Source: PURDUE UNIVERSITY submitted to NRP
MAMMALIAN RESPONSE TO AGING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0200788
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Nutrition Science
Non Technical Summary
An almost universally recognized major contributor to aging at the cellular level is increased production of reactive oxygen species The purpose of this study is to understand why cells of older individuals produce greater amounts of reactive oxygen species than cells of younger individuals.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
72360201040100%
Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
6020 - The family and its members;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
1. To characterize and clone the aging-related terminal oxidase of the plasma membrane and compare it to the terminal oxidase of the young individual. 2 To determine if arNOX is overexpressed in cells and transgenic mice where mitochondrial lesions have resulted in respiratory insufficiency. 3 To test potential botanical and nutritional supplement preparations for reduction of arNOX activity in cells and sera of aged humans and transgenic mice.
Project Methods
My laboratory has discovered an aging-related cell surface protein (ar-NOX) generates superoxide (based on reduction of cytochrome c) and this activity is inhibited by ubiquinone. The aberrant generation of reactive oxygen species is potentially important to propagation of oxidative stress. The ar-NOX protein will be characterized and cloned as an initial step toward developing the molecular reagents required to test the mitochondrial theory of aging and its relationship to the plasma membrane oxidoreductase system. Regions of the amino acid sequence deduced from the cDNA sequence will be analyzed and used to strategically generate peptide antisera. Peptide antibodies will be affinity-purified using immobilized peptide. The possibility of arNOX overexpression in respiratory-deficient aged cells will be ascertained at three levels: RNA message, arNOX protein amount and arNOX activity. Young and aged Namalwa cells will be fixed in 10% formalin at room temperature, dehydrated in an ethanol series, cleared in xylene and embedded in paraffin. In each instance, respiratory deficient cells will be identified on skipped serial sections using cytochemical methods for localization of cytochrome oxidase and/or succinic dehydrogenase. The increased production of superoxide is suggested to result in progressive oxidant damage to cellular components and particularly to mtDNA that encodes subunits assembled in respiratory complexes. Earlier studies of respiration in muscle mitochondria obtained from large cohorts of patients supported this notion by showing that either singly or in combinations, the respiratory complexes exhibited decreased activity in the elderly. Mice will be used as a source of cells and sera for animal studies. Since our laboratory has had experience with generating transgenic mice, experiments will be designed to generate transgenic mice that contain the human arNOX cDNA. By expressing the arNOX protein, the arNOX transgenic mice should exhibit increased reactive oxygen species production and evidence of aging-related atherogenesis. Finally, botanical and nutrition supplement preparations will be tested for inhibitory activity against arNOX as an analytical endpoint and potential target molecule contribution to imbalance between production of reactive oxygen species and antioxidant defense in cells and sera of aged individuals (greater than 65 year). Sera and cells from young (25 to 35 y), middle-age (40 to 50 y) individuals will be used for comparison. Samples from young (25 to 35 year), middle-age (40 to 50 year) individuals will be assayed for comparison.

Progress 10/01/04 to 09/30/09

Outputs
OUTPUTS: Retired, nothing additional to report PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
X

Publications

  • No publications reported this period


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: The overall goal of this project is to understand why greater amounts of reactive oxygen species is produced by cells of older individuals than by cells of younger individuals. arNOX, an aging-related cell surface protein is a member of a family of cell surface proteins designated as ECTO-NOX proteins. It is uniquely characterized by its ability to generate superoxide. arNOX was identified by our laboratory and is found in sera, saliva, skin, lymphocytes, perspiration and urine of older humans of 50 years or older as well as in older plants. By producing reactive oxygen species at the cell surface it acts to accelerate aging-related changes. The reduction of ferricytochrome c is utilized in our laboratory as the standard measure of superoxide formation. Compounds such as coenzyme Q10, selected botanical products inhibit the activity of arNOX PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nutritionist and geriatric specialists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our laboratory has identified an aging-related protein, arNOX, located on the cell surface whose activity can be inhibited by synthetic and natural products. This has the potential to decrease aging-related changes in, for example, skin and in diseases (i.e., atherogenesis) in the older individual.

Publications

  • Weaver, C.M., Barnes, S., Wyss, J.M., Kim, H., Morre, D.M., Morre, D.J., Simon, J.E., Lila, M.A., Janle, E.M. and Ferruzzi, M.G. 2008. Botanicals for age-related diseases: from field to practice. Am. J. Clin. Nutr. 87:493S-497S.
  • Tang, X., Tian, Z., Chueh, P.J., Chen, S., Morre, D.M. and Morre, D.J. 2007. Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface. Biochemistry 46:12337-12346.
  • Morre, D.M., Heald, S.M., Coleman, J., Orczyk, J., Jiang, Z. and Morre, D.M. 2007. Structural observations of time dependent oscillatory behavior of CuIICl2 solutions measured via extended X-ray absorption fine structure. J. Iorg. Biochem. 101:715-726.
  • Morre, D.J., Jiang, Z., Marjanovic, M., Orczyk, J. and Morre, D.M. 2008. Response of the regulatory oscillatory behavior of copperII-containing ECTO-NOX proteins and of CuIICl2 in solution to electromagnetic fields.
  • Kromkowski, J., Hignite, H., Morre, D.M. and Morre, D.J. 2008. Response to lithium of a cell surface ECTO-NOX protein with time-keeping characteristics. Neurosci Lett. 438:121-125.
  • Tang, X., Morre, D.J. and Morre, D.M. 2007. Antisense experients demonstrate an exon 4 minus splice ariant mRNA as the basis for expression of tNOX, a cancer-specific cell surface protein. Oncol. Res. 16:557-567.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The overall goal of this project is to understand why cells of older individuals produce greater amounts of reactive oxygen species than do cells of younger individuals. An aging-related cell surface protein (arNOX) identified by our laboratory is found in sera, skin, lymphocytes, perspiration and saliva of human individuals of 50 years or older as well as in older plants. arNOX is a member of a family of cell surface proteins designated as ECTO-NOX proteins and is uniquely characterized by an ability to generate superoxide. It can contribute significantly to the imbalance between production of reactive oxygen species and antioxidant defense by producing reactive oxygen at the cell surface that accelerate aging-relating changes. We utilize the reduction of ferric cytochrome by superoxides as the standard measure of superoxide formation. Our work involves the use of various compounds such as coenzyme Q, superoxide dismutase and others to investigate thir inhibitory potential on the activity of arNOX . Our laboratory is heavily involved in attempts to purify the arNOX protein catalyzing the aging-related cytochrome c reduction. We have some success but are continuing to work toward obtaining meaningful sequence of the protein PROJECT MODIFICATIONS: Medical and nutrition professionals interested in the aging process and it prevention

Impacts
An aging-related protein designated arNOX has been identified by our laboratory in individuals 50 years and older. Natural and synthetic compounds targeted to this protein are being tested in our laboratory as a potential to eliminate or decrease effects seen with diseases often associated with aging.

Publications

  • Yagiz, K., Wu, L-Y., Kuntz, C.P., Morre, D.J. and Morre, D.M. 2007. Mouse embryonic fibroblast cells from transgenic mice overexpressing tNOX exhibit an altered growth and drug response phenotype. J. Cell. Biochem. 101:295-306.
  • Morre, D.J., Yagiz, K., Balicki, A., Kim, C. and Morre, D.M. 2007. ECTO-NOX target for the anticancer isoflavene phenoxodiol. Oncol. Res. 16:299-312.
  • Cooper, R., Likimani, T.A., Morre, D.J. and Morre, D.M. 2007. Catechins and caffeine in tea: A review of health risks and benefits. In: Caffeine and Activation Theory. Effects on Health and Behavior. Smith, B.D., Gupta, U. and Gupta, B.S., eds., Chap. 14, pp. 351-364, CRC Press, Taylor & Francis Group, Boca Raton, FL.
  • Yagiz, K., Morre, D.M. and Morre, D.J. 2006 Transgenic mouse line overexpressing the cancer-specific tNOX protein has an enhanced growth and acquired drug-response phenotype. J. Nutr. Biochem. 17:750-759.
  • Morre, D.M. 2006 The role of nutrition in the older individual. Gerontology: Perspectives and Issues, 3rd ed., Wilmoth, J. and Ferraro, K., eds. Springer Publishing Co., NY.


Progress 10/01/05 to 09/30/06

Outputs
The overall goal of this project is to understand why cells of older individuals produce greater amounts of reactive oxygen species than cells of younger individuals. Our laboratory has identified an aging-related cell surface protein, arNOX, that is associated with sera, lymphocytes, perspiration and saliva of individuals of 50 years or older. arNOX is postulated to link the accumulation of lesions in mitochondrial DNA to cell surface accumulations of reactive oxygen species as one consequence of its role as a terminal oxidase in a plasma membrane electron transport chain. The protein is capable of directly reducing ferric cytochrome c through the generation of superoxide. Confirmation that reduction of ferric cytochrome c in the assay was due to superoxide was achieved by adding superoxide dismutase to remove the superoxide as it was generated. Previous work had shown that both sera and lymphocytes of older individuals contained the arNOX proteins. The findings have been extended to that of perspiration and saliva where arNOX activity based on assays of cytochrome c reduction and inhibition by superoxide dismutase is observed in individuals <50 years old but not in individuals >30 years old. Attempts to purify the arNOX protein has shown the protein to be present but no meaningful sequence has been obtained.

Impacts
An aging-related protein, arNOX, is being characterized in order to better understand its mechanism(s) of action at the molecular level. After it has been characterized and sequenced, it is possible that natural and synthetic compounds can be used to target it with the potential of decreasing the incidence of aging-related diseases, such as atherogenesis, in the older individual.

Publications

  • Morre, D.J. and Morre, D.M. (2006) Aging-related cell surface ECTO-NOX protein, arNOX, a preventive target to reduce atherogenic risk in the elderly. Rejuvenation Res. 9:231-236.
  • Morre, D.M. and Morre, D.J. (2006) Catechin-vanilloid synergies with potential clinical applications in cancer. Rejuvenation Res. 9:45-55.
  • Cooper, R., Morre, D.J. and Morre, D.M. (2005) Medicinal benefits of green tea: Part I: Review of noncancer health benefits. J Altern. Complement. Med. 11:521-528.
  • Cooper, R., Morre, D. J. and Morre, D.M. (2005) Medicinal benefits of green tea: Part II Review of anticancer properties. J. Altern. Complement. Med. 11:639-652.
  • Orczyk, J., Morre, D.M. and Morre, D.J. (2005) Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H. Mol. Cell. Biochem. 273:161-167.
  • Morre, D.J. and Morre, D.M. (2006). tNOX, an alternative target to COX-2 to explain the anticancer activities of non-steroidal anti-inflammatory drugs (NSAIDS). Mol. Cell. Biochem. 283:159-167.
  • Morre, D.M. and Morre, D.J. (2006). Anticancer activity of grape and grape skin extracts alone and combined with green tea infusions. Cancer Letts. 238: 202-209.
  • Morre, D.J. and Morre, D.M. (2006) Membrane redox as an essential component of how cells increase in size following cell division. Acta Biologica Szegediensis 50:75-77.
  • Morre, D.M. and Morre, D.J. (2006) Role of membrane redox in aging-related diseases. Acta Biologica Szegediensis 50:67-69.
  • Morre, D.M. and Morre, D.J. (2006) Anticancer activity of grape and grape skin extracts alone and combined with green tea infusions. Cancer Lett. 238:202-209.


Progress 10/01/04 to 09/30/05

Outputs
The overall goal of this project is to understand why cells of older individuals produce greater amounts of reactive oxygen species than cells of younger individuals. Our laboratory has identified a family of cell surface proteins (designated as ECTO-NOX proteins) which have both hydroquinone (NADH) oxidase and protein disulfide-thiol interchange activity. Preliminary work in our laboratory with sera and lymphocytes of individuals of >70 y has identified a protein of this family to be an aging-related NADH oxidase (arNOX). arNOX is a member of the ECTO-NOX family of growth-related and time-keeping proteins that is associated specifically with aging cells in culture and in skin and aging cells in general. The arNOX is uniquely characterized by an ability to generate superoxide and can, therefore, contribute significantly to the imbalance between production of reactive oxygen species and antioxidant defense by producing reactive oxygen at the cell surface to greatly accelerate aging-related changes including atherogenesis and other aging phenomena. A circulating form of arNOX increases markedly in sera and lymphocytes of individuals age 65 y or older. Reduction of ferric cytochrome c by superoxide was employed as a standard measure of superoxide formation. When superoxide dismutase was added to remove the superoxide as it was generated, the reduction of ferric cytochome c was prevented to confirm that the assay was due to superoxide. The arNOX activity of aging cells and in sera is inhibited by Coenzyme Q8, Q9 and Q10 but not by Q0, Q2, Q4, or Q7. A reactive band at a molecular weight of ca. 22 kD was observed in lanes by Western blot analysis from samples of sera of older individuals. Attempts at purifying the protein catalyzing the aging-related cytochrome c reduction has shown the protein to be present. However, no meaningful sequence was obtained when the target band on the PVDF membrane was excised and submitted for N-terminal amino acid sequencing, suggesting that the arNOX protein is blocked to N-terminal sequencing.

Impacts
A member of a protein family identified by our laboratory has been identified as being aging-related and is being termed as arNOX (aging-related NADH oxidase). The protein is being characterized in order to better understand its mechanism(s) of action at the molecular level. Once this is understood, it is feasible to use natural and synthetic compounds to target it with the potential of decreasing the incidence of atherogenesis in the elderly.

Publications

  • Morre, D.J. and Morre, D.M. 2004. Editorial. Plasma membrane electron transport. A metabolic process deserving of renewed interest. BioFactors 20:183-187.
  • Axanova, L., Morre, D.J. and Morre, D.M. 2005. Growth of LNCaP cells in monoculture and coculture with osteoblasts and response to tNOX inhibitors. Cancer Lett. 225:35-40.
  • Orczyk, J., Morre, D.M. and Morre, D.J. 2005. Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NADP+/NAD(P)H. Mol. Cell. Biochem. 273:161-167.
  • Kim, C., Layman, S., Morre, D.M. and Morre, D.J. 2005. Fourier transform infrared and circular dichroism spectroscopic analysis underlie tNOX periodic oscillations. Nonlinearity Biol. Toxicol. Med. 3:299-322.
  • Cooper, R., Morre, D.J. and Morre, D.M. 2005. Medicinal benefits of green tea. Part I: Review of noncancer health benefits. J. Alt. Comp. Med. 11:5211-528.
  • Cooper, R., Morre, D.J. and Morre, D.M. 2005. Medicinal benefits of green tea. Part II: Review of anticancer properties. J. Alt. Comp. Med. 11:638-652.


Progress 10/01/03 to 09/29/04

Outputs
This is a new project in which we are working with an aging-related cell surface protein that generates superoxide (based on reduction of cytochrome c) and this activity is inhibited by ubiquinone (Coenzyme Q10)

Impacts
We anticipate to understand at the molecular level why cells of older adults produce greater amounts of reactive oxygen species than cells of younger adults.

Publications

  • No publications reported this period