Epitalon Research Article – Part 2

This is the Epitalon Research Aticle – Part 2

Part 2 - Materials and methods

Animals

Female outbred Swiss-derived SHR 2-month-old mice (108 specimens) were purchased from the Rappolovo Animal Farm of the Russian Academy of Medical Sciences (St. Petersburg). The mice were kept in polypropylene cages (30×21×9 cm), 5 mice to a cage, at a temperature of 22±2◦ C. A regimen was followed of 12 hours of light and 12 hours of dark.

The animals received sterilized standard laboratory  feed (Anisimov et al. 2003) and tap water ad libitum. Mice were checked daily by animal care personnel and weekly by a veterinarian. The study was conducted in accordance with the regulations for ensuring the humane treatment of animals under the approval of the Committee on Animal Research of the N.N. Petrov Research Institute of Oncology.

Experiment

At the age of 3 months the mice were randomly divided into two groups, 54 animals in each, and they were individually marked. Mice of the control group were subcutaneously injected with 0.1 ml of 0.9% normal saline for 5 consecutive days every month, whereas the mice of the second group received subcutaneously 1.0 µg of Epitalon dissolved in 0.1 ml of saline. This treatment dosage and regimen were effective for the inhibition of spontaneous tumorigenesis in female CBA mice (Anisimov et al. 2001a).

Epitalon was synthesised in St. Petersburg, Institute of Bioregulation and Gerontology, by E.I. Grigoriev and was 99.8% pure. Four intact female SHR mice were euthanized at the age of three months to evaluate the initial level of chromosome aberrations. Additionally four mice from each group were euthanized at the age of 12 months for a cytogenetic study of chromosome aberrations in bone marrow cells (see below).

Once every 3 months, simultaneously with weighing, the amount of food consumed was measured. Thirty grams of food were given in each cage after cleaning and twenty-four hours thereafter the food that had not been consumed was collected from each cage and weighed. The mean amount of food (grams) consumed per mouse during this day was calculated for each group.

Once every three months, vaginal smears taken daily for two weeks from the animals were examined cytologically to estimate the phases of their estrous functions. In the same period, the rectal body temperatures of the mice were measured with an electronic thermometer, TPEM (KMIZ, Russia). Animals were observed until their natural death. The date of each death was recorded, and the mean life span, the age by which 90% of the animals died, and the maximum life span were estimated.

Cytogenetic study

Chromosomal aberrations in bone marrow cells were studied by a modified Ford’s method, described by Rosenfeld et al. (2001). Mice were sacrificed by etheranaesthesia. Both femurs of each mouse were dissected and bone marrow cells were flushed gently with 0.56% KCl solution into a centrifuge tube. Cells were treated for 20 min with hypotonic solution and fixed with an ethanol : acetic acid mixture (3:1). Slides were stained with 4% acetoorseine: 20–30 well spread anaphases were analyzedfor each animal and cells with chromosome breaks,acentric fragments, and other aberrations were evaluated at 1000×magnification under a light microscope (Leitz, Germany).

Pathomorphological examination

All animals that died, or were sacrificed when moribund, were autopsied and their skin and internal organs were examined. Neoplasias were classified according to the recommendations of the International Agency of Research on Cancer (IARC) as ‘fatal’ (i.e., those that directly caused the death of the animal) or ‘incidental’ (in cases where the animal died of a different cause) (Gart et al. 1986). All tumors, as well as tissues and organs with suspected tumors, were excised and fixed in 10% neutral formalin.

After routine histological processing, tissues were embedded in paraffin. Thin, 5–7 µm histological sections were stained with hematoxylin-eosine and examined microscopically. The experimental group to which the mouse belonged was blinded. Tumors were classified according to IARC recommendations (Turusov and Mohr 1994).

Statistics

Experimental results were statistically processed by the methods of variation statistics (Goubler 1978). The significance of discrepancies was defined according to Student’s t-criterion, Fischer’s exact method,χ2-analysis, and the non-parametric criterion of Wilcoxon–Mann–Whitney (Goubler 1978). To estimate discrepancies in neoplasm incidence, an IARC method of combined contingency tables calculated individually for the fatal and incidental tumors (Gart et al. 1986). For survival analysis, Cox’s method (Cox and Oakes 1996) was used. All reported test values for survival analyses are two-sided.

Survival models and estimations

The mathematical model used to describe survival is the Gompertz model with the survival function:

where parametersαandβare associated with demographic aging and initial mortality rate, respectively.

Parameters for the model were estimated from data using the maximum likelihood method implemented in the GAUSS statistical system (Gauss System 1994). Confidence intervals for the aging rate parameter estimates were calculated using log-likelihood functions (Cox and Oakes 1994).

How to Reverse Aging?

by Alex (Admin) 7 months ago 0 comments

Michio Kaku – How To Reverse Aging

Here’s a very interesting video you really need to see. It is about aging, or how to reverse aging. I am sure you are already familiar with Mr. Michio Kaku. If not, you should definitely watch this video now because there’s lots in common with what this website is all about, anti aging and life extension with the help of Pure Epitalon.

I am sure you will love it as much as I did. Enjoy!

And here’s the transcription of the “how to reverse aging” video if you prefer reading it:

Enzymes like Telomerase and Resveratrol, though not the Fountain of Youth unto themselves, offer tantalizing clues to how we might someday soon unravel the aging process.

Question: Do you think the enzyme Telomerase could be used to reverse the aging process in our lifetime? (Submitted by Paul Cellura)

Michio Kaku: Paul, Telomerase hit the headlines; however, I think we have to put it into perspective. It is not the fountain of youth; however, it is a significant breakthrough. We have to put it into a much larger perspective.

First of all, we know that DNA is sort of like a shoelace. It has plastic tips at the end.Every time a cell reproduces, the tips get shorter and shorter and shorter until finally they fray. And you know that your shoelace, without the plastic tips will simply fall apart. That’s what happens inside a cell. A cell, for example, your skin cell, will divide about 60 times, that’s called a Hayflick Limit. Then the cell goes into senescence and eventually dies.

Hayflick Limit

So in some sense, every cell has a biological clock. It is doomed to die after about 60 reproductions. However, Telomerase can eliminate some of the contraction of thechromosomes and the chromosomes can maintain their length. So at first you may say, “ah-ha! We can now defeat the biological clock.”

But not so fast, first of all, cancer cells also use Telomerase. Cancer cells are immortal. Cancer cells are immortal and that’s precisely why they kill you. Why are cancer cells so dangerous? Because they are immortal. They grow and they grow and they grow until they take over huge chunks of your body, meaning that your bodily functions cannot be performed and you die. So we have to make sure that when you hit ordinary cells with Telomerase that you don’t also trigger cancer in the process.

Now, also you have to realize that genes are also very essential for the aging process. It turns out that we know what aging is. Aging is the buildup of error. That’s all aging is. The build up of genetic and cellular error. And cells begin to age; they begin to get sluggish because genetic mistakes start to build up. Now cells; however, have a repair mechanism. They can repair damage to their cells; otherwise we would all basically rot very soon after birth.

However, even the repair mechanisms eventually get gummed up and then the cell really starts to get old as a consequence. So then the question is, can you accelerate cell repair? That is another branch of gerontology which is being looked at using genes and using chemicals to accelerate the repair mechanisms.

For example, if I take any organism on the planet Earth from yeast cells to spiders, insects, rabbits, dogs, and even monkeys now. And I reduce their caloric intake by 30%, they live 30% longer. In fact the only organism which has not yet been deliberately tested by scientists are homo sapiens. All the other species obey this basic rule. You starve them to death, they live longer. This is independent of Telomerase. This is a function of the wear and tear that we have on the cells. And this is the only known way of actually deliberately extending the lifespan of any organisms almost at will.

Now, what we want is a genetic way of mimicking this mechanism without having to starve yourself because how many people do you know would be willing to starve themselves in order to live 30% longer? Not too many. So then the question is, are there genes that control this process. And the answer is apparently, yes.

There’s something called the Sirtuin genes, Sir2 being the most prominent of them. They in turn stimulate certain enzymes, among them Resveratrol, which is found in red wine, for example. So this does not mean that drinking red wine or taking Telomerase is the fountain of youth. I don’t think that anyone has the fountain of youth yet.

Abstract

Sirtuin genes function as anti-aging genes in yeast, Caenorhabditis elegans, and Drosophila. The NAD requirement for sirtuin function indicates a link between aging and metabolism, and a boost in sirtuin activity may in part explain how calorie restriction extends life span. In mammals, one of the substrates of the SIR2 ortholog, SIRT1, is a regulator of mitochondrial biogenesis, PGC-1alpha. Indeed, the putative SIRT1 activator resveratrol has been shown to stimulate mitochondrial biogenesis and deliver health benefits in treated mice. I explore here how mitochondrial biogenesis may have beneficial effects on aging and, perhaps, diseases of aging. In particular, I speculate that SIRT1-mediated mitochondrial biogenesis may reduce the production of reactive oxygen species, a possible cause of aging, and offer two possible mechanisms for this effect. An understanding of how calorie restriction works may lead to novel drugs to combat diseases of aging. Source: http://www.ncbi.nlm.nih.gov/pubmed/18419308

What I am saying is, we are now finding pieces of the fountain of youth, tantalizing clues that mean that perhaps in the coming decades, we might be able to actually unravel the aging process. We don’t have it yet. Don’t go out to the drug store and stock up on these kinds of chemicals and enzymes thinking you’re going to live forever. However it is conceivable that in the coming decades we’ll come very close to finding it.

So, how to reverse aging naturally?

Pure Epitalon is very close to reversing aging, to the fountain of youth and now you too have a chance to try it and experience its numerous benefits.