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Post  quicksilvercrescendo on Tue 23 Mar 2010, 19:08

I will share with the semi-enlightened beings of the forum my considerations and recipe for my citrus-ginger-aid electrolyte anti-oxidant "sports" drink tonic a la extraordinaire.

I can remember back in the late seventies when Gatorade was being marketed as "the thing" to drink. A university in Florida supposedly did a study on athletes and their drink concoction was shown to be the space-age super drink of the decade providing the all-important "electrolytes". Why the comparison? Because my drink tastes a little like Gatorade and has a slight salty and off-limey taste.

Unfortunately, and much to the ignorance of the public, the Gatorade released on the market in glass bottles and powder form was not exactly the same as that used in the university study.
As the commercial brand contained refined white sugar, artificial colors and oleoresin. Oleoresin or esters are actual byproducts of petroleum manufacturing. Yummie perhaps, but not exactly healthy.

When it comes to store bought fruit juices, even not from concentrate, they have usually been pasteurized at temperatures which destroys their precious enzyme content. I believe anything over 114 F guarantees complete enzyme destruction.
Unless you are lucky enough to buy fresh squeezed or extracted juices that have not been pasteurized. In Florida, there was a brand that sold a four liter, or gallon, of orange juice that was fresh squeezed and kept refrigerated for purchase. This was convenient in times where one could not make their own in this amount. It would keep about a week in the fridge. Any longer and one would have to freeze a batch.

But it is even more ideal to make your own juices and drink them as soon as possible as oxygen exposure, light and heat all break down the nutrients of juices. Vitamin C in sliced fruit or fresh juice begins to break down and is significantly reduced in an hour or two due to oxygenation with the air. But without getting obsessive about it, making a big batch of juice for a three or four day stretch of storage in the fridge and in a good container is not a huge negative. Then you also have the astounding work of Linus Pauling with Vitamin C to consider as a benefit with this drink.

Orange and citrus juices are acid when they are ph tested. But when consumed and digested by your stomach's hydrochloric acid the mixture then transforms to high alkaline which is very beneficial for the body to have alkaline formed substances consumed. Orange juice also has an incredible cleansing action on the body and the lymph system. So much so, that someone that is internally toxic and not used to drinking fresh orange juice may feel bad at first due to cleansing reactions from the orange juice and the subsequent dumping of their body tissues toxic load into the lymph and circulatory system.

Now when you take a fruit and juice it. You are now separating it from some of its fiber. The liquid contains the sugars of the fruits. This means that when you drink just juice without its fiber, the sugar will release into the bloodstream quicker and spike it a bit. Whereas, a piece of fruit with its fiber will release much slower as the body will have to separate the sugar from the fiber. Those with sugar sensitivities may have to consider this. But, juice is also a concentrated form of the enzymes and vitamins which also will be delivered into the blood there is an advantage here if blood sugar is not a problem for you. But the fiber is also important. So juicing should never take the place of also eating the whole, unadulterated piece of fruit. Because the natural sugars in juice will release quicker and that one glass of juice may equal two to four pieces of fruit then one is advised to dilute the juice with 50% good water. This will also cut down on extreme cleansing reactions from occurring to quickly and also increases the amount of the beverage to be consumed. Also if one has diabetes or issues with candida, then fruit juices may not be the way to go.

Organically produce is always a plus as random test show that organic can contain three to six times more natural sugars and nutrients over conventionally grown. But, again, realize drinking conventionally grown fresh fruit juice will be light years better than drinking all the shit sold in stores.

So now for..."the drink"...

To make a batch that can last a few days or a small pitcher's amount...
You will need...

3 Large Oranges (or 2 oranges and three tangerines as a variation)
2 Lemons
2 Limes
One chunk of fresh ginger root about the size of half the length of your thumb, with rind removed.
One heaping tablespoon unfiltered raw honey
One eighth level teaspoon unrefined raw sea salt, or celtic sea salt.
A small bowl of ice cubes
Good water
, about the same amount as the amount of juice extracted from the fruit used in this dilute and double the mixture.

First. Finely chop the ginger root.
Place ginger root in a metal tea ball, preferably one made with steel screen material.
Place the tea ball filled with chopped ginger root, honey and salt in a medium-sized tea/coffee cup.
Boil water and pour water to fill the tea-coffee cup.
Let the ginger root, honey and salt steep in the hot water for about ten minutes. The salt will dissolve and the honey will melt.
And the goodness of the ginger will be extracted. Ginger has incredible properties for immunity and it has a cooling effect on the system when consumed in a beverage. Good for hot weather.
In the meantime, while this steeps, start with the next steps.

Cut in half all the citrus and juice the halves. I use a manual device and grind the halves over it to extract the juice. You can use a motorized device if you have.
Combine all the three or four types of citrus juices together in a tall glass or bowl.

So now I have my juices done and my tea mixture is done steeping. I remove the metal tea ball with ginger and toss the ginger.

Now in a blender I add the cup of hot tea mixture. I then add about a half to three quarter cup of ice cubes. I cover and blend until the ice has melted and this cools down the tea. So now, I can add my citrus juice without it being heated by hot tea for the tea is now quite cold. So I add my juices. I add another half cup of ice and I add the water to dilute the mixture. Use about as much water as you used fruit juice. This is a 100 percent dilution, and the ice also dilutes the problem it will still taste good. The use of tablespoon of honey will keep things sweet and you also want the salt used to be diminished in the flavor of the drink. I find that adding all the water will fill my blender too high so I just add enough water to cap it off at the top and blend. Then I add the drink to a covered pitcher and stir in the remainder of the water. Serve cold or with ice. It is cool because the blender will create a light foam that rises to the top of the drink when you pour a glass...somewhat reminiscent of the American product...the Orange Julius drink should anyone know what that is.

The good salt, not regular table salt, used in this beverage is added because when the salt hits the tongue and stimulates the salt receptors on the tongue, those receptors tell the nervous system and other parts of the body to get ready to absorb liquids and water. This increases the absorption of the drink. Salt also provides the sodium electrolyte.

Pure, raw unfiltered honey also has numerous health benefits...almost too many to list. Unless you are allergic to bees. Some don't like the taste of honey, but used in this recipe the final product usually has no taste of the honey in it. So most like it.

I sometimes steep a teabag of Chinese green, or better yet, Chinese white tea in the teacup with the ginger. To add to the anti-oxidant factors. The green tea will alter the flavor a bit and some may prefer just juice. The white tea is considered a step above green tea and it really does not alter the flavor of the beverage at all. Also, green tea can contain caffeine which is not always preferable, but the white tea does not. So better to go with a good quality white tea. This is good for immunity and for...the chi.

Don't be surprised when you first make this that you suck down the whole pitcher in a couple hours. Most people I have shared this recipe with do drink a lot right away. It is almost as if their body intrinsically needs a large dose of what is in the drink and that their bodies may be lacking and really craving some of the nutrients that are in it.


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Post  tgII on Tue 23 Mar 2010, 22:42

qsc, very cool and very useful, think I might wonder off to
the supermarket today for some oranges and grapefruit.

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Post  seraphim on Sat 27 Mar 2010, 23:03

Thanks for the drink quicksilvercrescendo.......aaaaahhh..

Here is a different kind of electrolyte drink, don't let the color fool you, your body will like to drink more than one cup of this. It's one of the most refreshing drinks.

Chlorophyll liquid
essential oil peppermint
ice cold water

Best made in a pitcher, about a gallon size. Add a dropper of chlorophyll. Around five drops of peppermint oil or taste to your likening.
Chlorophyll is the blood of plants, just switch the magnesium molecule for the iron and you have something like human blood.

But hands down the best electrolyte is fresh coconut juice, it is pretty much identical to human blood plasma.

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Post  seraphim on Tue 30 Mar 2010, 22:07

Randy Roach has written a remarkable book that provides a new dimension to our understanding of the history of physical culture by focusing on nutrition. Though it sprang to life outside of normal academic channels, Muscle, Smoke and Mirrors exhibits some of the most important qualities of scholarship - extensive research, comprehensive coverage, ample contextualization, and sound judgements. it is also intelligently written with an engaging conversational tone.

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Post  seraphim on Fri 02 Apr 2010, 21:16

Hi Blackbird, sorry to hear of that. You had a heck of a life? It's really sad to see all those children with no homes, and then there are so many one's that go hungry and are abused. It just can't go on.
Yeah it would be good to know if that is a chip! And what they did to you. Would you do hypnosis?

Flames, I have never gone to the doctor, only when young. My parents got me vaccinated when, luckily it was only a couple of injections back then, not the 30 plus they have now! I can take very good care of myself, the only way I would go is if I needed to be stitched up or repaired. Laughing
You know I remember there used to be a poster on the mtsar who had bad anxiety or panic attacks and he found a person that really helped him, he's referenced on the web, I'll see if I can find that.
Oh and yeah it really is all about the money for the doctors nowadays.

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Post  seraphim on Sun 04 Apr 2010, 07:54

I believe our lives are pre planned somewhat (the setups are constant) and we have very little choices. What to do about that??

But it's great that you were born, and want to break free. If you post on forums like these, which I never in a million years would have done, then you are so far ahead of the mass.

Oh I was going to say I also would like to find out how to make my own penicillin, because getting a bacterial infection (dirt in a cut, bacteria in a cavity) is very serious and calls for a doctors visit. Doing the antibacterial herbs are good but they are not always around.

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Post  quicksilvercrescendo on Sun 25 Apr 2010, 20:25

Nutrasweet...made from the excretion of genetically modified E. coli bioweapon strain...

Process for producing aspartame.
European Patent Application EP0036258
Kind Code:
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Recombinant Protein
Custom Recombinant Protein Expression in E.coli.

The artificial sweetener aspartame, a dipeptide with the formula Asp-Phe-me, is produced using a cloned micrcorganism. A DNA which codes for a large stable peptide comprised of the repeating amino acid sequence (Asp-Phe)n is inserted into a cloning vehicle which in turn is introduced into a suitable host microorganism. The host microorganism is cultured and the large peptide containing the repeating Asp-Phe sequence is harvested therefrom. The free carboxyl group of the large peptide is benzylated and then hydrolysed to benzyl Asp-Phe dipeptides. This dipeptide is methylated and then debenzylated to form aspartame.

1. A method for producing aspartame, comprising, synthsizinq double stranded DNA in which a coding strand has alternating codons for Asp and Phe, said codons being of a number sufficient to produce a polypeptide which is stable in a predetermined host microorganism, insertinq said DNA strand into a cloning vehicle so that resulting chimera directs the synthesis of said protein, introducing said chimera into said predetermined host microorganism and cultivating said microorganism to produce said stable polypeptjde, harvestinq said stable polypeptide from said host microorganism esterifying the free carboxyl group of aspartic acid by benzylation, fragmenting said polypeptide to produce benzyl-Asp-Phe dipeptides, methylating the carboxyl group of the Phe moiety, and debenzylatinq the aspartic acid carboxyl group by hydrogenolysis.

2. A method according to Claim 1 wherein the carboxyl group of the Phe moiety is methylated by protectinq the carboxyl group of Asp, breaking the Phe-Asp hond, methylating the carboxyl group of the Phe moiety, and deprotecting the carboxyl group of the Asp moiety.

3. A method according to Claim 2 wherein said carboxyl group is protected by esterification.

4. A method according to Claim 2 wherein said carboxyl group is protected with a benzyl group or substituted derivatives thereof.

5. A method according to Claim 2 wherein said deprotection is accomplished by hydrogenolysis.

6. A method according to Claim 1 wherein said polypeptide is fragmented by digestion in a medium which breaks specific peptide links not including Asp-Phe or Phe-Asp bonds.

7. A method'according to claim 6 wherein said medium contains CNBr.

8. A method according to claim 6 wherein said medium includes an enzyme which breaks said specific peptide links.

9. A method according to claim 8 wherein said medium contains trypsin.

10. A method according to claim 1 wherein said host is E.coli.

11. A method according to claim 1 wherein said host is B.subtilis.

12. A method according to claim 1 wherein said host is E.coli K12.

13. A method for producing aspartame, comprising, cultivating a microorganism which produces a protein with a segment having a sequence (Asp-Phe) n, harvesting said protein segment, esterifying the free carboxyl group of aspartic acid by benzylation, fragmenting said polypeptide to produce benzyl-Asp-Phe dipeptides, methylating the the carboxyl group of the Phe moiety, and debenzylating the aspartic acid carboxyl group by hydrogenolysise

14. A microorganism which produces a protein with a segment having a sequence (Asp-Phe)

15. A microorganism according to claim 14 wherein said microorganism is a strain of E.coli.

16. A microorganism according to claim 14 wherein said microorganism is a strain of E.coli K12.

17. A microorganism according to claim 14 wherein said E.coli contains a plasmid which directs the synthesis of said protein.

18. A microorganism according to claim 14 wherein said microorganism is a strain of B.subtilis.

19. A microorganism according to claim 14 wherein said B.subtilis contains a plasmid which directs the synthesis of said protein.


PROCESS FOR PRODUCING ASPARTAME This invention relates to the production of artificial sweeteners and more particularly to the production of sweet tasting peptides through the use of genetically manipulated microorganisms.

The most widely used and cheapest sweetener presently available is sucrose, generally derived from cane or beet. However, some people such as diabetics must severely limit or abstain from the consumption of sucrose. Moreover, medically deleterious effects of sucrose are being described increasingly in medical journals as well as in the popular press Not least of the harmful effects of sucrose is its contribution to the problem of obesity.

Various so-called artificial sweeteners have been developed as a substitute for sucrose. At one time such sweeteners as saccharine and the cvclamates were widely distributed and held promise as a means of limiting consumption of sucrose. Recent research, however, has led to the suggestion that saccharine and cyclamates may be carcinogenic and, consequently, the government is questioning their use for many purposes.

The prohihitions on use of such sweeteners have forced many food processors to return to the use of sucrose.

This has created significant inconvenience for those persons such as diabetics, who cannot tolerate sucrose, and for those persons trying to control their weight.

A dipeptide, having the structure Asp-Phe-me, is described in Patent No. 3,492,131 issued to J. H.

Schlatter. This dipeptide, aspartame, has been found to be from 100 to 200 times sweeter than sucrose.

Aspartame is not only sweeter than sucrose, but is preferable as a food to sucrose. While sucrose can provide the body with little more than energy, aspartame is composed of amino acids, the building blocks of body proteins, and like other proteins is broken down by the digestive enzymes in the stomach to its constituent amino acids thus providing nutritive value. This fact also makes it unlikely that aspartame will be found to have carcinogenic properties, such as have been wound in saccharine and cyclamates which are not simil¬arl digested.

For these reasons, aspartame holds significant promise in replacing sugar as a sweetener. However, because sucrose is a relatively inexpensive substance, aspartame, if it is to gain widespread commcial acceptance, must be produced for a price which is reasonably competitive with sucrose.

One way of producing aspartame is by using known peptide synthesis techniques. Hower, the synthesis of specific peptide chains is generally a tedious and expensive process. While amino acids such as aspartic acid and phenylalanine are re;Sily available and while the formation of peptide bonds 3 easily achieved, correct synthesis of peptide sex fences involves intensive protecting and deprotec ing of alpha amino, alpha carboxyl and side chain group,. Even the production of a simple dipeptide such as i:;p-Phe-me requires several protecting and deprotect:tg steps.

Thus, although relatively high priced aspartame may be accepted by those whose health requires i such a high price places a limit on the commercial poc:ntial of this product. It would, therefore, be highly Jeirable to have an inexpensive and convenient methods fcr producing aspartame.

Recent techniques have made possible the introduction of foreign genetic material into microorganisms which then produce the protein or proteins for which such foreign genetic material codes.

The genetic code, which is based on sequence combinations of four possible nucleotide bases on the reading strand of a double-stranded DNA molecule, is now well known. Each sequence of three nucleotide bases is called a codon and for each specific amino acid, one or more codons exist. The four possible nucleotide bases of DNA are thymine, adenine, guanine and cytosine, which will hereinafter be referred to by their common abbreviations T, A, G, and C. The non-reading strand or complementary strand contains bases which are "complementary" to those in the reading strand. In the DNA molecule, C complements G, T complements A, G complements C, and A complements T.

It is known that the nucleotide base sequence GAC comprises a codon for aspartic acid (Asp). It is also known that the nucleotide base sequence TTT comprises a codon for phenylalanine (Phe) . Inserting such codons in the DNA of a microorganism, preceded and followed by appropriate processing or termination codons, under appropriate control, and in the correct reading frame, would result in the microorganism producing the dipeptide Asp-Phe as part of its own protein producing processes.

Inserting a DNA segment coding for the dipeptide Asp-Phe as suggested above would, however, be commercially unsuited for the production of Asp-Phe.

Because the natural digestive enzymes of an organism degrade or destroy the unnatural dipeptide, the likelihood of substantial product recovery is low. This is compounded by the fact that the sequence coding for Asp-Phe would represent only a minute fraction of the organism's DNA and significant amounts of Asp-Phe would not be produced.

An object of the invention is to provide an improved process for producing aspartame.

Another object is to provide a method for producing commercial quantities of aspartame using recombinant DNA.

Another object of the invention is to produce a microorganism from which substantial amounts of the dipeptide Asp-Phe may be derived.

The above objects are achieved by inserting into a cloning vehicle a synthesized DNA segment which codes for a protein segment of the formula (Asp-Phe) where n is a large number. The resulting chimera is introduced into a living organism which in its changed form will produce a correspondingly large protein with the segment (Asp-Phe) n After benzylation of the free carboxyl groups, the protein is appropriately cut into dipeptide segments (Asp-Phe), methylated and debenzylated to form the peptide Asp-Phe-me which is useful as a sweetener.

So that the invention may be more fully understood1 the invention will now be described in greater detail.

For a microorganism to produce a peptide having a long chain of the repeating sequence (Asp-Phe) n the organism must have a strand of DNA which has alternating codons which code for aspartic acid and phenylalanine.

Furthermore, such a strand must be inserted in a DNA segment in an appropriate position relative to promoters and operators and in the correct reading frame so that the genetice code is transcribed to messenger RNA and translated to form the desired protein. The promotors and operators may be synthesized along with the sequence (Asp-Phe) n as part of the inserted strand, or may be part of the cloning vehicle in which the strand is inserted.

In the genetic code, the codons TTT and the codons TTC code of phenylalanine. The codons GAT and GAC code for aspartic acid. Thus, for example, a microorganism having a DNA segment in the reading strand (GAS-TTT) n properly located will produce a peptide with a long segment of the formula (Asp-Phe) n DNA segments suitable for cloning are obtained from other organisms or must be synthesized. As there is no known natural source for a DNA strand which codes for the repeating protein sequence Asp-Phe, an appropriate DNA chain must be synthesized.

Single stranded DNA chains may be built-up in a stepwise method. A preferred method is a modified phosphotriester method described by K. Itakura, C. P.

Bahl, N. Katagiri, J. Michniewicz, R. H. Wightman and S.

A. Narang. Can. J. Chem. 51,3649 (1973). Such a synthesis may be used to produce a DNA chain with the exact sequence of nucleotide bases required.

A limitation on the modified triester method is that it is usually quite difficult to produce nucleotide base chains over about 15 or 20 bases. As it is desirable to insert a chain which codes for (Asp-Phe)n where n is large enough to confer stability on the protein, it is usually preferable to synthesize shorter nucleotide sequences and join them together.

A double stranded segment of DNA having the six nucleotide base sequence (GAC-TTT) is polymerized (CTG-AAA) to end would result in a chain having the formula (GAC-TTT) . However, there is no suitable way to join CTG-AAA)n a number of such chains together in invariably the correct order. For example, two such chains could join GAC-TTT-AAA-GTC together to form the sequence CTG-AAA-TTT-CAG instead GAC-TTT-GAC-TTT of CTG-AAA-CTC-AAA Accordingly, for reasons hereinafter more fully discussed, it is preferable to synthesize a pair of single stranded 12-base nucleotide chains. The first is the coding sequence GAC-TTT-GAC-TTT and the second is the sequence AAA-CTG-AAA-CTG.

The 12-base coding nucleotide sequence GAC-T?T-GAC-TTT is not the only sequence which would alternately code for Asp and Phe because, as is well known in the art, there are two possible codons for both aspartic acid and for phenylalanine. However, the above 12-base coding chain is chosen for simplicity of synthesis.

The 12-base nucleotide coding chain may be formed by the stepwise addition of nucleotides as described by Narang, et al. However, it is simpler and thus preferable to form the six-base chains with the sequence GAC-TTT and join two such chains together. The dimerization of two six-base nucleotide chains is accomplished through the use of mesitylene sulfonyltetrazole as a coupling reagent, as described by J. Stawinski, T. Hozumi and S. A. Narang, Can. J. Chem., 54, 670 (1976). The six-base DNA chain is synthesized stepwise by the modified triester method.

The base sequence AAA-GTC-AAA-GTC is similarly formed. This sequence comprises a segment of the DNA strand complementary to the (GAC-TTT) n strand, but is offset in relation to the GAC-TTT-GAC-TTT segment. The reason for this will become apparent below.

DNA exists in nature primarily as double-stranded helical molecules. Base pair hydrogen bonding between adenine and thymine and between cytosine and guanine provide the binding force between a nucleotide chain and its complementary chain.

Similarly, an artificially produced strand of nucleotide bases will in an appropriate solution attract its complementary chain and attach thereto by hydrogen bonding of complementary base pairs. Thus, in an appropriate solution a segment GAC-TTT will bind to the segment AAA-GTC.

Because of the repeating sequence in both synthesized strands of 12 nucleotides described above, there are three ways that they can pair.

I e 5' GAC-TTT-GAC-TTT 3' 3' CTG- AA-CTG- AA 5' II. 5' GAC-TTT-GAC-TTT 3' 3 CTG-AAA-CTG-AAA 5' III. 5' GAC-TTT-GAC-TTT 3' 3' CTG-AAA-CTG-AAA 5' The manner of pairing is a random consequence of initial interaction of complementary nucleotides. The offset strands as in I and III may further bind with other 12-base single strands or polymerize with other offset double strands to form long hydrogen bonded nucleotide chains, i.e.: GAC-TTT (n) GAC-TTT GAC-TTT-GAC-TTT 'n) (n) CTG-AAA-CTG-AAA CTG-AAA (n) CTG-AAA The blunt end chain as in II above will not polymerize by hydrogen bonding.

Although there are methods to join such chains, as for example with T4 ligase, there is no way to assure that the chains so produced will invariably form in the correct order.

One could assure that the 12-base segments would join in the offset manner by changing the codons of the second set of 6 nucleotides to the alternate codons for Asp-Phe.

The polymerized double chain formed by the hydrogen bonding of the complementary 12-base chains is not a complete DNA double strand as there is typically a break in the deoxyribose phosphate backbone every 12 nucleotides on each chain. The missing deoxyribose phosphate bonds are formed with DNA ligase to give a double stranded DNA segment having the formula of the type: (GAC-TTT) n GAC-TTT CTG-AAA (CTG-AAA)n At each end of the double strand a 6 nucleotide base chain tail will be single stranded. This is converted to a double strand through the use of DNA polymerase in the presence of the appropriate deoxyribonucleotide triphosphates to achieve a blunt ended DNA chain.

(GAC-TTT) GAC-TTT GTP,ATP,CTP,TTP (GAC-TTT) CTG-AAA (CTG-AAA)n-2 DNA polymerase (CTG-AAA)n (GAC-TTT) The DNA segment has the correct (CAG-AAA) (CAG-AAA)n base sequence to direct the production of the protein sequence (Asp-Phe)n. However, in order for the protein to be Produced it is necessary to insert the segment in a cloning vehicle and insert the cloning vehicle into a living organism.

Cloning vehicles are generally relatively simple DNA molecules which may be introduced into a microorganism and which function in the microorganism to direct the synthesis of protein. Appropriate cloning vehicles include plasmids and viruses such as lambda phages or SV 40 virus. Plasmids are non-nuclear DNA which in a microorganism replicate and direct the synthesis of protein. Viruses are a simple type of organism composed largely of DNA which lack independent ability to metabolize and reproduce. Viruses infect cells and will in most cases take over and eventually destroy a cell. Certain viruses such as lambda phages, however, may exist as lysogens in microorganisms and may be carried from one generation to another in the microorganisms.

While most double helixes of DNA exist as straight chains, many simple DNA strands such as viruses and plasmids are closed loops of DNA. Closed loops of DNA are most suitable as cloning vehicles. Becuase the DNA in the cloning vehicle must be cut in order that the artificial or- foreign segment may be inserted, it is desirable that a small loop of DNA be used, so that the severed ends may remain in proximal relation to each other.

In order that the foreign DNA segment be inserted, the cloning vehicle must be cut. This is accomplished through the use of various restriction enzymes. Restriction enzymes recognize a particular nucleotide base sequence, usually a segment having a center of symmetry, and cut a double-stranded DNA chain in a predetermined manner. For example, the sequence 5' GAATTC 3' 3' CTTAAG 5 is cut by EcoRl to form two severed ends as follows: 5' G AATTC 3' 3' CTTAA G 5' The severed ends may rejoin by base pairing to each other or may join to chains having a single strand tail complementary to the single strand tail on the cut strand. Thus, for EcoRl the sequence -AATT is a recognition sequence for the EcoRl restriction site, the sequence -AATT being self complementary.

A restriction enzyme will cut the cloning vehicle wherever the recognized sequence appears. It is most desirable to use a restriction enzyme which cuts the cloning vehicle at a single site. If a circular cloning vehicle is cut at a single site, generally none of the genetic material of the cloning vehicle will be lost and hence will probably remain functional after insertion of the foreign segment and rejoining of the ends. A cloning vehicle may be useful which is cut by a restriction enzyme at more than one site providing that a remaining DNA fragment contains sufficient genetic material to be functional after insertion into a microorganism.

The virus SV 40 is an example of a virus which is cut by a restriction enzyme, i.e., EcoRl, at a single site. Plasmids have also been developed by genetic manipulation which are cut by a particular restriction enzyme at a single site. A suitable plasmid for insertion of an artificial DNA segment is-pBGP120 which was developed and described by B. Polisky, R. J. Bishop and D. H. Gelfand, Proc. Natl. Acad. Sci. U.S.x., 73, 3900-3904 (1976). The plasmid pBGP120 was developed to be split by the restriction enzyme EcoRl at a single site so that after insertion of a foreign DNA segment the ends could be rejoined to form a plasmid containing all the original genetic material as well as all the inserted foreign genetic material.

In order that protein synthesis be directed by an inserted DNA segment, the inserted DNA must be inserted so that it is under the direction of a promoter and operator for mRNA transcription to occur. The plasmid pBGP120 has its sole Ecori restriction site at the distal end of most of the beta-galactosidasegene.

Foreign genetic material inserted at the EcoRI restriction site is under the direction of the lac prompter and operator. Transcription reads through the beta-galactosidase gene into the inserted segment so that inserted foreign genetic material will direct the production of protein.

The inserted foreign segment must be in phase for correct transcription and translation as the genetic code is read in groups of 3. So that the foreign segment will be in phase, the foreign segment must be inserted 3n bases from the beginning of translation. If inserted 3n+l or 3n-1 bases from the beginning of translation, the foreign segment will be out of phase.

Thus, a sequence XXX-GAC-TTT where XXX is a codon will be read XXX, GAC, TTT. However, if the inserted segment is out of phase, as for example, in XXX-Y-GAC-TTT where XXX is a codon and Y is an additional nucleotide base, the sequence will be read XXX, YGA, CTT, T -- etc. Out of phase insertions of foreign genetic material will result in production of "junk" protein and/or termination of translation.

The EcoRI site in the plasmid pBGPl20 is in the middle of a pair of codons GAA-TTC which code for glutamic acid and phenylalanine and is split to form identical ends: 5' G 3' 3' CTTAA 5' A foreign segment may be inserted in an EcoRI cut pBGP120 plasmid if it has single stranded EcoRI recognition tail i.e. -AATT at eachs' end. To be in phase an additional 3n+l nucleotides must precede the coding sequence.

A preferred method for inserting DNA segments is through the use of adaptors for molecular cloning a described by C. P. Bahl, K. J. Marians, R. Wu, J.

Stawinski, and S. A. Narang, Gene., 1, 81 (1976).

AAA The polymeric DNA {5! TTT 3SllniS adapted for insertion into the EcoRI site of pBGPl20 by fusing a 12 nucleotide self complementary dodecanucleotide CATGAATTCATG using T4 ligase. The DNA is cut with EcoRI 5' AATTCATG 3' endonuclease, the resulting DNA has a 3I GTAC 5 sequence attached to each end of the polymeric DNA.

This sequence puts the polymeric DNA in the desired frame with promoter and ribosome binding sites of beta-galactosidase.

In an appropriate solution the adapted foreign segment attaches by hydrogen bonding of the single stranded recognition sequences to each end of the cut cloning vehicle. DNA ligase completes the deoxyribose-phosphate backbone to reform a circular DNA cloning vehicle which will direct the synthesis of a protein having a segment with the repeating sequence (Asp-Phe) n The cloning vehicle is inserted in a microorganism. For the plasmid pBGP120, a preferred microorganism is a strain of E. coli and particularly the well characterized strain of E. coli K12. Plasmids may be introduced into bacteria by methods such as those described by Cohen, et al., Proc. Natl. Acad Sci., 69 2110-2114 (1972).

The microorganism in which the cloning vehicle is inserted produces along with its other proteins the desired protein which contains a long (Asp-Phe) segment. An E. coli organism containing a chimeric plasmid is cultured by methods for culturing E.

coli well known in the art.

The desired protein is then harvested from the culture of cloned microorganism. If the desired protein is secreted by the microorganism, the protein may be drawn off in a solution such as a supernatant. If the protein is retained in the cells, the cells may be lysed and centrifuged to remove cell walls and other insoluble material. Small molecules are removed from the supernatant by appropriate methods such as dialysis or molecular sieve.

The (Asp-Phe) n protein segment is a long amino acid chain and is, of course, a repeating sequence. The repeating sequence is advantageously used in the protein purification. Several chemicals and enzymes are known which split protein chains at specific location. For example, CNBr splits protein on the carboxyl side of methionine. Trypsin splits proteins at the arginine or lysine moieties. Neither trypsin nor CNBr cuts the Asp-Phe or Phe-Asp bond. If the protein fraction is digested by either trypsin or CNBr, the proteins will be cut at each susceptible site.

Hybrid protein attached to the (Asp-Phe) n segment is substantially eliminated and the other proteins fragmented, but the (Asp-Phe) n segment is uncut and is significantly larger than any of the resulting peptide fragments. The long chain (Asp-Phe) n is removed from the short peptide fragments by methods such as ultra centrifugation or filtration through an appropriate sized molecular sieve.

Because the carboxyl group of the phenylalanine is to be esterified and because the aspartic acid has a free carboxyl group, the carboxyl group of the aspartic acid is protected with a benzyl group or a substituted benzyl group which is to be removed later by hydrogenation. The protected protein is digested with chymotrypsin which cuts the (Asp(B2)-Phe)n chain into the protected dipeptide (Asp)(B2)-Phe). The protected dipeptide is methylated with an excess of methanol to produce Asp(B2)-Phe-Me. Hydrogenolysis removes the benzyl group to produce the desired methylated dipeptide Asp-Phe-Me.

The described method of producing aspartame allows the artifical sweetener to be cheaply produced in large quantities. While the production of an altered microorganism as described above is a long and tedious procedure, once a microorganism which produces (Asp-Phe) n is developed, so long as the strain is kept alive, the microorganism forming procedure need not be repeated. The microorganism can be grown in large batches analogous to the production of yeast. The living cells do not need purified amino acids as is required in the stepwise production of peptides but only requires simple growth media providing a source of carbon, nitrogen, phosphorus and simple salts. The protein purification steps are relatively simple and are adaptable to industrial techniques known to those skilled in the art.

Although the invention has been described with regard to certain preferred embodiments, it is to be understood that the invention includes modifications obvious to one skilled in the art. For example, while the invention is described in terms of preferred cloning vehicles and in terms of preferred host organisms, the invention includes any suitable cloning vehicle and any suitable host.

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Post  seraphim on Wed 12 May 2010, 19:56

Glad your getting better sleep Sleep

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Post  quicksilvercrescendo on Sun 13 Jun 2010, 18:29

Obama Issues Executive Order Mandating “Lifestyle Behavior Modification”
June 12, 2010

White House Chief of Staff Rahm Emanuel is fond of saying, “You don’t ever want a crisis to go to waste; it’s an opportunity to do important things that you would otherwise avoid.” Well, the Obama Administration certainly has not let the British Petroleum (BP) Deepwater Horizon oil rig crisis go to waste, using it as a smokescreen to silently assault and further diminish American citizens’ personal freedom.

While the nation has its eyes and ears focused on the blame game ping-pong match between President Obama and BP top brass, President Obama on Thursday, June 10, quietly announced a new Executive Order establishing the “National Prevention, Health Promotion, and Public Health Council.”

You will "change" to my liking!

Claiming the “authority vested in me as President by the Constitution and the laws of the United States of America,” President Obama has truly gone off the deep end this time in his most atrocious attempt to date to control every aspect of Americans’ lives.

According to Sec. 5. of the Executive Order that details the President’s “National Prevention and Health Promotion Strategy,” the Council will be charged with carrying out “lifestyle behavior modification” among American citizens that do not exhibit “healthy behavior.”

The President’s desired lifestyle behavior modifications focus on:

* smoking cessation;
* proper nutrition;
* appropriate exercise;
* mental health;
* behavioral health;
* sedentary behavior;
* substance-use disorder; and
* domestic violence screenings.

Making matters even worse, if that is even possible at this point, President Obama will create an “Advisory Group” composed of experts hand-picked from the public health field and various other areas of expertise “outside the Federal Government.”

Let’s consider who the President has sought advice and mentoring from in the past:

* Rev. Jeremiah Wright, who the Anti-Defamation League calls a “Messenger of Intolerance,” and
* Bill Ayers, leader of the 1960′s domestic terrorist group ”Weatherman” that was “responsible for 30 bombings aimed at destroying the defense and security infrastructures of the U.S.”

Now, President Obama is going to seek medical advisors who will be charged with modifying lifestyles and behaviors of those citizens he deems unhealthy? “Paging Dr. Kevorkian! You’re wanted in the White House STAT by President Obama!”

Whether you are a child, a parent, a worker, or retired, the President’s approximately 25-member “Advisory Group” will soon be present in every aspect of Americans’ lives, as the Executive Order prescribes. Specifically, our new so-called lifestyle behavior modification advisors will be actively carrying out the President’s orders in:

* worksite health promotion;
* community services, including community health centers;
* preventive medicine;
* health coaching;
* public health education;
* geriatrics; and
* rehabilitation medicine.

President Obama’s sweeping plan to enforce “lifestyle behavior modification” is chock full of open-ended target areas, especially when it comes to issues of “mental” and “behavioral” health, “proper nutrition,” “sedentary behavior,” and “appropriate exercise.” The President’s Executive Order is a blatant and forceful attempt to adjust the way Americans young and old think, behave, eat, drink and whatever else free will used to entitle our nation’s citizens to enjoy as prescribed by the Founding Fathers.

If you are feeling stressed-out, sad, confused, hungry, thirsty, bored, or tired, do you honestly trust President Obama and his “Advisory Group” to act in your best interests?

You will be "modified"...or else!!!

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Post  tgII on Sun 13 Jun 2010, 23:25

Herr Qsc, ist das die einzige Mengle?

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Post  quicksilvercrescendo on Tue 15 Jun 2010, 21:57

Today was a bright, sunny and clear summer day.
I drove with my Somalian friend, Abdid, fifteen minutes up a mountain to a farm situated above town where I can look out over the vastness of Norwegian forests, farms and one of its largest fresh water lakes.
At this farm the grass was a super bright green and the cows were contently wandering along the landscape munching to their delight.
This farm has been in the same family since the 1300's and the family has lineage to a Norwegian king of the past.
A stout old lady emerged from the farmhouse and waved for me to come in.
I had with me my two containers in hand.
There in a huge and extremely cold metal tank was the treasure.
Fresh unadulterated milk from ranging grass fed cows.
I loaded up my ten liters, as did Abdid, and then hurried home.
Poured a huge glass and downed it. Quickly followed by another two glasses.

This milk has a much different consistency than the milk in the grocery store.
The milk in the grocery store has more viscosity when I drink it. It leaves a coating on my tongue and in my mouth.
The milk in the grocery store also has a slight smell of sour, as if it were beginning to not be fresh and start turning.
But this milk from the cow actually goes down like water. It leaves no coating on the tongue and no mucous in my throat.

When I first get a batch of this in late spring or early summer, I end up drinking a liter of it immediately and then feel satisfied.
I will drink the remainder within the next five days. Which is a lot of milk. But during this time I reduce my meat consumption significantly.

My friend Abdid from Somalia has taught me a lot about how his people deal with food.
Their is great wisdom still left in their traditions.
The west has so much to re-learn and re-establish back into their cultures...until then they will suffer greatly from the degenerative illnesses that modern medicine does not handle well.
Abdid has taught me where to buy good quality calf and sheep livers and sheep kidneys and how to prepare these valuable organ meats.

As a former experimenter into vegetarian and macrobiotic diets, I can tell you that my constitution demands good quality animal foods wisely acquired, prepared and consumed. It is not quantity that is important here, but using them in the diet as a form of concentrated fuel.

I would also try brains, sweetbreads (thymus glands), adrenal glands and hearts, but those are not legally allowed to be sold to the public in markets where I live. As these two have a traditional use as being highly nutritious amongst the more traditional peoples, particularly the Native Americans.

I would imagine haggis is one of these traditional dishes Kapis?

The Health Thread - Page 4 Bathur10
"I will have the Big Mac with fries and a Coke...supersized...."

Hunter-gatherer societies also tend to have relatively non-hierarchical, egalitarian social structures. This might have been more pronounced in the more mobile societies, which generally are not able to store surplus food. Thus, full-time leaders, bureaucrats, or artisans are rarely supported by these societies.[7][8][9] In addition to social and economic equality in hunter-gatherer societies there is often, though not always, sexual parity as well.[10][7] Hunter-gatherers are often grouped together based on kinship and band (or tribe) membership.[10]

Others, such as the Haida of present-day British Columbia, lived in such a rich environment that they could remain sedentary, like many other Native Americans of the Pacific Northwest coast. These groups demonstrate more hierarchical social organization.

Violence in hunter-gatherer societies is usually caused by grudges and vendettas rather than for territory or economic benefit.[10]

At the 1966 "Man the Hunter" conference, anthropologists Richard Borshay Lee and Irven DeVore suggested that egalitarianism was one of several central characteristics of nomadic hunting and gathering societies because mobility requires minimization of material possessions throughout a population; therefore, there was no surplus of resources to be accumulated by any single member. Other characteristics Lee and DeVore proposed were flux in territorial boundaries as well as in demographic composition. At the same conference, Marshall Sahlins presented a paper entitled, "Notes on the Original Affluent Society," in which he challenged the popular view of hunter-gatherers living lives "solitary, poor, nasty, brutish and short," as Thomas Hobbes had put it in 1651. According to Sahlins, ethnographic data indicated that hunter-gatherers worked far fewer hours and enjoyed more leisure than typical members of industrial society, and they still ate well. Their "affluence" came from the idea that they are satisfied with very little in the material sense. This, he said, constituted a Zen economy.

Mutual exchange and sharing of resources (i.e., meat gained from hunting) are important in the economic systems of hunter-gatherer societies.

Historically, people obtained food from hunting and gathering, farming, ranching, and fishing, known as agriculture. Today, most of the food energy consumed by the world population is supplied by the food industry operated by multinational corporations using intensive farming and industrial agriculture methods.

Food safety and food security are monitored by agencies such as the International Association for Food Protection, World Resources Institute, World Food Programme, Food and Agriculture Organization, and International Food Information Council. They address issues such as sustainability, biological diversity, climate change, nutritional economics, population growth, water supply and access to food.

There is archaeological evidence of roasted foodstuffs at Homo erectus campsites dating from 420,000 years ago.[25] Boiling as a means of cooking requires a container, and was practiced at least since the 10th millennium BC with the introduction of pottery.

Food marketing brings together the producer and the consumer. It is the chain of activities that brings food from "farm gate to plate."[46] The marketing of even a single food product can be a complicated process involving many producers and companies. For example, fifty-six companies are involved in making one can of chicken noodle soup. These businesses include not only chicken and vegetable processors but also the companies that transport the ingredients and those who print labels and manufacture cans.[47] The food marketing system is the largest direct and indirect non-government employer in the United States.

In the pre-modern era, the sale of surplus food took place once a week when farmers took their wares on market day, into the local village marketplace. Here food was sold to grocers for sale in their local shops for purchase by local consumers.[23][39] With the onset of industrialization, and the development of the food processing industry, a wider range of food could be sold and distributed in distant locations. Typically early grocery shops would be counter-based shops, in which purchasers told the shop-keeper what they wanted, so that the shop-keeper could get it for them.[23][48]

In the 20th century supermarkets were born. Supermarkets brought with them a self service approach to shopping using shopping carts, and were able to offer quality food at lower cost through economies of scale and reduced staffing costs. In the latter part of the 20th century, this has been further revolutionized by the development of vast warehouse-sized, out-of-town supermarkets, selling a wide range of food from around the world.[49]

Unlike food processors, food retailing is a two-tier market in which a small number of very large companies control a large proportion of supermarkets. The supermarket giants wield great purchasing power over farmers and processors, and strong influence over consumers. Nevertheless, less than ten percent of consumer spending on food goes to farmers, with larger percentages going to advertising, transportation, and intermediate corporations.

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Post  KapitanScarlet on Wed 16 Jun 2010, 00:02

Hi qsc, yeah i know a few people who munch haggis but they not so keen to dwell on the contents and preparation ...some of them.
Interesting on the subtlties of the milk, in junior school as ye say, we used to be given a small bottle of milk at the first early interval break supplied by the school, i spewed up mine one day, it tasted awfull and i have not drunk milk since apart from through indirect means like chocolate etc, although i love coconut milk when aquired

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Post  tgII on Wed 16 Jun 2010, 00:19

Qsc, brings back memories of being on my uncle's small
horse farm out in the rural area of the Midwest. Use to
spend some of my summers there. The neighbor was a
dairy farmer and always enjoyed going over there helping
out and for the work he would give me several gallons of
fresh milk from his humanely treated non-medicated grass
fed Guernsey cows. Always liked the cream that came to
the surface and scooped it off before drinking the milk.

  • "Cruelty to animals is one of the most significant vices of a low and ignoble people. Wherever one notices them, they constitute a sign of ignorance and brutality which cannot be painted over even by all the evidence of wealth and luxury." -- Alexander von Humboldt

Reminds me, did you happen to catch that news about
kosher slaughter being banned in New Zealand? It is now
mandatory all slaughtered animals be stunned first rather
than being slaughtered by the kosher (shechita) Jewish
method. This method is barbaric and brutal if anyone has
ever witnessed it.

This has pissed off da Jooz to no end in New Zealand.

Too bad Jews, suck it up.

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Post  tgII on Fri 18 Jun 2010, 00:19

Arnold presses?

When you did the presses were you sitting at a 90 degree
angle or were you slightly reclined?

It sounds like you compressed a couple of discs in your
spine and from the sound of the pain you might have
ruptured an intervertebral disc.

  • The Health Thread - Page 4 Spinalcordfracture

If it's ruptured it means it is putting pressure on your spinal
nerve and obviously it is extremely painful.

  • The Health Thread - Page 4 Images?q=tbn:L3kOt3seRLofaM::&t=1&h=229&w=220&usg=__2aji4WY0_V3TxJeNumOTqn_hDc4=

My suggestion would be to remain as immobile as possible for a few
days to take pressure off the spine and nerve.

If the pain continues you may want to have an X-ray taken to
determine how much the disc has slipped or has been damaged.

I suffered the same injury and it is recurring unless I am extremely
careful. I find that laying down with my knees up is the most

Stop lifting weights and when you recover start doing back bridges
which are far more beneficial for strength and flexibility.

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Post  quicksilvercrescendo on Fri 18 Jun 2010, 00:56

Stop all exercising immediately and do not begin exercising until you are completely pain-free and rid of this problem.

For self-treatment to immediately bring down inflammation in the neck area and reduce pain...
Buy yourself a flexible small-sized gel-filled icepack or coldpack (found just about anywhere).
Put this in the freezer so it gets cold.
Take it out of the freezer and wrap in a thin dish or kitchen towel that is moderately dampened with some water. As direct contact with the icepack and skin tends to be uncomfortable.
If it is comfortable for you to lie on your back with a small pillow behind your head, then place the towel-wrapped ice pack directly behind your neck. Best not to lie it to low on the trapezius muscle or shoulders as this can chill the body and tense the muscles, but higher up above the seventh vertebra directly behind the neck.
Lie on this icepack for no more than twenty minutes at a time.
Then put the icepack back in the freezer so it will be ready when you need it again.
You can use this ice treatment at least three times a day, or as often as once every hour for the next two days.
After a couple days, continue with a minimum of three times a day with ice for twenty minutes, or as often as once every two hours instead of every you want to allow a bit more time between ice treatments if doing it frequently for the next two days.
After four or five days cut back on the ice to just three times a day. You don't want to overuse ice beyond this time, so three times a day is fine...and keep using it.
After the twenty minutes with the icepack is finished, I like to slide the pack from under my neck and just lie there for another ten minutes just to let the cold muscles thaw a bit back to normal temperature before I get up off the floor.

Do not use a heat pack of any kind for now. But when in the shower you can let hot water flow down the back of your head and neck for quite a few minutes as a heat treatment. Good to do in the morning to warm up for the day.

Do take the Ibuprofin should you feel you need it even if you feel it isn't doing anything. Follow the directions on the bottle as I do believe there is a length of time where you may wish to discontinue its use for a while, maybe it is seven to ten days. Drink plenty of water during the day to flush this out and to also hydrate your muscles and proper hydration calms pain.

I can tell you right now that you have a problem with your neck that is causing the pain into the shoulder and down the back. So the shoulder and the back are secondary to the neck problem.

This is not typical workout soreness and there is an insult or injury to the neck that should be looked at.
I would try to find a competent chiropractor to do an exam and take an X-ray. Sometimes good to get a personal referral to a chiropractor should you not know of one. From a family member, friend, co-worker or anyone that has had good results with their doctor. This helps in finding a competent doctor. You want to rule out any damage to a cervical spinal disc at the C4, C5, C6 levels. Or if it just a case of malpositioning of the vertebrae in this area. If the chiropractors suggests the former, a more serious diagnosis, then they may suggest you get an MRI. But you cannot know until an exam and X-ray are first done.

No one should ever lift weights without getting a chiropractic screening first. The compression from weights is considerable and any pre-existing weaknesses in the spine may make themselves known after a few months of training. Many times it is an old trauma that it is related to, from falling to car accidents or head traumas....could be any reason.

The Arnold press is a dumbbell lift where you simultaneously lift the weight overhead while rotating the arms from a curled position to a press position. But chances are this was not the "cause" of your neck problem.

Chances are you already had something going on in the neck and training may have slowly made it worse. But in the future, drop the Arnold Press. It really does nothing more than an ordinary press other than complicate the move and increase potential impingement on the shoulder muscles and such.

I would see a Chiropractor for a sufficient diagnosis. You want to know what is really going on in the neck and they are specialists over regular doctors in this area. Do your best to find a good one and a good one always demands an X-ray before doing treatment. If a chiropractor wants to adjust your neck without an X-ray first, then find another doctor.

Good Luck. And send me a private message for anything else.

And when you are completely pain free and given clearance to workout again with weights...use the exercises and techniques from these books and only these books...the are the best out there.
He offers several books but I can tell you that everything he offers is contained within two of his books...they are all you will ever need.

My neighbor just came over this evening for ninety minutes of deep tissue massage therapy because he herniated his disc a couple weeks

ago. His tear in the disc doesn't allow him to get the benefits of good chiropractic treatment, as it is too damaged. He must let it naturally calcify and fuse. So everything done at this time is just for comfort and be able to function. Should he not improve over the next six months, then he may be a candidate for surgery depending on how much of a problem is still remaining and how miserable he may be.

Consider yourself fortunate if a Chiropractor rules out disc herniation. And if he can treat you, then do exactly what he says and go as often as he says. Many people assume the can stop Chiropractic when the pain is gone. No. You must continue for about eight weeks after that until proper alignment is achieved in the neck, not just a relief of symptoms. Don't cut it short, but wait until he discharges you from care. Another X-Ray taken at a later date to compare with your first X-Ray will be telling as to the improvement.

Some therapeutic massage may also be helpful based upon the Chiropractor's recommendation.

I go to a Chiropractor once every eight weeks for preventative my massage job is very physical and I workout with weights.
That is a minimum of six visits a year. I may go more if I wake up with a stiff neck or some kind of persistent pain my spine that lasts more than a couple days. I also try to get deep massage at least four times a year as maintenance, or perhaps more if I have a painful problem or injury. Although, I never get I know how to avoid it from my own doing at least.

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