examined mutant frequencies in liver of the very
short-lived Ercc1(*292) mouse. This mouse model lacks the last 7 amino acids of
Ercc1 and is deficient in both NER and DNA ICL repair. We observed a two-fold
increase in mutant frequency over control mice at 23 weeks, which is close to
their maximum life span. A comparison of mutation spectra in liver revealed that
the general NER-deficiency of XPA-mice resulted in point mutations,
predominantly consisting of one base pair deletions. The increased mutant
frequency in the Ercc1(*292) mice was due to both point mutations and
translocations, the latter presumably reflecting the additional cross-link
repair deficiency compared to Xpa-mice.
|
|
Falcon, Alaric A. |
-
56. DNA EPISOMES AND REPLICATIVE SENESCENCE IN
S. CEREVISIAE
-
- Alaric A.
Falcón*, Diego M. Ayo, Natalie Rios, and John P. Aris
-
- Department
of Anatomy and Cell Biology, Health Science Center, University of Florida,
Gainesville, FL 32610-0235
-
- We have shown that
plasmids containing an autonomously replicating sequence (ARS; yeast DNA
replication origin) reduce yeast replicative life span, most likely due to
their accumulation during replicative aging (Plasmid accumulation reduces
life span in Saccharomyces cerevisiae(Falcón and Aris, 2003). This raises
the question: how is plasmid accumulation mechanistically linked to
replicative senescence in yeast? One possibility is that accumulated DNA
exerts an effect on other cellular DNA, perhaps by influencing DNA
replication and/or inheritance. To explore this, we have studied
“interactions” between different types of episomes in yeast: recombinant
plasmids, extrachromosomal rDNA circle (ERCs), and the naturally-occurring 2
micron plasmid. A short-lived sir2∆ strain that contains elevated
ERC levels transmits recombinant ARS1-plasmids to daughter cells more
often than a SIR2 control strain. However, an ARS1-CEN4-plasmid
is transmitted to daughter cells equally well in sir2∆ and SIR2
strains. Levels of ARS1-plasmids are reduced in old sir2∆
cells, compared to old SIR2 cells. Unexpectedly, an ARS1-plasmid,
but not an ARS1-CEN4-plasmid, significantly increased the
frequency of loss of the 2 micron plasmid (i.e., curing of a cir+ strain
to cir0).
Consistent with this, levels of the 2 micron plasmid are reduced in old
cells compared to young cells. Thus, accumulation of different types of
nuclear episomal DNAs can have significant and unexpected effects on other
nuclear DNAs. These findings support a relationship between episomal DNA
accumulation, effects on cellular DNA metabolism, and cell senescence in S. cerevisiae.
|
|
Fiscus, Ronald R. |
-
57.
INHIBITOR OF APOPTOSIS PROTEIN-2 (IAP-2) EXPRESSION LEVELS ARE
DOWN-REGULATED BY CYCLIC GMP DEPLETION AND UP-REGULATED BY CYCLIC GMP
ELEVATIONS INDUCED BY NATRIURETIC PEPTIDES OR ANALOGS OF CYCLIC GMP IN
NG108-15 CELLS: MOLECLULAR MECHANISM OF CYCLIC GMP/PROTEIN KINASE G (PKG)-MEDIATED
NEUROPROTECTION
-
-
R.R.
Fiscus* and J.P. Yuen
-
-
Dept. Physiology (Fac. Medicine), Epithelial Cell Biology Res. Ctr. and Ctr.
for Gerontology & Geriatrics, The Chinese University of Hong Kong, Shatin,
NT, Hong Kong
-
-
Previously, we showed that cGMP elevations protect hippocampal neurons
against glutamate toxicity [1], inhibit apoptosis and prolong cell survival
in stressed PC12 cells [2] and protect NG108-15 cells against the
pro-apoptotic effects of nitric oxide (NO, used at high/neurotoxic
concentrations mimicking pathogenesis of Alzheimer’s and Parkinson’s
disease) [3]. Activation of cGMP/PKG signaling pathway in neurons may
represent an important physiological mechanism protecting against
neurotoxicity and neurodegeneration (reviewed [4]). Even basal levels
of cGMP appear sufficient (and necessary) to prevent spontaneous development
of apoptosis in some neural cells (e.g. NG108-15 cells), because depletion
of cGMP by ODQ triggers apoptosis [4]. Recently, cAMP was shown to
inhibit apoptosis in intestinal epithelial cells via protein kinase A (PKA)-mediated
phosphorylation of CREB and increased IAP-2 expression [5]. Because
PKG phosphorylates CREB at the same site as PKA [4], we hypothesized that
cGMP/PKG-mediated neuroprotection may involve IAP-2 expression. The
present study found that ODQ (10-100 micromolar, 24 h), which lowered basal
cGMP levels, greatly reduced protein content of IAP-2 (Western blot) in a
concentration-dependent manner in NG108-15 cells. Elevation of cGMP
levels with either atrial or brain natriuretic peptides (ANP or BNP, 10-100
nM) or with 8-pCPT-cGMP (1-100 micromolar, a cell-permeable direct activator
of PKG), all of which by-pass the ODQ block, completely reversed the ODQ-induced
down-regulation of IAP-2. Indeed, ANP and BNP up-regulated IAP-2
levels above normal control. The results suggest that up-regulation of
IAP-2 expression in neural cells may represent one of the molecular
mechanisms that mediates cGMP/PKG-induced protection against apoptosis.
1. Barger et al (1995) J Neurochem 64:2087-2096, 2. Fiscus et al
(2001) NeuroReport 12:185-189, 3. Chew et al (2003), Histochem Cell
Biol 120:163-171, 4. Fiscus (2002) NeuroSignals 11:175-190, 5.
Nishihara et al (2003) PNAS 100:8921-8926. (Support: Grant #
CUHK4169/02M from Research Grants Council of Hong Kong to RRF)
|
|
Gemma,
C |
58. nINHIBITION OF CASPASE 1 IMPROVES CONTEXTUAL FEAR CONDITIONING IN AGED RATS
C. Gemma (P)*, M. Fister, C.
Hudson, P.C. Bickford
James A Haley VAH, Tampa
Florida and Center for Aging and Brain Repair, USF, 12901 Bruce B. Downs BLVD,
Tampa, FL 33612
An interplay between the immune
system and the central nervous system may underlie the neuropsychological
changes occurring with aging. It has been long accepted that brain levels of
certain cytokines increase as a function of age, even in absence of a pathologic
stimulus. In aged rats there is an increase in IL1b that has been implicated in
declines of synaptic plasticity in the hippocampus and performance on cognitive
tasks. IL-1b receptor expression is high in the hippocampus, an area of the
brain, which plays a pivotal role in memory and learning, suggesting that the
effects of IL-1b may be specific to hippocampal-dependent memory processes.
However, although at pathophysiological levels IL-1b produces detrimental
effects on learning and memory processes, under physiological circumstances
IL-1b seems to be required for normal learning and memory processes. IL-1b is a
proinflammatory cytokine initially synthesized in an inactive precursor form
that is cleaved to generate the biological mature 17kDa form by a protease named
caspase-1. In the present study we chronically inhibit the cleavage of IL-1b
using a specific inhibitor of Caspase-1(Ac-YVAD-CMK, 10 pmol), both in old (22
months) and young (4 months) rats, and observe the effect on contextual fear
conditioning paradigm. Ac-YVAD-CMK was delivered for 28 days icv through a
brain infusion cannula connected to an osmotic minipump (Alzet, Model 2004
pumping rate, 0.25ml/h; total volume 200ml) implanted subcutaneously. On day 20
the animals were exposed on contextual fear conditioning and the memory for
context was tested on day 22. Chronic infusion of a specific Caspase-1
inhibitor in aged rats resulted in an improvement in the memory for context.
|
|
Gorbunova, Vera |
59.
EXPRESSION OF HUMAN TELOMERASE (hTERT) DOES NOT PREVENT STRESS-INDUCED
SENESCENCE IN NORMAL HUMAN FIBROBLASTS BUT PROTECTS THE CELLS FROM
STRESS-INDUCED APOPTOSIS AND NECROSIS
Vera Gorbunova1 (P),
Andrei Seluanov1, and Olivia M. Pereira-Smith2
1Verna
and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, Houston, TX 77030
2Department
of Cellular and Structural Biology, Sam and Ann Barshop Center for Longevity and
Aging Studies, University of Texas Health Science Center, San Antonio, TX
78229-3900
Cells subjected to sub-lethal
doses of stress such as irradiation or oxidative damage enter a state that
closely resembles replicative senescence. What triggers stress-induced
premature senescence (SIPS) and how similar this mechanism is to replicative
senescence are not well understood. It has been suggested that stress-induced
senescence is caused by rapid telomere shortening resulting from DNA damage. In
order to test this hypothesis directly, we examined whether overexpression of
the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS.
We therefore analyzed the response of four different lines of normal human
fibroblasts with and without hTERT to stress induced by UV, -irradiation, and
H2O2. SIPS was induced with the same efficiency in normal and hTERT-immortalized
cells. This suggests that SIPS is not triggered by telomere shortening and that
nonspecific DNA damage serves as a signal for induction of SIPS. Although
telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were
more resistant to stress-induced apoptosis and necrosis. We hypothesize that
healing of DNA breaks by telomerase inhibits the induction of cell death, but
because healing does not provide legitimate DNA repair, it does not protect
cells from SIPS.
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Guerin, John C. |
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60. ROCKFISH: RETARDING AGING WITH "NEGLIGIBLE SENESCENCE"
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J.
Guerin
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Portland, OR
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Field observations have suggested for quite some time that certain fish,
turtles and invertebrates have extremely long maximum lifespan potential.
Age validation techniques have since confirmed these observations.
Negligible senescence is defined in part as no observable age-related
increase in mortality rate or decrease in reproduction rate after maturity,
and no observable age-related decline in physiological capacity or disease
resistance. Recent data compiled on rockfish (genus Sebastes) have
documented ages exceeding 200 years. However, many rockfish species
are reported under 30 years maximum observed lifespan, raising the
intriguing possibility of intra-species lifespan comparison. The
Centenarian Species and Rockfish Project has 14 total pilot studies.
The three studies reported in this poster are: “Rockfish liver microarrays
using existing 16,000+ zebrafish gene chips”, a collaboration between Glenn
S. Gerhard, Dartmouth Medical Center, and Renee Malek, TIGR (The Institute
for Genomic Research); “Heat Shock Protein comparison between younger and
older rockfish”, Marcelle Morrison-Bogorad, Associate Director, Neuroscience
and Neuropsychology of Aging Program, NIA; and “Electron transport
abnormalities and mitochondrial DNA mutations in rockfish heart tissue”,
Judd Aiken, University of Wisconsin-Madison.
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Halaschek-Wiener, Julius |
- 61. ANALYSIS OF LONG LIVED C. ELEGANS DAF-2 MUTANTS USING SERIAL
ANALYSIS OF GENE EXPRESSION
J Halaschek-Wiener*, S McKay, S Jones, M Marra, D Riddle, A
Brooks-Wilson
Genome Sciences Centre, BC Cancer Agency, 600 West 10th
Avenue, Vancouver, BC V5Z 4E6, Canada
Serial Analysis of Gene Expression (SAGE) was used to identify
longevity-associated genes in a long-lived daf-2 C. elegans mutant. The daf-2
gene encodes an insulin/IGF-1 receptor-like protein and mutations therein lead
to a 100% increase in mean life span. SAGE is a method to efficiently count
large numbers of mRNA transcripts by sequencing short tags. We prepared adult C.
elegans SAGE libraries of days 1, 6 and 10 for daf-2 and days 1 and 6 for
control worms. Analyses of gene expression profiles within daf-2 libraries and
between daf-2 versus control SAGE libraries (day 6 vs. 6 and day 10 vs. 6)
identified not only single genes but also whole gene families that were
differentially regulated. Furthermore, daf-2 mutants at day 6 show a strong
hypometabolic phenotype when compared to equally aged control worms that
diminishes at advanced age. Identified gene families regulate important
metabolic processes including stress response, lipid-, DNA/RNA-, protein, and
energy metabolisms as well as intracellular signaling and cell structure.
Identical expression patterns of various members of several gene families
emphasize the importance of these types of genes in longevity-related processes.
Our results suggest that long-lived daf-2 mutants are severely hypometabolic in
mid-life and we present evidence that specific gene families are involved in
these metabolic changes.
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Harper, James M. |
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62. SERUM IGF-I AND LIFESPAN IN CROSSES BETWEEN WILD AND LABORATORY STOCKS OF
MICE
J.M. Harper
(P)1, S.N. Austad1, R.A. Miller1, R.C.
Dysko2
1Department
of Pathology and Geriatrics Center, University of Michigan School of
Medicine, 2Unit for Laboratory Animal Medicine, University of
Michigan-
-
Using F2 hybrid stocks
constructed as a cross between two stocks of wild-derived mice (Id and Ma)
and the C57BL/6J inbred strain, we have assessed the ability of a suite of
life-history and physiological traits to act as predictors of lifespan, as a
prelude to mapping genes that may regulate lifespan in mice. The Id
parental strain, the Ma parental strain, and the (Id x B6)F2 hybrid stock
are all significantly longer-lived than a laboratory-derived control stock
(DC), generated as the offspring of (BALB/cJ x C57BL/6J)F1 females and (C3H/HeJ
x DBA/2J)F1 males (log-rank test p £ 0.02). The lifespan of the (Ma x B6)F2
hybrid stock is indistinguishable from the control (log-rank, p = 0.84).
Because serum IGF-I levels measured in 6-month old mice are lower in both
the Id and Ma stocks than in the DC control (p < 0.001), we tested to see if
IGF-I levels could predict life expectancy in mice of the two segregating F2
stocks. We found in each case that low IGF-I levels at 6 months of age
were associated with longer life span (r2 £ 0.19, p = 0.02). IGF-I
levels did not predict life span in either of the wild-derived parental
stocks Ma and Id, nor in the laboratory control DC mice (p > 0.2 for all).
These data suggest that endogenous levels of early life IGF-I may play a key
role in the regulation of lifespan, and that there are polymorphisms between
at least the Id and C57BL/6J stocks of mice that influence lifespan.
The QTL mapping of the loci responsible for these effects will provide
valuable insight into the hormonal and genetic control of mammalian aging.
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Kirchman, Paul A. |
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63. ANALYSIS OF THE INFLUENCE OF MITOCHONDRIAL DNA POINT MUTATIONS ON
LONGEVITY IN SACCHAROMYCES CEREVISIAE
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P.
Kirchman
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Harriet L. Wilkes Honors College, Florida Atlantic University, 5353 Parkside
Dr., Jupiter, FL 33458
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Evidence from several species points to mitochondrial DNA (mtDNA) as one
determinant of longevity. Interpretation of these studies is hampered
by nuclear DNA heterogeneity of the test subjects and, in some cases, by the
variable environments in which subjects live(d). To eliminate
variability in both the nuclear genome and the environment, the budding
yeast, Saccharomyces cerevisiae, was used to examine the influence of mtDNA
variation on longevity. Mitochondrial DNA’s containing
point mutations in the COX1, COX3, or CYTB gene were transferred to a yeast
strain that lacks mtDNA (rho0). The created strains vary only in the
point mutations on the mtDNA. These strains were analyzed for
variation in longevity relative to a control strain containing the
non-mutated mtDNA. No statistically significant variation in longevity
has been found between any of the strains tested. To influence
longevity, variation in mitochondrial DNA may need to be more extensive than
the single nucleotide mutations tested. Analysis of additional
mitochondrial mutant strains is ongoing and additional results will be
reported.
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Lee, Yongwoo |
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64. MICROARRAY ANALYSIS OF GENE EXPRESSION CHANGES IN
INTERLEUKIN-4-STIMULATED HUMAN VASCULAR ENDOTHELIAL CELLS
-
-
YW
Lee*, M Toborek
-
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Department of Surgery/Division of Neurosurgery, University of Kentucky
College of Medicine, Lexington, Kentucky 40536
-
-
Oxidative stress-mediated
inflammatory reactions within vascular endothelium have been implicated in
the development of age-related human diseases including atherosclerosis. We
have reported that interleukin-4 (IL-4) can induce the pro-oxidative and
pro-inflammatory pathways in human vascular endothelial cells. The cellular
and molecular regulatory mechanisms underlying this process, however, are
not fully understood. In the present study, we performed GeneChip microarray
analysis to investigate global gene expression patterns in human vascular
endothelial cells after treatment with IL-4 using the Affymetrix GeneChip®
Human Genome U133A Arrays, which contain more than 22,000 human genes. Our
results showed that mRNA levels of a total of 106 genes were significantly
up-regulated and 41 genes significantly down-regulated with more than a
twofold change. Majority of these genes are critically involved in the
regulation of inflammatory responses, apoptosis, signal transduction,
transcription factors, metabolism; functions of the remaining genes are
unknown. The changes in gene expression of selected genes related to
inflammatory reactions such as VCAM-1, E-selectin, MCP-1 and IL-6 were
verified by quantitative real-time RT-PCR and ELISA, respectively. IL-4
treatment also significantly increased the adherence of inflammatory cells
to endothelial cell monolayers in a dose-dependent manner. These results may
help determine the molecular mechanisms of action of IL-4 in human vascular
endothelium. In addition, a better understanding of IL-4-induced vascular
injury at the level of gene expression could lead to the identification of
new therapeutic strategies for atherosclerosis. (This work was supported by
the American Heart Association, Ohio Valley Affiliate and University of
Kentucky Microarray Facility Program)
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Li, Hong |
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65. GENDER DIFFERENCES IN NEURONAL CELL DEATH AFTER OGD INJURY AND NMDA-MEDIATED
EXCITOTOXICITY IN ORGANOTYPIC HIPPOCAMPAL CULTURES
-
-
Hong
Li*, MD, Katrin Andreasson, MD, Louise McCullough, MD, PhD
-
-
Department of Neurology, Johns Hopkins University, Baltimore, Maryland,
21287
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-
Background and Objective:
Increasing evidence has demonstrated striking sex differences in outcomes
after acute neurological injury. Females are less vulnerable to acute
insults associated with experimental cerebral ischemia. It is believed that
the greater neuroprotection seen in females is due to circulating estrogens.
However, this endogenous female neuroprotection may not be due solely to
hormonal influences. The objective of this study was to determine if there
are gender differences in neuronal cell death in organotypic hippocampal
cultures after oxygen glucose deprivation (OGD) injury and
M-Methyl-d-Aspartic Acid (NMDA)-mediated excitotoxicity. Methods:
Organotypic hippocampal slice cultures were prepared from individual
postnatal day 8 Sprague–Dawley rat pups. Gender genotyping was accomplished
by PCR analysis from pup tail DNA. The cultures were exposed to 10 mM NMDA
for 1 h or subjected to OGD for 45 min, in presence or absence of 30 mM 7NI
(7 Nitroindazole; a selective nNOS inhibitor), or 10nm 17b-estradiol (E2)
for 7 days prior to OGD. Neuronal death was quantified with propidium iodide
(PI). Data were analyzed by one-way ANOVA followed by Newman–Keuls post hoc
test. Results: Baseline neuronal cell death in control, 7NI, and E2 treated
groups was equivalent in males and females. Cell death markedly increased
after stimulation with NMDA or OGD (P < 0.001) in both groups, but there was
a significant increase in cell death in male cultures (P < 0.001 ~ 0.05)
after both OGD and NMDA treatment. Treatment with7NI and E2 reduced neuronal
damage induced by NMDA and OGD (P < 0.001 ~ 0.05). Interestingly, this
effect was more notable in males than in females after OGD injury (P <0.01).
Conclusions: There is a consistent gender difference after OGD and NMDA
toxicity, demonstrating neuroprotection in female cells. Dimorphisms in cell
survival may underlie enhanced neuronal survival (or decreased apoptosis) in
female brain.
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Lopez-Cruzan, Marisa |
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66. ROLE OF MITOCHONDRIAL CASPASE-2 ACTIVITY IN OXIDATIVE STRESS-INDUCED
APOPTOSIS
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Marisa Lopez-Cruzan, Victoria Centonze, and Brian Herman
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Cellular and Structural Biology Department, University of Texas Health
Science Center at San San Antonio. 7703 Floyd Curl Drive, San Antonio, Texas
78229-3900
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The role of mitochondrial
caspase-2 in oxidative stress-induced apoptosis was examined by first
determining if mitochondria contain oxidative stress-inducible capase-2
activity and second, what the effect of loss of mitochondrial caspase-2
would be on mitochondrial-specific oxidative stress-induced apoptosis.
Oxidative stress-induced mitochondrial-specific caspase-2 activation and
apoptosis were assayed in murine NIH-3T3 fibroblasts or fibroblasts obtained
from caspase-2 knockout mice using Florescence Resonance Energy Transfer
(FRET)/optical microscopy (mitochondrial caspase-2 activity) and uptake of
propidium iodide (apoptosis). Mitochondrially targeted FRET caspase-2 fusion
protein containing the preferential caspase-2 substrate flanked by CFP and
YFP, as donor and acceptor fluorophores respectively, were used as an
in-situ sensor of mitochondrial caspase-2 activity. FRET efficiency,
measured using acceptor photobleaching, demonstrated a time-dependent loss
of FRET after exposure of cells to tert-butyl hydroperoxide, indicative of
mitochondrial specific caspase-2 activation. Inhibition of complex I or III
of the mitochondrial electron transport chain is known to induce reactive
oxygen species (ROS) within the mitochondria. Treatment with the complex I
inhibitor rotenone, the complex III antimycin A or staurosporine (STS), a
non-ROS inducer of apoptosis, produced a significant amount of cell death in
fibroblasts isolated from wild type mice. However, cells isolated from
caspase-2 deficient mice were highly resistant to rotenone and antimycin A,
but not STS-induced apoptosis. These results indicate that mitochondria
contain oxidative stress-inducible caspase-2 and that caspase-2 activation
is required for mitochondrial oxidative stress-induced apoptosis.
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Mamczarz, Jacek A. |
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67.
PATTERN OF GENE EXPRESSION IN LIVER OF YOUNG AND AGED RATS AFTER SHORT-TERM
DIET RESTRICTION OR 2-DEOXY-D-GLUCOSE INJECTION: cDNA
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Jacek Mamczarz*, Min Zhu, Jonna Bowker, Kara Duffy, Donald Ingram
-
-
Lab. Exp. Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock
Drive, Baltimore, MD 21224
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Diet restriction (DR) in
rodents increases lifespan, reduces age-related disease and pathology,
increases stress responses, and maintains better function later into life
compared to conventional ad libitum (AL) feeding. We have been investigating
DR regimen and also DR mimetics that stimulate stress response pathways that
are activated by DR. By inhibiting glycolysis, feeding or injection of
2-deoxy-D-glucose (2DG) has been proposed as a DR mimetic. In the current
study we investigated the pattern of gene expression using cDNA microarray
in liver of young (4 mo) and aged (26 mo) male Fischer-344 rats subjected to
3 weeks of 40% DR or 2DG injections once a day. 40% DR reduced initial body
weight (BW) around 15% in young and 14% in old rats, respectively. 2DG in
the dose of 125 mg/kg attenuated food intake and developmental gains in BW
in young rats, while in aged rats 2DG decreased BW and food intake below
initial levels. 2DG at a dose of 250 mg/kg also decreased BW and food intake
in both young and aged rats; however, in aged rats the decrease was at the
level of DR. In general, DR and 2DG treatment up-regulated more genes
in aged rats than recorded in young rats (around twice in DR, 6 times in
2DG-125, and 3.5 times more in 2DG-250). Down-regulation of gene expression
was similar in young and old DR rats, twice higher in young 2DG-125, and
twice lower in young 2DG-250, respectively, compared to aged rats. DR and
2DG-125 shared much more common patterns of gene expression in aged rats
than in young, but for 2DG-250 there was a generally common pattern shared
in both age groups. Analysis of gene expression patterns can be used to
assess potential effectiveness of DR mimetics.
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