Blackberry's fruit prevent aging

M.Khayami[1] , E.Nikkhah[2]

Department of biology, urmia university, Iran

The fruit of blackberry (Morous nigra L.) use in ethno botany as a medicinal plant in Iran. It could prevent aging by cleaning the liver from toxin and refine the blood.

Blackberry contains anthocyanins which are strong antioxidant. Anthocyanins are a group of naturally occurring phenolic compounds responsible for the attractive color of many fruits and vegetables. Extracts of these natural pigments (e.g., red grape, blackberry, blackcurrant and elderberry) are used as food colorants and components of pharmaceuticals. Blackberry originated from Iran and turkey; grow also in Europe and Middle Asia, South Asia and North Africa. the tree of blackberry has a height up to 15m.the trunk has a dark grey color and the leaves are dark green with short petiol, the flowering time is in march to April and fruit maturation is in august to September.

In this research project we used the methods of Chiriboga and Francis (1970) using of 0.1% acidified ethanol with hydrochloric acid.

The filtered product then placed in a balloon in a vacuum evaporator at 35 C to separate the ethanol-acid solvent. The product then transferred to a 1000ml balloon and brought the volume to 1000 ml using distilled water. The product then centrifuged at 8000 rpm, the supernatant was separated and kept for further analysis.

The total content of monomeric anthocyanins in blackberry extract was determined using the pH-differential method. (R.E. Wrolstad et al., 2001) Absorbance was measured in a spectrophotometer at 510 and 700 nm, in buffers at pH=1.0 and 4.5,                          using A= [(A510–A700) pH=1.0– (A510–A700) pH=4.5] with the molar absorption coefficient of cyanidin-3-glucoside of 29 600 mol^–1L cm^–1.

The result was expressed as milligram of cyanidin-3-glucoside equivalents (CGE) per gram of extract.

Total anthocyanin =A×MW×DF×10³ / є×L = 274.1mg/L

MW=Molecular weight

DF=Dilution factor

L=Path length

Є=Molar absorption coefficient of cyanidin3-glucosid.

Living to the advanced age of eighty-five and beyond without developing age-related diseases is strived for by many but achieved by few. Fortunate individuals who achieve this goal may either lack susceptibility factors that contribute to age-related diseases in the majority of people, or may possess resistance factors that enhance their ability to resist disease and prolong lifespan. Healthy aging is considered to be a complex phenotype that is determined intrinsically by genes for which expression and physiological consequences are modified by extrinsic factors such as lifestyle and environment. Variation in such genes may contribute to a variety of processes that result in long-term good health.

A multidisciplinary team of researchers with expertise in genomics, genetics, gerontology, biostatistics, bioinformatics and cancer research has been formed to study healthy aging. Our objective is to identify “healthy aging” genes that contribute to exceptional health in old age. We will assess whether genetic variation including single nucleotide polymorphisms (SNPs) and haplotypes in these genes are associated with healthy aging by performing case/control based genetic association tests comparing exceptionally healthy seniors to ordinary middle-aged controls. The cases are healthy seniors over eighty-five chosen for freedom from cancer, cardiovascular disease, pulmonary disease, diabetes and Alzheimer disease who have a good quality of life.

            Candidate genes are selected based on gene expression analysis (SAGE) of long-lived C. elegans daf-2 mutants as well as from the literature. Exons and gene regulatory regions of candidate genes will be re-sequenced in forty-seven healthy seniors for discovery of novel genetic variations (SNPs, Insertions, and Deletions). A subset of variations will be chosen based on inter-marker linkage disequilibrium and putative functional relevance, and genotyped in 500 cases and 500 controls.

Aging is characterized by a breakdown of many fundamental cellular and system-wide processes. DNA-damage repair efficiency has been linked to aging in many organisms; additionally, many model systems for accelerated aging involve mutations in the DNA-damage repair machinery.  Here, we examine the efficiency of DNA damage repair in muscle stem cells residing in old versus young environments.  Myoblasts (or muscle fiber progenitor cells) damaged by UV irradiation (causing single-strand breaks) or X-ray irradiation (causing double-strand breaks) were grown in culture under conditions mimicking a young or aged environment.  We find that the aged environment significantly reduces a cell’s ability to repair both double-strand and single-strand breaks. Thus, the "age" of the environment in which a cell exists, rather than the age of the cell itself, determines its ability to repair DNA damage. 

These results suggest that stem cells maintain their inherent ability to repair DNA with age, but are strongly influenced by the particular microenvironment in which they reside.  For stem cell therapeutic purposes, this suggests that the age of the systemic environment (such as the circulating blood supply) needs to be considered when applying treatments.

The etiology of breast cancer is still poorly understood. The observation that breast cancer recurrences can occur more than ten years after primary surgery suggests that a subset of cells, possibly stem cells may be of central importance in the pathophysiology of breast cancer. Indeed, in the last few years, cancer stem cells have been identified in a several human neoplasms including leukemia, breast and brain tumors. The central characteristics of stem cells are their capacity for unlimited self-renewal and the retention of pluripotency to differentiate. While the mechanisms underlying stem cell differentiation have been well examined, and at least two families of proteins, Bmi-1 and Wnt, have been identified to be involved in the maintenance of the tumor stem cell phenotype, less is known about how stem cells avoid cell death and resist genetic damage. The aim of this study is to identify and characterize putative cancer stem cell genes that correlate with the ability to avoid cell death and to determine their role in tumor development and progression. Two of the major mechanisms of cell death, apoptosis and senescence, are regulated by a series of tumour suppressor genes often associated with a response to DNA damage. We therefore analyzed a panel of DNA repair and tumour suppressor proteins in a population of breast cancer stem cells derived from the MCF7 breast carcinoma cell line. Our results show that the stem cells express markedly lower levels of p53, p21 and pRB, key proteins required for signaling apoptosis and senescence. Moreover, the expression of p21 and pRB was further reduced in the stem cells one hour after irradiation with 1 Gy g-radiation. Thus, compared to the general population of MCF7 cancer cells, the cancer stem cell immortality may be linked to a reduced tendency to undergo apoptosis and senescence.

Recent advances in physical activity (PA) monitoring have allowed researchers to address questions about the amount of PA obtained by certain populations as well as how that PA is accumulated. However, there has been limited work done using objective tools to profile the activity patterns of older adults. Therefore, the goal of this study was to assess the PA profile of a sample of older adults.  First we conducted a laboratory-based assessment of the relationship between output from a commonly used accelerometer (Actigraph 7164) and measured energy expenditure while walking in 38 older adults (20 women and 18 men; mean age 70.8 (3.5) years). Then PA was objectively assessed in 33 of the participants with 7 consecutive days of minute-by-minute accelerometry measurements. The raw accelerometer counts were categorized as moderate to vigorous physical activity (MVPA) based on the mean counts (1041) associated with a walking speed of 2 mph and a mean oxygen uptake of 13 ml∙kg^-1∙min^-1. Total minutes of MVPA (counts>1041) were further examined to determine how active minutes were accumulated. Long bouts of activity were defined as 20 or more consecutive minutes, short bouts 10-19 minutes, and all remaining minutes of MVPA were labeled sporadic. Men obtained on average 75 (±39) minutes of MVPA per day, with 62% of those minutes occurring as sporadic activity, 11% as short bouts and 27% as long bouts. Women obtained 63 (±26) minutes of MVPA per day with 70% of that as sporadic activity, 9% as short bouts, and 21% as long bouts. Males accumulated on average 8.9 hours of sedentary time (excluding sleep) per day while females accumulated 7.4 sedentary hours per day. These results demonstrate the detailed information that can be obtained with time-stamped, minute-by-minute accelerometer data, including not just total amount of PA but also when and how that PA is accumulated. This tool has important implications for understanding older adult activity patterns and developing appropriate PA guidelines.

The inevitable process of senescence is characterized by a multitude of conditions ranging from muscular wasting to increased incidence of disease.  However, the primary interest within the context of this research is the consequences of aging on brain function.  The Free Radical Theory of Aging suggests oxidative stress caused by endogenous metabolic processes accumulated over the lifetime of an organism is to blame for the age-related degradation of cells.  This research focuses on the redox modulation that occurs within neurons as an organism senesces, which may be responsible for the cognitive decline often observed in the aged.

The action potential is the fundamental unit of information in the central nervous system. Its generation, waveform characteristics, and frequency are integral to neural network health and function. Ion channels determine an individual neuron’s excitability, or capacity to generate action potentials (APs). Interestingly, not all neurons within a single organism’s CNS age at the same rate, suggesting the (dys)function that accompanies aging occurs at the sub-cellular level – namely the ion channel.

 As lipid-embedded proteins, these channels are subject to the same oxidative stress as all cellular components exposed to reactive oxygen species (ROS).  Voltage gated potassium channels play a particularly important role in AP frequency and generation, and are known to be subject to redox modulation (Ruppersberg et al., 1991; Kourie, 1998).As such, we propose intracellular redox modulation of ion channels and neuronal excitability are inextricably linked within the context of aging. By employing electrophysiological techniques to identified molluscan neurons in vivo, the effect of various oxidizers and reducers on neuronal excitability has been examined.

       Bone marrow stromal cells (BMSC) are the adherent cells of the bone marrow and contain a subpopulation of mesenchymal stem cells. BMSC can be used in the treatment of a multitude of genetic and acquired diseases after gene transfer ex vivo, followed by autologous transplantation. BMSC has the potential to substitute for many tissue types and to be used in the treatment of many diseases due to their ability to regenerate and differentiate. Genes for osteoinductive growth and differentiation factors have been used to genetically modify BMSC in order to enhance their osteogenic differentiation and promote the release of proteins involved in bone remodeling. Genetic modification of BMSC has been primarily achieved by using viral carriers including retroviruses, lentiviruses, or adenoviruses, but oncogenic and immunogenicity-related problems still exist. Non-viral gene delivery to BMSC can eliminate these virus-related problems. Polymers are safe alternatives to viral-based carriers, since they do not integrate into the cellular genome. They are easier to manufacture and have large DNA carrying capacities. The positively-charged polymers can interact electrostatically with negatively-charged DNA molecules and facilitate passage of DNA through cell membrane, which is negatively-charged under physiological conditions and impedes the uptake of naked DNA.

The objective of the current study is to demonstrate the BMSC transfection efficiency of a new polymeric gene carrier, Poly(L-Lysine)-Palmitic Acid (PLL-PA) conjugate, by using a model plasmid that expresses Enhanced Green Fluorescent Protein (pEGFP). We hypothesized that Palmitic Acid (PA) conjugation could enhance the gene delivery capacity of the cationic polymer PLL due to increased membrane interactions created by the lipophilic PA and we demonstrated the promising transfection ability of PLL-PA as a gene delivery agent.

Calcium (Ca) plays an essential role during development but also during aging. The maintenance of cellular Ca gradients represents a major energetic expense, with SERCA function observed to decrease with age (Squier, 2001). The rate of refilling of ER stores declines with age (Pottorf et al., 2000b; Vanterpool et al., 2005). Loss of intracellular Ca homeostasis contributes to generation of reactive oxygen species and protein aggregation, both of which are responsible for numerous aging phenotypes, such as damage to cellular proteins and formation of amyloid deposits. Aging in the nervous system is associated with increased amounts of oxidative stress (Mattson, 2007). The ubiquitous involvement of Ca in multiple pathways has led investigators to suggest that dysregulation of intracellular Ca homeostasis may be the primary mediator of age-related neural degeneration (Pottorf et al., 2000a). As well, decline in chaperone function may well contribute substantially to Alzheimer’s disease and may underlie the physiological deficiencies of aging (Erickson et al., 2006).

Calreticulin is a ubiquitously expressed calcium (Ca) binding chaperone of the endoplasmic reticulum (ER). Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Calreticulin-deficient embryonic stem (ES) cells can not be efficiently differentiated into cardiomyocytes and demonstrate impaired myofibrillogenesis. Cardiac differentiation of calreticulin-deficient cells can be restored by expression of full length calreticulin or the calreticulin domain involved in Ca buffering in the ER. This indicates that ER Ca stores may play an important role in cardiac differentiation of ES cells. Molecular analysis of calreticulin-deficient ES cells revealed that they have impaired ER–dependent Ca homeostasis. This in turn, affects Ca-dependent transcriptional processes. MEF2C and NF-AT transcription factors are not efficiently translocated to the nucleus. In this study, we show that this is due to low or no activity of the Ca–dependent phosphatase calcineurin and to a reduced activity in the MAPK signaling pathway in calreticulin-deficient ES cells.

In the early stages of cardiac development, calreticulin is required to ensure normal Ca release from the ER and thus proper activation of calcineurin and its associated transcriptional pathways. Over-expression of activated-calcineurin renders calcineurin-dependent pathways less reliant on sustained elevation of cytoplasmic Ca and therefore calcineurin-dependent pathways should be active even in the absence of Ca release from the ER. These observations provide a molecular explanation for the embryonic lethality witnessed in calreticulin-deficient mice and indicate that calreticulin is a key upstream player in the Ca signaling cascades which regulate calcineurin activity. It also highlights the importance of both calreticulin and calcineurin in Ca-dependent signaling cascades during early cardiac development. In the absence of calreticulin, expression of activated-calcineurin in the heart permits adequate progression of cardiac development during embryogenesis. Combined, these findings show that calreticulin and calcineurin play important roles in cardiogenesis of ES cells.

The Endoplasmic Reticulum (ER) is the site where the critical function of posttranslational modification, protein synthesis, and protein folding take place [1].  Aberrant or incorrect folding of proteins leads to congenital and adult pathologies such as Alzheimers, Huntingtons and Parkinsons[2].  Additionally, a new paradigm associates a specific loss of the capacity of the ER to fold proteins with the aging process [3].  Calnexin (CNX) is a type 1 integral membrane protein found in the ER that acts as a chaperone to correctly fold newly synthesized glycosylated proteins [1].  Targeted deletion of CNX results in live mice with severe motor defects [4].  In order to investigate the role CNX plays in the aging process, we looked at the activation and expression of CNX throughout development as well as the function of CNX as an ER chaperone.  The beta-galactosidase reporter gene was inserted into the CNX gene to create mice expressing the reporter gene under control of endogenous CNX promoter. This provided us with an experimental model where expression of beta-galactosidase reported the activation of the CNX gene during development. Embryos containing beta-galactosidase transgene were harvested at different times of gestation, prepared for histological analysis and stained for beta-galactosidase activity. A relatively high activity of beta-galactosidase activity was found in the heart and neuronal tissue at E.11, E.12, and E.13. High expression in the eye, neuronal tissue and the liver was also detected at E.15, E.17, and E.18. We also carried out RT-PCR analysis as well as immunostaining to confirm activation of the CNX gene. To determine if activation of the CNX gene, as reported by measuring of the beta-galactosidase activity, corresponded to expression of the CNX protein we carried out Western blot analysis of mouse tissue extracts with anti-CNX antibodies. In the adult mouse there was high expression of CNX in the liver but low expression in the heart. Additionally, CNX deficient mouse fibroblasts have increased cellular stress relative to wild-type as shown by an increase in chaperone proteins GRP78 and GRP94, 44% and 83% to wild-type, respectively. Cellular stress at the embryonic stage in CNX deficient mouse embryonic fibroblasts (MEFs) as relative to wild-type MEFs is not as pronounced. We concluded that at early stages of embryonic development the CNX gene is activated in neuronal tissues suggesting that CNX must be essential for neuronal differentiation during embryonic development. The ataxic features presented in the CNX-deficient mouse model may be explained by a chaperone function of CNX in neuronal differentiation.  When CNX is absent during embryonic development cellular stress is not as obvious, however, postnatally CNX may have an important chaperone function necessary for neuroprotection against misfolding diseases such as Alzheimers.

1.         Ellgaard, L. and A. Helenius, Quality control in the endoplasmic reticulum. Nature Rev. Mol. Cell Biol., 2003. 4(3): p. 181-191.

2.        Pind, S., J.R. Riordan, and D.B. Williams, Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem., 1994. 269: p. 12784-12788.

3.         Erickson, R.R., L.M. Dunning, and J.L. Holtzman, The effect of aging on the chaperone concentrations in the hepatic, endoplasmic reticulum of male rats: the possible role of protein misfolding due to the loss of chaperones in the decline in physiological function seen with age. J Gerontol A Biol Sci Med Sci, 2006. 61(5): p. 435-43.

4.         Denzel, A., et al., Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression. Mol. Cell. Biol., 2002. 22(21): p. 7398-7404.

Standing and reaching are critical components of many activities of daily living, but as a functional pair they may present both motor and cognitive challenges to older adults.  These challenges can be amplified in demanding contexts, such as standing on tiptoes or an elevated platform while making the reach.  Furthermore, successful reaches made in demanding contexts may both require and elicit increased control of postural kinetics.  While much research has examined the antero-posterior kinetics and kinematics for reaches directed along the A/P axis, less investigation has been done on the medio-lateral kinetics of forward reaching.  Adequate control in the M/L dimension is important for successful reaches, including those made in demanding contexts.  In this study, we examined displacement of the centre of pressure in the M/L direction relative to displacement in the A/P direction for 8 healthy older adults (69.5 +/- 7.7 years) and 5 healthy younger adults (22.0 +/- 1.9 years).  All reaches were to full arm’s length in an anterior direction, and subjects performed reaches while standing in contexts with either low or high demands on postural control.  Specifically, in the high demand context, participants had no vertical surface in front of their starting position on which to plant a recovery step.  We found that older adults made medio-lateral movements of greater amplitude, relative to their antero-posterior movements, than younger adults, regardless of contextual demands.  This increased relative magnitude of off-axis movement may be indicative of postural control deficits in standing reaching, and may contribute to the low confidence levels many older adults report for reaching activities in demanding contexts.