AGING NEURON MITOCHONDRIAL FUNCTION AND REJUVENATION WITH ESTROGEN OR MITOSIS

 

G. Brewer, J. Nash, T. Jones, J. Reichensperger, M. Parihar

 

PO Box 19626, Springfield, IL 62794-9626

 

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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ACE inhibition intervention: Implications for improving age-related declines in physical performance and longevity.

 

CS Carter, M Cesari, M Pahor

 

Wake Forest Univeristy School of Medicine, J. Paul Sticht Center on Aging, 1 Medical Center Blvd. Winston-Salem NC, 27157

 

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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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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THE EFFECTS OF 6-MONTHS OF CALORIE RESTRICTION ON BIOMARKERS OF AGING IN NON-OBESE HUMANS.

 

Leonie K Heilbronn*, Eric Ravussin and the Pennington CALERIE group.

 

Pennington Biomdeical Research Center, 6400 Perkins Rd, Baton Rouge, LA

 

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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THE PRESENCE OF AN OPTIMAL DOSE RANEGE MAY EXPLAIN DISCREPANCIES IN THE EFFECTS OF (-)DEPRENYL (D) ON SURVIVALS OF ANIMALS IN PAST REPORTS

 

K.Kitani*1, S.Kanai2, K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4

 

Tokyo Metropolitan Institute of Gerontology

 

THE PRESENCE OF AN OPTIMAL DOSE RANGE MAY EXPLAIN DISCREPANCIES IN THE EFFECTS OF (-)DEPRENYL (D) ON SURVIVALS OF ANIMALS IN PAST REPORTS

 

K.Kitani*1, S.Kanai2, K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4

*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

 

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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AGE-ASSOCIATED CELLULAR STRESS IN HSP-40 MOLECULAR CHAPERONE MUTANT MICE

 

S Knoblaugh, J Morton, G Moore-Sanders, A MacAuley, W Ladiges*

 

Department of Comparative  Medicine, University of Washington, Seattle, WA 98195

 

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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PHENOTYPIC, FUNCTIONAL AND GENETIC PROFILES OF THE AGING T- LYMPHOCYTES IN PRIMATES AND THEIR ALTERATION BY CALORIC RESTRICTION

 

Janko Nikolich-Zugich, Jessica Warner, Bree Fisher, Dragana Nikolich-Zugich

and Ilhem Messaoudi

 

Vaccine and Gene Therapy Institute and the Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA

  

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.

 

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.

 

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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cAMP forms a neurochemical correlate of tinnitus

 

A, Shaikh; P, Finlayson

 

Department of Otolaryngology, Wayne State University, Detroit, MI 48201 

 

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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HIPPOCAMPAL GH AND IGF-1 EXPRESSION IN GH-DEFICIENT MICE

 

Liou Y. Sun*, Khalid Al-Regaiey, Michal M. Masternak, Jian Wang and Andrzej Bartke

 

Geriatrics Research, Department of Medicine and Department of Physiology

Southern Illinois University, Springfield, IL 62794, USA

 

Liou Y. Sun*, Khalid Al-Regaiey, Michal M. Masternak, Jian Wang and Andrzej Bartke

Geriatrics Research, Department of Medicine and Department of Physiology

Southern Illinois University, Springfield, IL 62794, USA

 

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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PASSIVE AMYLOID IMMUNOTHERAPY CLEARS AMYLOID AND TRANSIENTLY ACTIVATES MICROGLIA IN A TRANSGENIC MOUSE MODEL OF AMYLOID DEPOSITION

 

Donna M. Wilcock1, Amyn Rojiani2, Arnon Rosenthal3, Jennifer Alamed1, David Wilson1, Nedda Wilson1, Melissa J. Freeman1, Marcia N. Gordon1, Dave Morgan1.

 

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.

3: Rinat Neuroscience Corp. 3155 Porter Drive, Palo Alto, California, 94304, USA.

 

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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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 &#947;/&#950; 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.
 

44.  AGING RETARDATION BY CALORIE RESTRICTION: ROLE OF REGULATORS OF ENERGY METABOLISM.

 

R. Anderson (1), J.Barger (1), T. Pugh (1), S. Park (2), T. Prolla (2) and R. Weindruch (1).

 

(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.

 

University of Wisconsin Madison, GRECC, VA Hospital, Madison WI 53705, USA.

 

     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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45.  REGULATION OF THE RENAL VITAMIN D 1ALPHA-HYDROXYLASE CYTOCHROME P450(CYP27B1) BY DIETARY PHOSPHORUS CHANGES WITH AGE

 

H.J. Armbrecht*, M.A. Boltz

 

Geriatric Center, St. Louis VA Medical Center, St. Louis, MO  63125

 

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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46.  SENEX: INTEGRATING GERIATRICS AND MOLECULAR GERONTOLOGY

 

S Ball, A Nhalil and V Mah

 

Department of Veterans Affairs, Greater Los Angeles Healthcare, 11301 Wilshire Boulevard, GRECC 11G, Los Angeles CA 90073

 

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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47.  THE AGE-RELATED ACCUMULATION OF DOLICHOL IN TISSUES SATISFIES ALL CRITERIA TO BE QUALIFIED A BIOMARKER OF AGING

 

E. Bergamini, G. Cavallini, A. Donati, Z. Gori, A. Manfrini, I. Parentini

 

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.

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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

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.

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.

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)

 

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50.  GENE EXPRESSION CHANGES IN NEURODEGENERATIVE MICE AND CULTURED CELLS
 

B.A. Citron*^1,2, A. Surguchov^3,4, I. Surgucheva^3,4, and B. W. Festoff<sup>3,4,5

1</sup>Lab 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.

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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.

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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.

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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.

 

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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.

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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. 

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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)

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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

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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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60.  ROCKFISH: RETARDING AGING WITH "NEGLIGIBLE SENESCENCE"

 

J. Guerin

 

Portland, OR

 

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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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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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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63.  ANALYSIS OF THE INFLUENCE OF MITOCHONDRIAL DNA POINT MUTATIONS ON LONGEVITY IN SACCHAROMYCES CEREVISIAE

 

P. Kirchman

 

Harriet L. Wilkes Honors College, Florida Atlantic University, 5353 Parkside Dr., Jupiter, FL 33458

 

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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64.  MICROARRAY ANALYSIS OF GENE EXPRESSION CHANGES IN INTERLEUKIN-4-STIMULATED HUMAN VASCULAR ENDOTHELIAL CELLS

 

YW Lee*, M Toborek

 

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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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

 

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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66.  ROLE OF MITOCHONDRIAL CASPASE-2 ACTIVITY IN OXIDATIVE STRESS-INDUCED APOPTOSIS

 

Marisa Lopez-Cruzan, Victoria Centonze, and Brian Herman

 

Cellular and Structural Biology Department, University of Texas Health Science Center at San San Antonio. 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900

 

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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67.  PATTERN OF GENE EXPRESSION IN LIVER OF YOUNG AND AGED RATS AFTER SHORT-TERM DIET RESTRICTION OR 2-DEOXY-D-GLUCOSE INJECTION: cDNA

 

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

 

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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68.  BLUEBERRY STUDY WEB SITES FOR COGNITIVE PERFORMANCE MEASUREMENT APPEAR SUFFICIENTLY PRECISE TO DETECT A 0.1% CHANGE IN PERFORMANCE DURING AGING

 

R. Martin*, MMT Corp., Sherman, CT 06784, R.J. Coppings, Lane College, Jackson, TN 38301, K.E. Gerstmann, NY, NY 10014, William Holme, Bethel, CT 06801, K. Hull, New Fairfield Senior Center, New Fairfield, CT 06812, J.A. Joseph, Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, A.C. Kokesh, Charleston, WV 25301, B. Kristal, Weill Medical College-Cornell University Medical Center, NY, NY 10021 and Burke Medical Research Institute, White Plains, NY 10605, M. Luzi and T. Millard, Sherman IGA Supermarket and B. Sachs, HR Herbs, Sherman, CT 06784, H.A. Raphaelson, Mansfield Senior Center, Mansfield, CT 06268, A. Pruchnicki, Mount Sinai Medical Center, NY, NY 10029, R. Schnoll, Brooklyn College, Brooklyn, NY 11210, and A. Wetherell, Defence Science & Technology Laboratory, UK. Contact email: BlueberryStudy@aol.com

 

1 Barlow Farm Road, Sherman, Connecticut 06784

 

To evaluate methods for reducing age-associated cognitive decline during 3-year study periods, measurement precision should detect with 95% confidence differences of 0.1% or more between treatment and control groups having ~1% average annual decline. To develop such high precision for our 2000, 2002 and 2004 blueberry studies, self-calibrating web sites were developed for online measurement of 4-choice and 2-choice decision speed and immediate and delayed word recall (patent no. 6,712,615). These sites enable participants to measure their performance repeatedly and thereby obtain very precise average measures of these aspects of cognition. Annual test-retest reliability values for decision speed during 2000-2004 ranged from 0.95 to over 0.99. Results collected from these sites were used to develop computer simulations which indicate that both our blueberry study design, with two major types of performance measurement (decision speed and recall), and Alex Comfort's (1970) approach to measuring human aging, with 60 different types of physiological and performance measurement, are capable of identifying agents that can reduce a 1% rate of annual decline to 0.95% for studies involving 10,000 or more participants. The major problem of accurately monitoring food and supplement intake over long study periods can be solved in part with supermarket purchase logs and weekly food sharing and spoilage reports. Purchase logs received to date indicate that 18 participants purchased 2,845 items at the Sherman IGA Supermarket with electronic discount cards that provide automatic "health discounts" of ~40% on blueberries and 35 other health-related foods. Automatic discount cards can significantly aid recruitment and retention and provide a high-density data stream to help examine whether local ad campaigns or health news reports cause unbalanced diet or health supplement shifts in either treatment or control groups. Blueberrystudy.com measurement and diet-tracking software are expected to be available to other research groups later this year.

 

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69.  THE EFFECT OF GROWTH HORMONE RECEPTOR KNOCKOUT AND CALORIC RESTRICTION ON EXPRESSION OF GENES RELATED TO INSULIN SIGNALING IN MICE

 

Michal M Masternak, Khalid A. Al-Regaiey, Michael S. Bonkowski, Andrzej Bartke

 

Departments of Internal Medicine and Physiology, Geriatrics Research, Southern Illinois University School of Medicine, 801 N. Rutledge, Springfield IL 62794

 

Growth hormone receptor knockout (GHR-KO) mice are long-lived, hypoinsulinemic and hypoglycemic and exhibit enhanced sensitivity to injected insulin (Ins). Using Real-time PCR (RT-PCR) we analyzed hepatic and muscle levels of insulin receptor (IR), insulin receptor substrate 1 (IRS1), IRS2, peroxisome proliferators-activated receptor gamma (PPARg) glucose transporter 4 (GLUT4), insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) mRNA expression in GHR-KO and in normal (N) animals from the same strain. We have also analyzed gene expression in GHR-KO (KO) and normal mice subjected to 30 % caloric restriction (CR). In the liver, gene expression was affected by genotype but not diet. The expression of IR, IRS1 and IRS2 mRNA was increased (P<0.0044, P<0.024, P<0.0003, respectively) in GHR-KO animals. PPARg mRNA expression was also increased in KO mice (P<0.0042). However, IGF1 mRNA expression in the liver was decreased in GHR-KO mice as expected (P<0.0001). Different effects were detected in the skeletal muscles of these animals. The mRNA expression was not affected by genotype but not by CR. The expression of IR, IRS1 and IRS2 was decreased by CR in N (P<0.02, P<0.0034, P<0.025, respectively) and in KO mice (P<0.0005, P<0.0003 and P<0002, respectively). PPARg mRNA was decreased by CR in normal and KO animals (P<0.0096 and P<0.0009, respectively). The expression of GLUT4 mRNA in muscle showed significant down regulated by CR in both N and KO mice (P<0.0044 and P<0.0003 respectively). The expression of IGF1 mRNA was decreased by CR in normal and KO animals (P<0.0053 and P<0.0001). Moreover, the expression of IGF1 mRNA was reduced in KO vs. normal mice (P<0.0001). In contrast, the expression of IGF1R mRNA was not affected by CR in N mice, but was up regulated in KO in comparison to normal mice (P<0.014) and reduced by CR in KO mice (P<0.0005).

Supported by NIA

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70.  THE INFLUENCE OF SODIUM INTAKE ON CARDIOVASCULAR RESPONSES IN RHESUS MONKEYS

J. A. Mattison, A. Bagrov, H. Spurgeon, P. Pullen, M. A. Lane, G. S. Roth, D. K. Ingram, E. G. Lakatta

NIA, NIH Animal Center, Poolesville, MD 20837

Although there is considerable evidence linking salt intake to hypertension, how this dietary variable is involved in remodeling of the vascular wall to affect arterial stiffness is still unknown. The effect of an incrementally increased salt load on vascular stiffness and modulation of this response by production of an endogenous ligand was studied in nine old normotensive male rhesus macaques (mean age at start = 19.1 +/- 0.67 years). Serial measurements of aortic pulse wave velocity (PWV), blood pressure (BP), urine and sodium output, and marinobufagenin (MBG) were made before and following each stepwise increase in dietary sodium chloride (NaCl) from 0.8% to 6.0% of daily intake. Sodium excretion significantly increased with each incremental increase in dietary intake. PWV significantly increased as a direct nonlinear function of Na intake independent of changes in BP.
Although there was considerable variability among monkeys in BP response, there was an immediate pressure increase that was attenuated at higher dietary Na loads. MBG excretion was positively correlated to Na excretion at baseline and with a 1.7% and 2.6% NaCl diet. This direct relationship was not evident at intakes of 3.5% and 6% NaCl. These results suggest that old monkeys are susceptible to NaCl dependent vascular changes that can be partially compensated by increased MBG to enhance Na excretion.

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71.  ABNORMAL SPLICING OF LMNA CAUSES PROLIFERATIVE DEFECTS LEADING TO A HUTCHINSON GILFORD PROGERIA SYNDROME PHENOTYPE IN MICE

 

Leslie C. Mounkes*, Lidia Hernandez, Serguei Kozlov, Colin L. Stewart

 

Cancer & Developmental Biology Laboratory, National Cancer Institute—Frederick, MD

 

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder resulting in the acceleration of age-related phenotypes, including shortened stature, craniofacial disproportion, parchment thin skin, alopecia, and osteoporosis, with death predominantly due to premature atherosclerosis in the early teens1.  We derived mice carrying a splicing defect in the Lmna gene and found that mice homozygous for the mutation display defects remarkably similar to progeria1.  Lmna encodes the A-type lamins, major components of the nuclear lamina with putative functions in nuclear structure, chromatin organization, gene regulation, and intracellular signaling.  Mutant mice (LmnaHGPS) showed marked reduction in growth rate and died by 4 weeks of age.  Pathology of the heart, bone, muscle and skin are consistent with phenotypes associated with aging of these tissues.  The Lmna mutation resulted in nuclear morphology defects and decreased lifespan of homozygous fibroblasts from postnatal, but not embryonic tissues, suggesting a developmentally regulated mechanism of premature cell death.  Furthermore, in a background deficient for p16/p19ARF, the premature death of the mutant progeric cells is rescued in vitro, suggesting the nuclear lamina plays a role in the control of cell growth, possibly through pathways involving pRB and/or p53.  Muscle myoblast cultures from progeric mice displayed a slower response to differentiation cues and made myotubes, which were shorter with disorganized nuclei compared to myotube differentiation of wild type myoblasts.  Defects in proliferation and differentiation could contribute to the incomplete development of muscle and other mesenchymal tissues in progeria.

 

1LC Mounkes, S Kozlov, L Hernandez, T Sullivan & CL Stewart.   A progeroid syndrome in mice is caused by defects in A-type lamins.  Nature 425: 298-301 (2003).

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72.  PHYSIOLOGICAL RESPONSES TO VERY MILD AND STANDARD LEVELS OF CALORIC RESTRICTION IN RATS

 

J. M. Overton, A.D. Parsons, S.A. Evans

 

Department of Nutrition, Food and Exercise Sci., Florida State Univ., Tallahassee, FL 32306-4340

 

Very mild caloric restriction (CR) is commonly used as a control intervention to assess the effects of more substantive CR on lifespan.  The purpose of this study was to compare the physiological effects of short-term, very mild CR to standard levels of CR in rats.  Male FBNF1 rats weighing about 360-380 g were instrumented with telemetry devices for measurement of mean arterial pressure (MAP) and heart rate (HR) and continuously housed in room calorimeters for assessment of oxygen consumption (VO2) and respiratory quotient (RQ).  Rats were acclimated to thermoneutral (TMN; 30°C) conditions prior to imposing very mild (5-10%) or standard levels of caloric restriction (CR; 40% restriction of ad lib) for two weeks.  Controls continued to consume food ad libitum (5-6 rats/group).  Over the two-week period, controls gained 15 grams, while mild CR reduced weight by about 3 grams and 40% CR reduced weight by 30 grams.  As expected, 40% CR significantly reduced body weight, VO2, HR and RQ; while increasing HR variability.  Unexpectedly, mild CR, which had no effect on body weight, RQ and HR variability, produced significant reductions in HR and VO2.  Weight maintenance produced 50% of the physiologic effect associated with 40%CR.  The results suggest that in rats housed at thermonuetrality, the cardiovascular and metabolic responses to CR are engaged by very mild stimulus that does not require weight loss.

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DC Paik

Department of Ophthalmology, Columbia University, New York, New York and Department of Surgery, North General Hospital. New York, New York

Introduction: Increased collagen cross-linking is one of the hallmarks of human aging and can contribute to the functional decline of aging organ systems. Nitrite ion, whose sources include inflammation and smoking, can mediate damaging nitration, nitrosation, and hydroxylation reactions. The in vitro neutral pH reaction of nitrite with collagen produces changes that mimic collagen aging and include increased cross-linking, UV absorbance, and fluorescence. The current study was undertaken in order to identify covalent collagen cross-links produced specifically by reaction with nitrite. Methods: Model studies were performed using gelatin. Samples (10mg/ml) were incubated with 0 to 200mM sodium nitrite at neutral pH for up to 10 days. The reaction was stopped with ammonium sulphamate. UV/vis and fluorescence spectra were obtained on the protein mixtures. Cross-link enrichment was performed on acid hydrolysates using Skinner’s (1982) cellulose mini-column method. Isodesmosine was added as an internal standard (0.5ug/mg) pre-hydrolysis. The water fraction was collected and analyzed by HPLC (C18) with diode array and fluorescence detection. Gradient conditions using 20mM heptafluorobutyric acid and 2-20% acetonitrile over 60 min were employed. Pump speed was 1ml/min. Results: Nitrite modification of gelatin produced spectroscopic changes similar to those observed previously for collagen types I and IV and include increased UV absorbance ca. 350nm and increased fluorescence with excitation peaks ca. 290 and 340, and an emission peak ca. 430. Cross-link analysis revealed 6 unique products of the reaction as detected by UV at 280nm. Five of the compounds also showed fluorescence (ex290/em410). The unknowns elute prior to pyridinoline and isodesmosine. Conclusions: This is the first identification of cross-link compounds specific to the nitrite/collagen reaction. The detected compounds may serve as biomarkers of the reaction in future studies.

74.  NOREPINEPHRINE RELEASE IN CEREBELLUM DURING DELAY CLASSICAL EYEBLINK CONDITIONING DECLINES IN AGED RATS

D. A. Paredes, A.N. Samec, M. Fister and P.C. Bickford

Center for Aging and Brain Repair, USF, 12901 Bruce B Downs Blvd, Tampa, Fl 33612

Delay Classical Eyeblink Conditioning is an important model of associative, cerebellar dependent learning. Aged animals and humans show deficits in learning on this paradigm. Our laboratory has been investigating the role of norepinephrine (NE) in this paradigm. We have demonstrated that blocking either the &#946;-noradrenergic receptor or protein kinase A with local infusions into cerebellar lobule HVI and interpositus nucleus can interfere with learning. The goal of this study was to determine whether NE release is observed in HVI during delay eyeblink conditioning and if this is altered in aged rats. In vivo microdialysis coupled to eyeblink conditioning was performed on three month old or twenty month old F344 rats. Dialysate samples were collected every 10 minutes. After 5 baseline samples were collected rats received 50 training trials (45 paired with a 12 psi airpuff and 5 tone only [3kHz 85 dB] trials), or Rescorla type pseudo conditioning. Rats showed a significant release of NE during and after training, for young rats with 47.3 ± 5.0 nM of NE released as a max value reached 30 min after training started; while aged rats showed a max of NE release of 23.2 nM at 80 min after training started. This data clearly shows a decline on NE release in aged rats compared with young, this could explain some of the deficiencies of aged animals to learn new motor tasks which could be linked with the NE reuptake system deficiencies.
Support Contributed By: NSF 019674

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75.  Mitochondrial function, organelle degradation, and cataract in lenses from aging: normal mice, GPX-1 KO mice, and Ghr-KO mice.

B Pendergrass,P Penn, A Bartke, H Van Remmen, and N Wolf

1Department of Pathology, University of Washington School of Medicine, Department of Pathology, University of Washington School of Medicine, Seattle, WA. 98195. USA

Cataract incidence was increased dramatically in old mice of each genotype relative to genotype matched young mice. Old Glutathione Peroxidase-1 knock out mice (GPX-1 KO), had an increased cataract incidence relative to age-matched controls. Older long lived growth hormone receptor knock out mice (GHr-KO mice) had significantly less cataract than age-matched controls. Mitochondrial oxidative cycling (a measure of O2 use) in lens epithelial cells (LECs) on the surface of mouse lenses was determined from the rate of oxidation of reduced Mitotracker red (H2MTR) to Mitotracker Red (MTR). This was significantly reduced in old compared to young control mice lenses. It was even more reduced in old GPX-1 KO mice lenses compared to age-matched controls. The reduction in oxidation was apparent whether measured relative to cell surface area, or to DNA in the cells. We also found a parallel decrease in total mitochondrial mass in old mouse LECs. The relative mitochondrial membrane potential (MMP) in the LECs, was determined from the fluorescence ratio of the vital stains Mitotracker Red (sensitive to MMP) to Mitotracker Green (sensitive to mitochondrial mass). This MMP ratio was slightly higher in older LECs than young LECs. Young GHr-KO mice had significantly reduced MMP relative to young controls, possibly indicating mild uncoupling of oxidative phosphorylation. We also monitored the degradation of DNA and mitochondrial proteins using specific fluorescent stains in internalized LEC fiber cells. The lens fiber cells in the interior of the lens from old mice were deficient in the ability to degrade nuclear and mitochondrial organelles. Cataractous areas of the lenses were especially heavily stained for DNA and mitochondrial debris. Dichlorofluorescein (DCF) staining of ROS was also significantly increased in lens fibers from old mice and this was frequently associated with cataractous foci in old lenses.

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E. Pepper, B. Yeiser, MF Goodman, and SE Finkel

Department of Biological Sciences, SHS 172, University of Southern California, Los Angeles, CA. 90089-1340

Escherichia coli encodes three SOS-induced DNA polymerases: pol II, pol IV, and pol V. We show here that each of these polymerases confers a competitive fitness advantage during the stationary phase of the bacterial life cycle, in the absence of external DNA-damaging agents known to induce the SOS response. When grown individually, wild-type and SOS pol mutants exhibit indistinguishable temporal growth and death patterns. In contrast, when grown in competition with wild-type E. coli, mutants lacking one or more SOS polymerase suffer a severe reduction in fitness. These mutants also fail to express the “growth advantage in stationary phase” (GASP) phenotype as do wild-type strains, instead expressing two additional new classes of GASP. These polymerases contribute to survival by providing essential functions to ensure replication of the chromosome and by generating genetic diversity.

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77.  NANOG KEEP EARLY EMBRYO AND TROPHOBLASTIC INVINCIBILITY FROM AGING

Quanjian Yan (P), Xiangmei Chen, Yumei Zhang, Daqiang Li, Quan Hong, Bo Fu.
State Key Laboratory of Nephrology, Kidney Department of 301 Hospital Fuxing
Road 28. Haidian, 100853, Beijing. P.R.China.

In our co-culture system, we found that early embryo and trophoblastic cell of KM mouse can invade malignant tumor cells and keep youth. Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells. So we investigate whether Nanog is necessary for early embryo and trophoblastic cell keep invincibility invasion and youth.  Morphological analysis showed that mouse blastocysts have the invincibility of adhesion, migration and invasion after implant in many normal cells or tumor cells. The normal cell strains include L929 and NIH3T3 mouse fibroblasts, 9HTE human tracheal epithelial cells and the ECV-304 human umbilical vein endothelial cells. The malignant tumor cell strains include differently histological origins, the HNE1 human nasopharyngeal carcinoma cells include BGC-823 and SGC-7901, human kidney carcinoma cells 786-0, human bladder carcinoma cells BIU-87 and BADM-60, human osteoblastoma cells Ros17/2.8 and Saos-2, human endometrial carcinoma cells RL95-2, rat breast carcinoma cells SHZ-88, human hepatocarcinoma cell strains HepG2, SMMC-7721, QGY-7703 and MHCC97-H; human breast carcinoma cell strains T47D, MCF-7, ZR75-30, Bcap-37, MDA-MB-231 and MDA-MB-435s. After 72 h of co-culture, integrin alpha V, FAK, MMP-9, Cyclin D1, CD44 protein and Ki-67 mRNA were lower expressed in the trophoblastic cells in the group without the inner cell cluster or Nanog expression embryonic stem (ES) cell cluster with immunocytochemical and in situ hybridization than that with Nanog expression ES cell cluster. And the outgrowth area in the group without the inner cell cluster or Nanog null ES cell cluster is smaller than that with the inner cell  or Nanog expression ES cell cluster (P>0.05).

These results strongly suggest that Nanog activation keep the inner cell or ES cell cluster from aging, promote trophoblastic adhesion, migration and invasion (etc).

78.  GENETIC DISSECTION OF DIETARY RESTRICTION

 

Brad A. Rikke*, Thomas E. Johnson

 

University of Colorado at Boulder

 

Dietary restriction (DR) is the most validated method of extending health and longevity n mammals, extending the life span of rodents by as much as 50%.  Many physiological responses to DR have been put forth as causal factors underlying DR’s benefits.  As a first step towards testing some of these factors, we’ve examined 83 strains of mice for significant differences in early responses to DR.  These responses include lowered body temperature, lowered body weight, reduced growth rates (tail and hair), and reduced motor activity (runwheel and home cage).  We’ve also examined the extension of female fertility after extended DR.  For each response, we find that there is a phenomenal amount of strain variation, with surprisingly little covariation among responses.  These findings could be used to critically test whether any of these responses covary with DR-induced longevity.  Responses validated to covary with life extension could be used as a tool to screen for mutations that are likely to affect DR-induced longevity.  In addition, we are mapping quantitative trait loci (QTLs) specifying each response by studying strains from the largest murine panel of recombinant inbred strains ever developed, the LXS panel of 77 strains.  This mapping makes it possible to subsequently identify the genes underlying these QTLs by positional cloning, ultimately leading to a better understanding of the molecular mechanism by which DR extends mammalian life span.  (Supported by the Ellison Medical Foundation and NIH R01 AA11984)

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79.  ANTIOXIDANT CAPACITY OF HONEY TYPES ACCORDING TO BOTANICAL AND ENTOMOLOGICAL ORIGIN

E. M. Pérez-Pérez¹,  A. J. Rodriguez-Malaver¹(P), P. Vit^{2  1}Lab. de Bioquímica Adaptativa, Dep. de Bioquímica, Fac. de Medicina; ²Dep. Ciencia de los Alimentos, Fac. de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida 5101, Venezuela.

At the present, there is overwhelming evidence that free radicals cause oxidative damage to lipids, proteins, carbohydrates and nucleic acids.  Therefore, reactive oxygen species (ROS) such as O2-&#9679;, OH&#9679;, or lipid peroxyl radical (LOO&#9679;) might lead to many biological complications including carcinogenesis, mutagenesis, aging,

Meliponinae bee subfamilies, on superoxide anion and hydroxyl radical. We found that honey samples inhibited the formation of superoxide anion and hydroxyl radical, under a range from 86 to 25% depending on the sample. This inhibition capacity was, for some samples, higher than quercetin and melatonin. On the other hand, we measured the antioxidant activity (AOA) values of honey samples in comparison to uric acid at 1 mM. In this case, honey samples had AOA values between 0.32 and 0.84 Mm which were considerably high as compared to melatonin and quercetin.

80.  ANTIOXIDANT CAPACITY OF VENEZUELAN BLACKBERRY (RUBUS BOGOTENSIS KUNTH) WINE

Pérez-Pérez EM¹, Rodríguez-Malaver AJ¹(P), Vit P²    

¹Laboratorio de Bioquímica Adaptativa, Departamento de Bioquímica, Facultad de Medicina, ²Departamento Ciencia de los Alimentos, Facultad de Farmacia y Bioanálisis; Universidad de Los Andes, Mérida 5101, Venezuela

The reported health benefits of moderated wine drinking, especially in theprevention of chronic diseases associated with oxidative stress, may be related to the antioxidant activity of flavonols; quercetin and myricetin, which can readily reduce free radical by donating an unpaired electron.  Rubus Bogotensis Kunth (Rosaceae) is the blackberry cultivar in the Venezuelan Andes, where a fermented product of the berries is marketed as blackberry wine. We studied the antioxidant capacity of 12 samples of blackberry wine produced in Mérida state, on the formation of superoxide anion and hydroxyl radical. Wine samples inhibited the formation of superoxide anion and hydroxyl radical under a range from 17% to 53%. For some wine samples, this inhibition capacity was close to those of quercetin (71%, for superoxide anion, 53% for hydroxyl radical) and melatonin (79%, for superoxide anion, 54%, for hydroxyl radical). We also measured the antioxidant activity (AOA) value of wine samples in comparison to 1 mM uric acid. Wine samples had AOA values between 0.95 and 0.80 mM, which were, for some samples, actually high if were compared to those of melatonin (0.83 mM) and quercetin (0.86 mM), used as reference in this study. This antioxidant activity can be due to the phenolic content of wine samples, which are very efficient scavengers of free radicals because its molecular structure includes an aromatic ring with hydroxyl groups containing mobile hydrogens, and they have the capacity to reduce or chelate divalent ions which catalyze redox reactions.

81.  HONEY AND LUTEOLIN EFFECT ON STREPTOZOTOCIN, SELENITE AND OSMOTIC INDUCED CATARACTS

P. Vit¹,5, R. De Jesus², M. Gudiño³,  E. Perez⁴, A. Rodriguez-Malaver⁴(P), A. Melendez⁵

¹Dep. Food Science, Fac. Pharmacy, ² Dep. Biology, Fac. Sciences, ³BIOULA Animal House, ⁴Dep. Biochemistry, Fac. Medicine, Universidad de Los Andes, Mérida, Venezuela, ⁵Dep. Physiology, Fac. Medicine, National University of Singapore, Singapore

Ocular cataracts are of epidemiological interest in human populations. The intraocular lens implantation is a successful technique to improve vision by removing the damaged ocular lens. However, a medical therapy to prevent, retard or reverse lens opacity is not available. In the Neotropics, stingless bee (Meliponinae) honey eyedrops are instilled to treat cataracts.  Trigona Tetragonisca angustula angustula honey is currently used for this purpose, therefore this honey type was harvested from a hive and refrigerated until use. Antioxidant capacity, luteolin and luteolin derivatives were detected in previous studies with honey extracts. From a screening with commercial flavonoids, luteolin tetra-methyl ether, luteolin 4’-glucosyde, luteolin 3’-7-diglucosyde reduced osmotic induced opacification in an ovine lens culture model assessed by digital image analysis. We further investigated two in vivo rat models of cataracts induced by streptozotocin and selenite, with slit lamp evaluations. Luteolin tetra-methyl ether and honey eyedrops were applied before and after cataract onset, to explore preventive and therapeutic effects. A preliminary 20 % of rats receiving the honey therapeutic treatment in the selenite model presented delay of opacification.

82. CLINICAL EXPERIENCE OF A DIET DESIGNED TO REDUCE AGING

R Rosedale, E Westman 

Rosedale Center, Denver CO - ronrosedale@comcast.net

The neuroendocrine theory of aging is associated with elevated levels of glucose, insulin and leptin.  The objective of this study is to describe the metabolic effects of a nutritional program designed to reduce these correlates of aging.  A retrospective chart review was performed of patients attending an outpatient metabolic management program utilizing instruction in a high-fat, adequate-protein, low-carbohydrate diet, the use of nutritional supplements, and periodic individual visits.  The general dietary recommendation was approximately 15% carbohydrate, 25% protein, and 60% fat.  Recommended sources of fat included raw nuts, avocados, olives and olive oil, flax and cod liver oil.  The intake of protein was limited to 1.0 - 1.25 grams/kg lean body mass per day (increased for exercise to 1.25 grams/day).  Recommended sources of protein included sardines, fish, eggs, tofu, poultry, wild meats, non-fat cheeses (cottage, ricotta, cream), and seafood.  Only non-starchy, fibrous vegetables were acceptable.  Nutritional supplements recommended daily were: L-carnitine 2000mg, alpha-lipoic acid 400mg, coenzyme Q10 100 mg, 1 tbsp cod liver oil, magnesium 300mg, potassium 300mg, vitamin C 1000mg, vitamin E 800mg, and a multivitamin. Medications were adjusted if needed.  The mean duration of follow-up was 91.5 days (range 36-211).  Thirty-one patients were identified with baseline and follow-up body weight, and fasting laboratory tests.  The mean age of patients was 57.6 years, 53% were female.  Over a mean follow-up of 91.5 days, body weight decreased 8.2% (p<0.01), fasting serum glucose decreased 8.3% (p=0.001).  There were approximate 50% reductions in insulin, leptin, fasting serum triglyceride, and triglyceride/HDL ratio (p<0.001).  Free T3 decreased 7% (p<0.001), while TSH did not change significantly.  We conclude that a high-fat, adequate-protein, low-carbohydrate diet with nutritional supplementation led to improvements in serum factors related to the aging process

83.  MITOCHONDRIA, SEX, AND MORTALITY

 

I. Ross

 

Dept. of Molecular, Cellular, and Developmental Biology

University of California, Santa Barbara, CA 93106-9610

 

Is a universal cause of senescence in healthy organisms the expression of both alleles of nuclear-coded proteins that form part of the oxidative-phosphorylation complexes of the mitochondria? Could this explain why mitochondria are inherited from only one parent?  And why clones (e.g. triplets) die of different, random, causes?  Prior to the evolution of sex, the endosymbiotic relationship between mitochondria and nuclear genomes would have selected mechanisms that maintained the optimum interaction between the two genomes. Once sex evolved, mating would introduce different, competitive, mtDNA and /or nDNA  gene products which could well upset the balance.The selection of mechanisms, such as the specific degradation of one mitochondrial genome, would have prevented part of such competition. Unlike most protein complexes in the cell, the proteins of the multi-enzyme complexes of the ox-phos system are derived from both nuclear-genome-coded genes and mitochondrial-genome coded genes. Minor mutations in either mtDNA or nDNA coding for these proteins are known to lead to major and catastrophic diseases of humans, suggesting that very tight and precise interactions are required. To maintain the evolutionarily established balance after mating, monoallelic expression of the nuclear-coded genes would be advantageous and prevent subtly different competitive proteins from interacting with the resident mitochondria. This would require regulation of the expression of those specific nuclear genes, possibly under the control of the resident mitochondria. I have postulated that nuclear genes coding for these ox-phos complex proteins are normally expressed monoallelically and that biallelic expression of these genes with age results in adverse effects in different tissues and organs leading to senescence and mortality.  I will be presenting a fungal model to explore this hypothesis that as already produced evidence of monoallelic expression of at least one of these genes in diploids

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84.  THE RELATIONSHIP BETWEEN THE ACTIVITY IN "AMCHA" AND THE HEALTH STATUS OF ELDERLY HOLOCAUST SURVIVORS

 

AbstractAuthors: D.Sagie(P)*, A. Biderman**

 

Department of Gerontology* and Department of Family Medicine**,Ben-Gurion University of the Negev, Beer-Sheva, Israel; dsagi@bgumail.bgu.ac.il

 

About 40% of the total elderly population in Israel, are Holocaust survivors*. Previous studies have shown that this population of elderly survivors, is in great risk for different health problems. "Amcha" – The Israeli center for Psychosocial support of Holocaust survivors, was established in 1987, in order to alleviate the emotional suffering of elderly Holocaust survivors. The treatment involves building a framework of mutual aid, memory processing and grief resolution. The aim of the current study was finding a relationship between being an active member in "Amcha", and the improvement of different health measures that are supposedly purely biological.

We sampled a study group that included "Amcha" members, and a control group composed of Holocaust survivors who had similar socio-demographic and health status background, but were not "Amcha" members.

The results indicated that "Amcha" members evaluated their health status more positively)F=5.64, p<0.05). This measure of self-evaluation was proved in previous studies to be strongly related with future morbidity and mortality. "Amcha" members also tended to utilize less health services (F=.70, p<0.05).

This research contributes not only to social sciences, but also to health and biological studies. It shows that an emotional treatment of the elderly can be related to an improvement in their health status, which is commonly associated with medical-biological factors and treatments.

 

* A person who used to live in Europe in the years 1939-1945, in the time of the Nazi regime.

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85.  EFFECT OF AGE AND LPS ON p69- SHCC INDUCTION IN MOUSE BRAIN

 

Sagi O*., Wolfson M., Fraifeld V

 

Department of Microbiology and Immunology, CMRA, Ben-Gurion University of the Negev, Beer-Sheva, Israel; fraifeld@bgumail.bgu.ac.il

 

Recently, Shc (Src homology 2 domain containing) proteins were implicated in the oxidative stress-induced apoptosis and longevity in mice. These proteins act as adaptors coupling activated receptors with tyrosine kinase activity to downstream signaling molecules, including the MAPK-associated pathway. The family of Shc proteins includes ShcA, ShcB, and ShcC. ShcC family includes two isoforms of 55- and 69-kDa, and their expression appears limited to neural tissue. The brain is one of the targets for Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, which is a classic trigger of inflammatory reactions accompanied by a generation of reactive oxygen species and activation of MAPKs. The response to LPS is altered with age. The present study was aimed at examining the expression of ShcC in the mouse brain with respect to animal’s age and the effect of LPS. Male CD-1 mice aged 4 days to 12 months old, were injected intraperitoneally with 1 mg/kg LPS and at different times post-injection, their brains were removed for preparation of tissue extracts. The latter were processed through Western blot analysis using specific anti-ShcC antibodies. We found that (i) the basal level of ShcC in mouse brain increased gradually during the postnatal period, reaching the maximum at 2 months. Middle-age mice had a lower level of ShcC compared to the young adult and young animals; (ii) both p55 kDa and p69 kDa isoforms of ShcC displayed similar age-related patterns; (iii) LPS differentially affects the expression of ShcC isoforms, resulting in a marked up-regulation of p69 and an insignificant effect on the level of p55; (iiii) the effect of LPS on ShcC was age-related, being more pronounced in the younger mice compared to the older animals.

 

This study was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

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86.  A DNA DAMAGE CHECKPOINT-MEDIATED RESPONSE IN TELOMERE-INITIATED SENESCENCE

 

F. d’Adda di Fagagna, G. Saretzki*, T. von Zglinicki, S.P. Jackson

 

University of Cambridge UK, The Wellcome Trust Sanger Institute Cambridge, UK

 

Replicative senescence in human fibroblasts can be triggered by telomere uncapping, however, the signalling pathways leading to telomere-initiated activation of cell cycle checkpoints were not clear.  Specifically, it was unknown whether uncapped telomeres could signal arrest directly or whether chromosomal breaks following telomeric fusions were main triggers of senescence.

Here we show that senescent human fibroblasts display molecular markers characteristic of functional DNA damage foci including phosphorylated histone H2AX in colocalisation with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. Importantly, these senescence-associated DNA damage foci (SDF) associate preferentially with telomeres both after ‘normal’ telomere erosion and after telomere uncapping by induction of a dominant-negative TRF2. SDF remain present for many months in senescent cells. Presumably, they remain active because inactivation of DNA damage checkpoint kinases in senescent cells can restore cell cycle progression into S phase.

Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint that is activated with a direct contribution from uncapped telomeres. Replicative senescence appears to be a state that is actively maintained by  sustained activation of  a DNA damage response. Thus, DNA damage foci are not only indicators of acute DNA damage, but might much more resemble novel markers for the presence of senescent cells in vivo.  

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87.  DNA END JOINING BECOMES LESS EFFICIENT AND MORE ERROR-PRONE DURING CELLULAR SENESCENCE

 

A. Seluanov*, D. Mittelman, O. Pereira-Smith, J. Wilson, and V. Gorbunova

 

Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas

 

Accumulation of somatic mutations is thought to contribute to the aging process.  Genomic instability has been shown to increase during aging, suggesting an aberrant function of DNA double-strand break (DSB) repair.  Surprisingly, DSB repair has not been examined with respect to cellular senescence.  Therefore, we have studied the ability of young, presenescent, and senescent normal human fibroblasts to repair DSBs in transfected DNA using a fluorescent reporter substrate.  We have found that the efficiency of end joining is reduced up to 4.5 fold in presenescent and senescent relative to young cells.  Sequence analysis of end junctions showed that the frequency of precise ligation was higher in young cells, whereas end joining in old cells was associated with extended deletions.  These results indicate that end joining becomes inefficient and more error-prone during cellular senescence.  Furthermore, the ability to use microhomologies for end joining was compromised in senescent cells, suggesting that young and senescent cells may use different end joining pathways.  We hypothesize that inefficient and aberrant end joining is a likely mechanism underlying the age-related genomic instability and higher incidence of cancer in the elderly. 

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88.  BLOCKADE OF ION CONDUCTANCE IN AUDITORY NEURONS MIMIC DISORDERED CENTRAL PROCESSING IN AGED

A, Shaikh (P); P, Finlayson

Department of Otolaryngology, Wayne State University, 550 E Canfield, Room # 327, Detroit, MI 48201

Presbycusis, a senile hearing-loss, is an inevitable sequelae of aging.  About one-third of Americans older than age 60 suffer from hearing disorders.  In addition, the elderly also have a diminished ability to understand time-compressed speech, which is likely due to a reduction in temporal resolving power of auditory neurons. Based on auditory brainstem evoked-responses (ABR), affected auditory temporal processing in the aged has a central origin, possibly in Superior Olivary Complex (SOC). Yet the precise mechanism for its pathogenesis is not defined. There is mounting evidence that excitability in the central auditory neurons is affected in presbycusis.  A range of ion conductances shape the auditory neural excitability. Hyperpolarization-activated-cationic-conductance (Ih) is one of the

predominant conductances determining resting membrane potential in the SOC neurons and could regulate their excitability. In order to understand the pathophysiology of disordered central processing in presbycusis, it is important to examine the influence of Ih on the SOC neural excitability. Auditory neurons also exhibit an important property, post-stimulatory suppression, to aid echo-suppression and thereby adequate processing of the time-compressed speech. As Ih is an important regulator determining the resting membrane potential, its role in the post-stimulatory suppression, particularly in the SOC neurons needs to be investigated. Here we document Ih has a predominant role in shaping the SOC neural excitability in vivo. We also report, selective blockade of Ih by ZD7288 (100microM) significantly increases the magnitude of post-stimulatory suppression for shorter inter-tone intervals and raises the neural-response threshold to the second of the sequential-tones. As Ih-blockade decreases the neural excitability and enhances the post-stimulatory suppression for short inter-tone interval, in the SOC neurons; incompetent function of Ih could be a possible neurophysiological correlate of decreased amplitude of ABR “wave”.

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89.  THE VESTIBULAR PROSTHESIS IN POSTURAL CONTROL AND BALANCE

 

A. Shaikh, G. Auner

 

Wayne State University, Detroit, MI 48202

 

In older age groups, spatial disorientation and falls are one of the most common reasons leading to morbidity, mortality and seeking for a physician-help. Bilateral vestibular end-organ failure is a major cause of spatial disorientation, which, in aged patients, is mainly due to Meniere’s disease, ototoxicity or age induced vestibular hair-cell loss. Idiopathic vertigo is also a frequent malady in geriatric age group. Treatment of vestibular end-organ failure is still unknown and existing therapeutic principles for chronic vertigo, such as central vestibular suppression, are just symptomatic. We have pioneered the state-of-art technology that will enable us to treat the vestibular end-organ failure and chronic vertigo by means of vestibular electrical-stimulation. The novel, prototype sensor is devised, which encodes head-angular-position in real time and thus computes head-velocity, a primary function of the semicircular canals. The sensor is implantable in the mastoid antrum and has three degrees of freedom. The sensor is linked to the decoder, processor, and sixteen channels stimulator chip; which are implantable subcutaneously on the squamous temporal-bone. The information encoded by the sensor is processed to compute component of the head velocity in a plane of each of the three semicircular canals. Results of this computation are further utilized to modulate frequency of biphasic square wave electrical pulses, which are delivered by the sixteen channels stimulator chip to nano-electric arrays. These arrays are developed using the novel “Self-Assembled Nano Array Platform Approach” and provide the best resolution for the artificial-nerve-stimulation. The arrays are attached to self-retaining probe adapting the shape of the ampullae and providing the electrode array a close contact with the neuroepithelia for specificity of the individual three-canal-stimulation. Integration of the sensor feedback with multiple similar sensors implanted at different body axes provides basis of functional-neural-stimulation to reinforce the muscular control of posture. 

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88.  BLOCKADE OF ION CONDUCTANCE IN AUDITORY NEURONS MIMIC DISORDERED CENTRAL PROCESSING IN AGED

 

A, Shaikh; P, Finlayson

 

Department of Otolaryngology, Wayne State University, 550 E Canfield, Room # 327, Detroit, MI 48201

 

Presbycusis, a senile hearing-loss, is an inevitable sequel of aging. About one-third of Americans older than age 60 suffer from hearing disorders.  In addition, the elderly also have a diminished ability to understand time-compressed speech, which is likely due to a reduction in temporal resolving power of auditory neurons. Based on auditory brainstem evoked-responses (ABR), affected auditory temporal processing in the aged has a central origin, particularly in Superior Olivary Complex (SOC). Yet the precise mechanism for its pathogenesis is not defined. There is mounting evidence that excitability in the central auditory neurons is affected in presbycusis. A range of ion conductances shape the auditory neural excitability. Hyperpolarization-activated-cationic-conductance (Ih) is one of the predominant conductances determining resting membrane potential in the SOC neurons and could regulate their excitability. In order to understand the pathophysiology of disordered central processing in presbycusis, it is important to examine the influence of Ih on the SOC neural excitability. Auditory neurons also exhibit an important property, post-stimulatory suppression, to aid echo-suppression and thereby adequate processing of the time-compressed speech. As Ih is an important regulator determining the resting membrane potential, its role in the post-stimulatory suppression, particularly in the SOC neurons needs to be investigated. Here we document Ih has a predominant role in shaping the SOC neural excitability in vivo. We also report, selective blockade of Ih by ZD7288 (100microM) significantly increases the magnitude of post-stimulatory suppression for shorter inter-tone intervals and raises the neural-response threshold to the second of the sequential-tones. As Ih-blockade decreases the neural excitability and enhances the post-stimulatory suppression for short inter-tone interval, in the SOC neurons; incompetent function of Ih could be a possible neurophysiological correlate of decreased amplitude of ABR “wave V” (decreased SOC neural activity) in aged patients with speech understanding problems.

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90.  MODULATION OF MRNA EXPRESSION PATTERNS IN MURINE CNS BY AGE AND DIETARY MELATONIN: FOCUS ON IMMUNITY
 

E. Sharman*, V. Perreau, K. Sharman, C. Cotman, and S. Bondy

Center for Occupational and Environmental Health, Department of Community and Environmental Medicine, and Institute for Brain Aging and Dementia, University of California Irvine, CA 92697-1825, U. S. A.

Age-dependent decline in brain immune response is a component of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. To assess the impact of age on CNS immune response, gene array analysis was used to identify changes in functional patterns and levels of mRNA expression induced by i.p. injection of lipopolysaccharide (LPS) in the cerebral cortex of both young (4-month) and old (27 month) CB6F1 male mice. Modulation of the LPS-induced CNS immune response by the pineal secretory product melatonin was assessed by supplementing the diet of aged animals with 40 ppm melatonin for 8 weeks preceding LPS injection. A wide range of age-related changes in basal levels of genes within the brain was found. Dietary melatonin administered to aged mice failed to reverse most of these changes. LPS treatment elevated the expression of many genes in the younger animal. However, with senescence, the response to such an inflammatory stimulus was attenuated. Administration of melatonin was able to restore this reaction to a level more closely resembling that of the younger animal.

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91.  SHORT-TERM COENZYME Q10 SUPPLEMENTATION ENHANCES COGNITIVE PERFORMANCE IN AGING MICE.

 

1) R. A.Shetty

2)N. Sumien.

3)R.Sohal.

4)M. J. Forster.

 

Dept of Pharmacology and Neuroscience, UNTHSC, FortWorth, TX-76107.

 

Brain aging is accompanied by an increase in oxidative stress and mitochondrial dysfunction. Coenzyme Q (CoQ) is an important component in the electron transport chain and is thought to serve as a potent antioxidant. It is also known that CoQ content declines with aging and the levels can be restored in older mice by supplementation. However the effects of CoQ10 supplementation on behavior are not known. Therefore the purpose of this study was to determine the short-term effects of CoQ10 supplementation on psychomotor and cognitive performance in young and old mice. Separate groups of young (4 months) and old mice (18 months) were fed a control diet or a diet supplemented with low or high concentrations of CoQ10 for a period of 15 weeks. The low and high supplemented diets yielded a daily CoQ10 intake of approximately 148- or 654-mg/kg, respectively. After 6-weeks on the diets the mice were subjected to a battery of age-sensitive behavioral tests for cognitive and psychomotor performance. These tests include locomotor activity, coordinated running performance, swim maze and startle response. CoQ10 treated mice tended to exhibit faster learning of the swim maze task, an aging-sensitive measure of cognitive performance. Diet supplementation with CoQ10 also improved the ability of the old mice to retain information in swim maze task, an effect that was not evident in the younger mice. In tests for auditory and shock startle reflex and coordinated running ability, CoQ10 supplementation failed to improve performance. These results suggest that short-term CoQ10 supplementation improves impaired cognitive function in older mice, but fails to ameliorate age-impaired psychomotor function.  

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94.  DIFFERENTIAL EXPRESSION OF METABOTROPHIC GLUTAMATE RECEPTORS (mGluRs) IN ALZHEIMER DISEASE 

 

H-G Lee, X. Zhu, M.J. O'Neill, G. Perry, M.A. Smith*

 

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

 

Although the mechanism underlying the loss and/or dysfunction of susceptible neurons in Alzheimer disease (AD) is unknown, it has been hypothesized that excitotoxicity may play a pivotal role. Since glutamate has long been thought to be important in the pathogenesis of AD, we hypothesized that select glutamatergic receptor populations (e.g., metabotropic glutamate receptors (mGluRs)) may correlate with those neurons vulnerable to AD. mGluRs are directly coupled to the intracellular signaling system via GTP-binding proteins and are thought to be related with synaptic transmission and neuroprotection. Since synaptic loss is a prominent feature of AD, in this study, we investigated the role of mGluR in AD. Specifically, we determined the expression of the family of mGluR receptors including group I, II and III in the brains of AD and age-matched control cases. We found a differential expression of mGluRs that related directly to the pattern of neuronal susceptibility in AD. mGluR2 is specifically increased in pyramidal neurons in the hippocampus of AD and often co-localizes with neurofibrillary pathology. mGluR5 showed a similar expression pattern to mGluR2 but was also found in astrocytes surrounding amyloid plaques. The specificity of these findings is demonstrated by the fact that mGluR1 expression, which, like mGluR5, is a group I mGluR, is not different between AD and control cases. Our data suggest that specific mGluRs such as mGluR2 and mGluR5 are differentially regulated in AD and that this expression is directly related to neuronal populations vulnerable to degeneration and to amyloid-beta and tau protein lesions. Therefore, the differential regulation of specific mGluRs may explain the selective neuronal degeneration in AD and provide a unique therapeutic target for the treatment of AD.

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92.  ACETYLATION: A NOVEL POSTTRANSLATIONAL MODIFICATION IN ALZHEIMER DISEASE

 

A.K. Raina, X. Zhu, A.D. Cash, L.M. Sayre, G. Perry, M.A. Smith*

 

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

 

The mechanism leading to the selective dysfunction of neurons in Alzheimer disease is incompletely understood. Nonetheless, the formation of the characteristic neurofibrillary pathology in Alzheimer disease is thought to play a pivotal role in this process. Associated with the formation of neurofibrillary pathology, proteins are subject to a number of posttranslational modifications with phosphorylation being the best studied. In this study, we report an entirely novel protein modification, namely acetylation of lysine, in vulnerable neurons that parallels the temporal pattern of neuronal vulnerability in cases of Alzheimer disease. While the exact role of protein acetylation in Alzheimer disease is unknown, it is likely, given the physiological roles of acetylation, that acetylation promotes neuronal survival by stabilizing the cytoskeleton in the face of occult, and otherwise destabilizing, tau phosphorylation events. This novel finding of a stabilizing modification in vulnerable neurons in Alzheimer disease will provide new insights for therapeutic interventions that could alter the natural history of this dementia.

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93.  A MECHANISTIC STUDY ON AMYLOID-BETA-INDUCED JNK ACTIVATION

 

X. Zhu, C.A. Rottkamp, Z. Kubat, A.K. Raina, S.L. Siedlak, C.S. Atwood, G. Perry, M.A. Smith

 

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

 

Amyloid-&#946; protein (A&#946;), the main constituent of neuritic and diffuse plaques as well as cerebrovascular amyloid deposits, characterizes the neuropathology of Alzheimer disease (AD). While A&#946; is neurotoxic in vitro, the actual biochemical mechanism remains to be established. In this study, we found that when A&#946; is pretreated with the iron chelator deferoxamine, neuronal toxicity is significantly attenuated while conversely, incubation of holo-A&#946; with excess free iron restores toxicity to original levels. These data suggest that the toxicity of A&#946; is mediated, at least in part, via redox-active iron. The in vivo significance of these findings is amplified by consideration of the enrichment of iron in AD neuropil, and especially in association with A&#946;. To understand the mechanistic consequence of these findings, we also found that JNK/SAPK is activated in susceptible regions of the brain in AD. The co-localization of activated JNK/SAPK and A&#946; deposits, as well as concomitant iron accumulation, suggests that JNK/SAPK pathway may play an important role in metal-mediated A&#946; toxicity. In this regard, we found that although both A&#946;PP (K670N/M671L) transgenic mice and A&#946;PP (V717I) transgenic mice develop amyloid senile plaques, iron accumulates only in the former and strikingly, activated JNK/SAPK is also only found in the former. Since such mice show cognitive alterations, we hypothesize that neuronal dysfunction is, in part, mediated through alteration in signal transduction pathways such as JNK/SAPK pathway. Supported by the NIH (NS38648) to MAS and the Alzheimer’s Association (NIRG-02-3923) to XZ.

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95.  TELOMERE LENGTH AS A POSSIBLE-AGE MARKER IN FISH

 

*Justine, C.Y. Tsui, Stephen, B. Pointing and Kenneth, M.Y. Leung

 

The Swire Institute of Marine Science, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

 

Telomeres are nucleoprotein structures at the ends of linear chromosomes consisting of DNA sequences arranged in tandem repeated units (TTAGGG). They act to prevent aberrant recombination and degradation of the chromosomal ends, and thus maintain chromosome integrity. Since DNA polymerases are unable to copy chromosomal DNA completely, telomere sequences typically shorten with the number of cell divisions and hence with age. Previous research has demonstrated a negative relationship between telomere length and age for various vertebrate species. In this study, we describe for the first time an assessment of telomere length as a function of age for the mangrove snapper, Lutjanus argentimaculatus, a commercially important teleost species in Asia and the Middle East. Individuals with known chronological ages (2.5-36 months) were obtained from a fish farm in Shenzhen, China. Genomic DNA was extracted from whole blood and dorsal muscle tissues. Telomere length was quantified using the Telo TAGGG Telomere Length Assay (Roche Molecular Biochemicals), which is based on Southern blot-hybridization with terminal restriction fragments of the telomere. Telomere length in blood was significantly reduced with age of individuals (r2 = 0.2484, F1,22 = 6.942, n = 23, p < 0.05), suggesting a telomere shortening rate of 0.39 kbp/yr. Telomere length in muscle tissues also decreased significantly in relation to fish age (r2 = 0.1513, F1,26 = 4.459, n = 27, p<0.05), resulting in a slightly faster shortening rate of 0.44 kbp/yr. The results suggest that patterns in fish telomere shortening are similar to that in other vertebrates such as mammals and birds. Measurement of telomere length in blood may have potential as a non-destructive method for estimating age in fish.

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Harry van Steeg1, Susan Wijnhoven1, Rudolf Beems1, Martijn Dollé1, Jan Vijg2, Paul Lohman1, Jan Hoeijmakers3 and Bert van der Horst3

1Natl Inst 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, Texas, USA; 3MGC department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands

The accumulation of somatic DNA damage is considered to be a major cause of the aging process in various species including mice and humans. Among the sources of DNA damage, reactive oxygen species (ROS) are often thought to be the ultimate cause of aging. However, the mechanisms involved remain obscure. To counteract the effects of DNA damage, an intricate network of DNA repair pathways has evolved. One major pathway is nucleotide excision repair (NER), which removes a broad range of bulky lesions including some forms of oxidative damage. Patients with a defect in NER proteins like CSB and XPD, both involved in repair as well as transcription of DNA, appeared to have a decreased life span.

In order to investigate whether defects in genome maintenance are correlated to accelerated aging, we have successfully conducted several longevity and cross sectional studies with mice having defect in DNA repair and/or RNA transcription (i.e. Xpa-, Csb-, XpdTTD- deficient mice as well as C57Bl/6 wild type controls). The mean survival of female XpdTTD as well as Xpa mice appeared to be much shorter (appr. 90 weeks) than those found for Csb and wild type littermate controls (104-110 weeks). Full histopathology has been performed in aged female mice, and gross examination at autopsy revealed small posture, kyphosis, large spleen, small thymus and abnormal skin and hair especially in XpdTTD mice. The terminal body weights in XpdTTD and Csb females were decreased, with an increase in the relative weights of several organs of XpdTTD mice, especially in the kidney, spleen and the heart. Furthermore, lipofuscin pigmentation (an aging feature, correlated to oxidative damage) was found to be accumulated in the liver of XpdTTD mice as compared to the other phenotypes. Finally, a variety of immune parameters were determined in mice of the various genotypes and ages.

98.  VARIATION IN TELOMERE SHORTENING RATE CAUSES HETEROGENEITY IN REPLICATIVE LIFESPAN

 

T.von Zglinicki, C. Martin-Ruiz, J. Petrie, G. Saretzki

 

Biogerontology Laboratory, Newcastle University, UK

 

The replicative lifespan of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a non-telomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing clones by flow cytometry. These senescent cells had shorter average telomere length than their cycling counterparts at all population doubling levels both in mass culture and in individual sub-clones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilised telomere length in the majority of cells and rescued these from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells which showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. The data show that heterogeneity of human fibroblast replicative lifespan can be caused by significant stochastic cell-to-cell variation in telomere shortening.

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97.  TELOMERE SHORTENING IN HUMAN FIBROBLASTS IS NOT DEPENDENT ON THE SIZE OF THE TELOMERIC 3’-OVERHANG

 

T. von Zglinicki, B. Keys, V. Serra, G. Saretzki

 

Biogerontology Laboratory, Newcastle University, UK

 

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semi-conservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3’ overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3’ overhangs and the rate of telomere shortening. Measuring overhang length by in-gel hybridisation, we could confirm shortening of overhangs as human MRC5 and BJ fibroblasts approach senescence. However, a large study comprising of fibroblast strains from 21 donors under conditions leading to two orders of magnitude of variation in telomere shortening rate ruled out any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither causal for nor indicative of telomere shortening.

 

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Zhihui Wang, K.A. Al-Regaiey, M.M.Masternak, A.Bartke

Southern Illinois University, School of Medicine, Geriatrics Research, 801 N. Rutledge Springfield, IL 62794

Ames dwarf (df) mice display primary deficiency of growth hormone (GH), prolactin (PRL), and thyroid simulating hormone (TSH), improved insulin sensitivity, diminutive body size, and prolonged longevity. Young adult df mice have higher % body fat compared with their normal littermates (Wild type, WT). In order to study the mechanism of improved insulin sensitivity in these mice, we have used real time PCR to examine gene expression of adipocytokines related to insulin sensitivity including adiponectin, leptin, resistin, TNF-&#61537;, and interleukin (IL)-6, and genes related to fat metabolism including fatty acid synthase (FAS), hormone-sensitive lipase (HSL), uncoupling protein (UCP)-2, and &#61538;3-adrenergic receptors (&#61538;3AR) in white adipose tissue (WAT). We also analyzed expression of the same genes in df and WT animals subjected to 30% caloric restriction (CR). In WAT from df mice fed ad libitum (AL), the expression of adiponectin, TNF-&#61537; , FAS, HSL, and UCP2 were significantly diminished, and the expression of IL-6 was dramatically suppressed, whereas the expression of leptin, resistin, and &#61538;3AR were not changed. CR increased gene expression of resistin, FAS, and &#61538;3AR, and diminished gene expression of UCP2 and IL-6 in WT mice compared with their AL littermates. CR also diminished expression of leptin in df mice compared with AL df mice. In both WT and df mice, CR did not alter the expression of adiponectin, HSL, although their expression and the expression of resistin, FAS, UCP2, &#61538;3AR in CR df mice were much lower than in CR WT mice. These results suggest that decreased expression of TNF-&#61537;, FAS, and IL-6 might play a role in improving insulin sensitivity in df mice. CR might down-regulate the gene expression of leptin via increasing expression of &#61538;3AR, and down-regulate the expression of UCP2, thus maintaining the energy balance of the body. (Supported by NIA and Ellison Medical Foundation)
 

100.  BLUEBERRY-DERIVED PHYTONUTRIENTS CAN AFFECT AGING AND STRESS TOLERANCE IN THE NEMATODE, CAENORHABDITIS ELEGANS

 

M. Wilson, B. Shukitt-Hale, G. Lee, D. Ingram, J. Joseph, C. Wolkow

 

Laboratory of Neuroscience, National Institute on Aging, IRP, 5600 Nathan Shock Drive, Baltimore, MD 21224

 

Plant-derived antioxidants have been shown to provide beneficial health effects. In order to explore the mechanisms behind these observations, we investigated the effects of blueberries on lifespan and stress resistance in the nematode, C. elegans. Either crude extracts or partially purified polyphenol fractions of blueberries increased mean lifespan and slowed age-related declines in pharyngeal pumping rates in wild-type animals. Resistance to thermal stress was also increased significantly. One possibility is that blueberry extracts inhibited bacterial growth, a treatment known to extend mean C. elegans lifespan. We examined this possibility by comparing the effects of antibiotics and blueberry. While treatment with either ampicillin or blueberry extract extended C. elegans lifespan, there were no additive effects on longevity when these treatments were combined.  However, while blueberry significantly increased thermal tolerance, ampicillin had no effect. The differential effect on stress tolerance suggests that blueberry can directly affect C. elegans physiology, independent of effects on bacterial growth. Through what genetic pathways can blueberries alter stress tolerance?  Reductions in insulin/IGF-like signaling are correlated with increased stress resistance and lifespan in a wide range of organisms.  Longevity and stress resistance in C. elegans insulin-like pathway mutants requires the DAF-16/FOXO transcription factor. To determine whether blueberry affects insulin-like signaling, we examined whether the beneficial effects of blueberry extracts required daf-16 activity.  Fractional increases in lifespan, and absolute increases in thermotolerance, were similar in wild-type and daf-16(mgDf50) animals.  In untreated daf-16(mgDf50) animals,  pharyngeal pumping declined more rapidly with age than in wildtype animals. This decline was slowed to the wild-type rate with blueberry extract.  These experiments indicate that blueberry's longevity-promoting effects do not require DAF-16/FOXO activity, and probably occur independently of insulin-like signaling. We hypothesize that treatment with blueberry extract increases C. elegans longevity by increasing intrinsic resistance to environmental and/or metabolic stresses that limit mean lifespan.

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101.  AGING ALTERS THE COORDINATION OF SENSORY INPUTS WITH MOTOR RESPONSES IN THE NEMATODE, CAENORHABDITIS ELEGANS

 

C. Wolkow, D. Chow, C. Glenn, L. David, I. Goldberg

 

Laboratory of Neuroscience, National Institute on Aging, IRP, 5600 Nathan Shock Drive, Baltimore, MD 21224

 

Many behavioral responses require the coordination of sensory inputs with motor outputs.  Aging is associated with progressive declines in both motor function and muscle structure.  However, the consequences of age-related motor deficits upon behavior have not been clearly defined.  To investigate this question, we have examined the effects of aging upon behavior in the nematode, Caenorhabditis elegans.  We first analyzed the effect of age on responses to sensory stimuli.  When touched gently with a hair, both young and aged animals responded appropriately by backing away from the stimulus.  When presented with an aversive odor, octanol, both young and aged animals stopped forward movement similarly.  However, aged animals exhibited defects in backing away from the odorant source and these defects were exacerbated when octanol was diluted 100-fold.  Aged animals displayed significant deficits in chemotaxis behavior, as older animals failed to progress fully to the source of an attractive stimulus.  Together, these results indicate that age was not associated with significant declines in sensory ability.  However, locomotory responses to stimuli were severely compromised.To determine the basis for these behavioral deficits, we analyzed the patterns of movement in aged animals.  Older animals moved 50% slower than young animals and failed to maintain forward movement for sustained intervals.  Examination of muscle structure at these ages showed that reduced muscle tissue integrity was correlated with age-related declines in locomotory behavior, although widespread muscle deterioration was rare at the ages examined.  Finally, treatment with a muscarinic agonist improved locomotory behavior in aged animals, indicating that improved neuromuscular signaling may be one strategy for reducing the severity of age-related behavioral impairments.

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102.  GENETIC SELECTION AND INITIAL CHARACTERIZATION OF MOUSE LINES FOR HIGH AND LOW STRESS SUSCEPTIBILITY

 

DS. Xu*,1,3, T. Lambert 1, D. Paul 2,  Laura Meyerle 1 , C. Nekl 1 , J. Potts 2, M. Nielsen 2, Y. Zhou 1

 

1 Department of Veterinary & Biomedical Sciences, 2 Department of Animal Sciences, University of Nebraska - Lincoln;  3 Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, Omaha

 

Two mouse lines were generated by genetic selection for high and low stress susceptibility (SH and SL lines, respectively), from baseline populations of mice selected for high (base for SH) and low (base for SL) rates of metabolism, which also showed differences in locomotor activity and stress responses. Corticosterone concentrations in sera collected at various time points from the SH and SL mice with or without exposure to acute or repeated restraint stress were determined by radioimmunoassay and used as one of the key criteria for selection. The SH mice showed significantly (P < 0.05) higher levels of serum corticosterone, at all tested time points, than the SL mice. Initial reverse transcription polymerase chain reaction (RT-PCR) analysis using brain RNA revealed differences in expression of three major stress-response regulatory genes: corticotrophin-releasing hormone (CRH), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Expression of CRH was higher while GR and MR expressions were down-regulated in the SH mice as compared to SL mice. Microarray assays were used to compare gene expression in the brains of the two lines of mice under stressed and non-stressed conditions, and revealed significant differential expression patterns of many genes (up or down regulated) between the two lines as well as within the same line of mice.  Subsequent RT-PCR experiments using specific primers for 25 genes of interest, selected based on the microarray data, confirmed the genetic and stress-induced alterations in gene expression between the SH and SL mice, such as microtubule-associated protein-2, P21-activated kinase-3, calcium/calmodulin-dependent protein kinase IIa, and calcium channel proteins.  Further establishment of the SH and SL mouse lines, divergent through selection for altered stressed susceptibility, may become a useful tool to study stress-induced early aging and neurodegeneration.

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103.  AGE-RELATED DIFFERENCES OF GENE EXPRESSIONS AND DNA DAMAGE IN HUMAN FIBROBLASTS AFTER EXPOSURE TO COBALT CHROME PARTICLES

 

Yin Z.R., Papageorgiou I., Clerkin J.S., Learmonth I.D., Case C.P.

 

Bristol Implant Research Centre, University of Bristol, BS10 5NB, U.K.

 

Wear debris from worn cobalt chrome orthopaedic joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the levels of DNA damage and certain gene expressions (TGF-ß2, p38 MAPK, Integrin&#946;1, SOD1, Caspase 10, PURA, FRA-1 and VNR) have been compared in human fibroblasts of different age (10 and 35 population doublings, PD) after exposure to different doses of cobalt chrome particles and at different times of exposure (6 and 24 hours). The 35 PD fibroblasts showed significantly more immunostaining of the senescence-associated &#946;-galactosidase (57.4%) than the 10 PD fibroblasts (1.4%).  The level of DNA damage, as detected with alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in younger (10 PD) fibroblasts. No significant change in cell viability was noted using MTT assay or LDH assay. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expressions, like the increase in DNA damage was greater at higher dose and at longer time of exposure. Interestingly these changes in DNA damage and gene expression were more pronounced in younger than in older fibroblasts but not until after 24 hours of exposure. These results show that levels of gene expression of TGF-ß2, p38 MAPK, Integrin&#946;1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts.

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104.  Assessment of motor performance in rhesus monkeys on long-term calorie restriction

J. Young1, Zhiming Zhang2, D. Gash2, G. Gerhardt2, G. Roth3, M. Lane,3 J. Mattison,3 D. Ingram3

1Sobran, Inc, 4401 Dayton-Xenia Road, Dayton, OH  45432; 2Dept. of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, KY 40536; 3Laboratory of Experimental Gerontology, National Institute on Aging, NIH, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224;

In numerous rodent studies, calorie restriction (CR) has been demonstrated consistently to increase lifespan, reduce the incidence and onset of age-related chronic disease, and attenuate age-related decline in many physiological functions.  Emerging evidence from studies in rhesus monkeys suggests that CR can also retard aging processes in a species closely related to humans.  An important question regarding the effects of long-term CR in monkeys is whether behavioral function will be impacted, either negatively or positively.  We have initiated studies to evaluate motor performance in male and female rhesus monkeys involved in a long-term study of CR.  Control monkeys were provided a highly nutritious diet while monkeys on CR have had their intake of this diet reduced 30 percent from the level of comparable aged controls.  Monkeys were introduced to the study at different stages in the lifespan, including juvenile, adult, and aged groups of monkeys.  Fine motor and coarse motor performance were measured in an automated movement assessment panel attached to the home cage of the monkey.  During trials of 45 sec duration, monkeys are given access to the apparatus during which they must reach with their hand through an opening and then must maneuver their hand at a 90o angle from entry into an elevated center chamber to retrieve a visible food treat (soft lifesaver candy).  During different sessions, the treat is positioned either on a flat platform, a straight rod, or a hook in the center chamber.  Performance on each task is measured as the time required to retrieve the treat once the hand has entered the apparatus.  Age-related decline in performance (longer retrieval times) of rhesus monkeys on all three tasks has been previously documented (Zhang et al. J.Gerontol. Biol. Sci. 55:B473, 2000), and we have replicated these findings in the current study.  To assess effects of CR on performance, comparisons of fine motor retrieval time were made for male and female monkeys between the ages of 12.1 - 24.3 yr that had been on CR for 12 -18 yr.  For males, we found that retrieval time was equivalent between diet groups on the easiest tasks (platform and straight rod), but times on the more difficult hook task were significantly faster for CR group.  For females, CR monkeys had significantly faster retrieval times on both the platform and the hook tests, but had significantly slower times on the straight rod task.  Considering performance on only the most difficult task, we can conclude that CR improves motor performance.

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105.  BOTULINUM TOXIN TYPE A (BoNT/A) FOR CHRONIC HEADACHES IN ELDERLY

 

S.Zachariah,M.D, T.Ranjan,M.D and Aveen Zachariah.

  

Background: Headache is reported to be the tenth and fourteenth most common symptom in elderly women and men respectively. Five to ten percent of patients over 65 develop chronic daily headaches. BoNT/A has dramatically improved the treatment of a variety of neurological disorders, including headache.

      Methods: Data reviewed of 30 patients (average age-60.8year) involved in an open label trial showed 21 had typical vascular headaches, 7 had chronic daily headaches [>20 headaches in a month] and 2 had tension headaches. They received BoNT/A for headaches (average duration 10 yrs) between 1995 and 2003.  All patients met the following criteria:  headache duration of >2 yrs; absence of known allergy to BoNT/A; severity of headache > 5 [on 0-10 scale]; failure to Triptans, Beta blockers, Calcium Channel blockers, Anticonvulsant and Antidepressants.  Forty to seventy-five unit intra muscular injections were given in corrugators and bilateral upper & lower frontalis and temporalis at the depth of 1-3 mm using number 30 needle.  Patients with tension headache were given semispinalis and splenius capitis injections, following the path of pain.  Injection was repeated after 3 months. Evaluation entailed: headache scale, satisfaction survey, medication use, life style questionnaire and neurological exam.

     Results: Most of the patients had effect in 3-5 days with optimum benefit in 2 weeks. The average pain intensity reduced from 8.66 to 1. First injection effects lasted from 45 to100 days with later injections lasting longer. Twenty-four patients out of 30 (80%) had clinical response, of which 20 patients were very satisfied, 4 patients were satisfied and 6 patients [20%] had no response to therapy. Respondents had long-term benefits like pain relief, stoppage or reduction of medications, better life style, and improvement in productivity.

     Conclusions:  Older patients with chronic headache with polypharmacy failures tolerate and benefit from BoNT/A therapy with minimal acute side effects.

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106.  UNDERSTANDING THE GENETICS OF AGING: A CANINE MODEL

 

S. Zienko, K.Greer, M. Breen, K. Murphy

 

Dept. of Veterinary Pathobiology; Texas A&M Univ. College of Veterinary Medicine; College Station, Texas, U.S.A.; Dept. of Molecular Biomedical Sciences, North Carolina State Univ. College of Veterinary Medicine; Raleigh, North Carolina, U.S.A.

 

The biology of aging is extremely complex, and is, therefore, poorly understood.  Research pertaining to aging has many potential benefits, the most important of which are extension of life spans and improvement of the quality of life in aged populations.  Select human populations have significantly longer life expectancies than others, suggesting an underlying genetic component associated with the aging process.  The genetic factors governing longevity are numerous, difficult to define, and may possess regulatory functions.  The hereditary component of longevity applies to animal populations.  Our interest in the dog as a model stems from the fact that the dog serves as excellent model animals for many human hereditary conditions, and that average life expectancy of purebred dogs decreases as both height and weight increase, as shown by preliminary regression analysis of 117 breeds.  As a first step to understanding the potential genetic factors associated with longevity in dogs, comparative gene mapping data is being generated by cataloging and mapping 56 candidate genes.  Variation in 23 of these genes is associated with either extended or truncated life spans in the human or mouse.  The additional 23 genes, that give approximately 0.5Mb coverage of a 22Mb region of human chromosome 4, were selected because a microsatellite marker, D4S1564, shows strong association with excessive longevity in an Italian population.  Following mapping of these genes, detailed analysis will be conducted to obtain polymorphic markers in close vicinity of the mapped genes.  These markers will be analyzed across a range of breeds with contrasting degrees of longevity.  Additionally, comprehensive analysis will be carried out to identify variations within selected genes (from the group of 56) across breeds with diverse longevity.  These pilot studies will serve as a platform to initiate broader research aimed at understanding genetic factors associated with longevity in dogs and other animals.

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