Friday, February 25, 2011

The Origin of the Lipid Hypothesis -- And Proposal of a New Term

by Chris Masterjohn

In my last post (The Proper Use of the Term "Lipid Hypothesis"), I traced the origin of the term "lipid hypothesis" to Edward Ahrens in 1976, who defined it as the hypothesis that reductions in blood cholesterol levels will lead to reductions in heart disease risk.  This can be seen as a testable prediction of the underlying hypothesis that high concentrations of cholesterol in the blood cause heart disease.

Ahrens popularized the phrase and published what was apparently the first full-length paper devoted to defining it, but was not the first to use the phrase.  I included a footnote in the last post with a reference to Daniel Steinberg using it in print in 1974 with the exact same meaning.  There may be earlier references.

These authors used the term to refer to a hypothesis quite distinct from the diet-heart hypothesis, or the "diet-heart question," terms that had first been used in the early 1960s and defined more clearly in the late 1960s under the supervision of a panel chaired by Ahrens.

While these events represent the origin of the term "lipid hypothesis," they do not represent the origin of the hypothesis itself.  After all, while the lipid hypothesis does not in any way depend on the diet-heart hypothesis, the diet-heart hypothesis is directly dependent on the lipid hypothesis because it proposes that dietary fat, or dietary saturated fat, causes heart disease precisely by raising the concentration of cholesterol in the blood.  If the diet-heart hypothesis existed in the 1960s, certainly the lipid hypothesis also did.

The actual lipid hypothesis itself pre-dates the diet-heart hypothesis by decades and traces to Anitschkov's cholesterol-fed rabbit.  I have discussed this model in detail in my article, "High Cholesterol and Heart Disease — Myth or Truth?"

Anitschkov and his partner Chalatov never intended to investigate cholesterol and were following up on earlier work suggesting that dietary protein accelerated aging.  They were investigating atherosclerosis because it was seen as a sign of aging.  

They found something quite different, and Anitschkov abandoned the idea of a dietary model of atherosclerosis in favor of a metabolic model.  Anitchkov was very clear that his work suggested nothing about dietary cholesterol in humans.

In a 1932 review,* cited in my Myth or Truth article, Anitschkov wrote the following (I will use bold for emphasis but all of the italics are his):
[I]n human atherosclerosis the conditions are different. It is quite certain that such large quantities of cholesterin are not ingested with the ordinary food. In human patients we have probably to deal with a primary disturbance of the cholesterin metabolism, which may lead to atherosclerosis even if the hypercholesterinemia is less pronounced, provided only that it is of long duration and associated with other injurious factors. . . . [O]n the basis of the experimental results we are certainly justified in stating that atherosclerosis belongs to the "metabolic diseases," and that cholesterin is the "materia peccans." [the "ill-making substance"]  It is not possible to deny that some other aspects of the metabolism can be disturbed at the same time, but in this respect hardly anything is known. 
Anitschkov rejected the myopic view that cholesterol is "the cause" of atherosclerosis, as if any one molecule could be the single cause of a complex disease.  Nevertheless, he considered it essential to the disease.  A pre-requisite, but not the only factor.
It would be entirely wrong if, on the basis of these conclusions, we were to describe cholesterin or rather hypercholesterinemia as "the cause" of atherosclerosis.  But that cholesterin plays an important part in this process, as far as its etiology is concerned, has now been definitely established as a fact by these experiments. . . . In brief, atherosclerosis never develops without cholesterin. 
Anitschkov was very decisive in arguing that atherosclerosis is a multi-factorial disease:
But, to say it again, it would be wrong if, on the basis of these experimental results, we were to regard this factor as of exclusive etiologic significance.  On the contrary, all pertinent observations recorded in these experiments point to the probability that several factors contribute to the genesis of atherosclerosis  — factors both of a general and of a local nature.
 Anitschkov reviewed experimental evidence that the following factors also contribute to atherosclerosis:
  • High blood pressure
  • Local arterial injury
  • Inflammation 
  • Other dietary factors (in the rabbit, protein)
He reviewed evidence that the following factors protect against athrosclerosis:
  • Sex hormones
  • Thyroid hormone
  • Iodine
 And he considered it likely that the following factors may also play a role:
  • Infection
  • Disturbances in the nervous system
He concluded thus:
If we now survey all the experimental results in their entirety, we find that quite a number of different factors are involved in the etiology of atherosclerosis. . . . The views here set forth concerning the etiology of atherosclerosis constitute what I have called the "combination theory"of its origin.
The "lipid hypothesis," then, from the beginning, never claimed exclusivity for cholesterol, and never denied the role of inflammation, hormones, infection, and other dietary and metabolic factors.  Nevertheless, it did give primacy to elevated levels of blood cholesterol.

Evaluating the lipid hypothesis is somewhat difficult, not only because misuse of terminology and conflation with the diet-heart hypothesis is so widespread (indeed, conflating these two hypotheses was exactly what led to the mass media's condemnation of fatty animal products in the 1980s, which will be the subject of the next post).  It is difficult because theories change over time as they attempt to accomodate new evidence.

Someone hell-bent on destroying the lipid hypothesis might accuse its proponents of creating a "moving target," but the reality is that when a "beautiful hypothesis" confronts an "ugly fact," its two legitimate choices are to change or die.

Hardly any mainstream scientists would accuse evolutionary theory of creating "moving targets" because it has undergone a great deal of revision and expansion since Darwin.

I have described in my Myth or Truth article, and in my last Wise Traditions lecture, "Heart Disease and Molecular Degeneration: The New Paradigm," what I would consider overwhelming evidence that the degeneration of lipids in the blood is essential to the initiation of atherosclerosis and plays a less prominent role in its progression.  I will provide further evidence of this several blog posts from now.

Is this a form of the lipid hypothesis?

There is nothing about the term "lipid hypothesis" that would suggest it is not.  After all, it is a hypothesis that involves lipids. 

However, it is a hypothesis of a fundamentally different character from that of Anitschkov, Ahrens, and Steinberg, even though it heavily incorporates the experimental contributions of all of them.

Anitschkov considered one of the defining characteristics of his theory of atherosclerosis to be that it considered atherosclerosis infiltrative instead of degenerative.  He focused on the amount of cholesterol, as did Ahrens and Steinberg after him.
It may therefore be regarded as certain that in these experimental animals large quantities of the ingested cholesterin are absorbed, and that the accumulation of this substance in the tissues can only be interpreted as deposits of lipoids circulating in large quantities in the humors of the body.  The establishment of this fact is of importance, because it shows that atherosclerosis is not essentially of degenerative nature, but rather of an infiltrative character.
In fact, he even called his theory the "infiltration theory."
Above all it has made it possible definitely to demonstrate that the initial stages of the process are in the nature of a lipoid infiltration or imbibition of the intima, and that the lipoids enter into the arterial wall from the lumen.  This has greatly strengthened the "infiltration theory" of atherosclerosis.
Anitschkov saw his "infiltration theory" as running in close partnership with his "combination theory."
The views here set forth concerning the etiology of atherosclerosis constitute what I have called the "combination theory" of its origin.  This theory is closely related to the "infiltration theory" of the formal genesis of this disease, because both theories are in part based on the operation of identical factors as established mainly by recent experimental research.
I think that Anitschkov's belief that atherosclerosis was infiltrative rather than degenerative (that is, that any degeneration followed infiltration of the lipid driven by its high concentration in the blood) was the most reasonable theory at the time, given the evidence available to him.

However, our understanding of the molecular mechanisms of atherosclerosis has progressed dramatically since his time, and we have definitive evidence that it is degeneration of lipids rather than their high concentration that drives atherosclerosis.

Yet proponents of the "lipid hypothesis" have nevertheless subjugated this evidence to constitute a mechanism for a theory that remains fundamentally infiltrative in character, based fundamentally on the amount of lipid in the blood.

I think, then, that the term "lipid hypothesis" is insufficient.  Anitschkov, brilliant as he was, used much better terminology.  If only it involved the term lipid or cholesterol, it would have been perfect.

I propose, then, that my view — that degeneration of lipids plays an essential role in the initiation of atherosclerosis and a less prominent role in its progression — is a lipid hypothesis, but it is not THE lipid hypothesis.

I propose a new terminology to distinguish between these two related but fundamentally different hypotheses:
  • The infiltrative lipid hypothesis is the hypothesis of Anitschkov, Ahrens, Steinberg, and the medical establishment, focused on the amount of cholesterol in the blood driving its infiltration into the arterial wall, and considering this infiltration to drive any subsequent degeneration.

  • The degenerative lipid hypothesis is the hypothesis that considers the amount of cholesterol largely or perhaps entirely irrelevant, and considers the degeneration of lipids in the blood to drive their infiltration into the arterial wall, and this infiltration to actually be partly protective against worse forms of degeneration that would have occurred in its absence.
Lest we become myopic, we should fully retain Anitschkov's combination theory as a powerful and intimate partner of the degenerative lipid hypothesis.

Read more about the author, Chris Masterjohn, PhD, here.

* Anitschkow N. Experimental Arteriosclerosis in Animals. In: Cowdry EV. Arteriosclerosis: A Survey of the Problem. New York: Macmillan. 1933.

Tuesday, February 22, 2011

The Proper Use of the Term "Lipid Hypothesis"

by Chris Masterjohn

There seems to be a lot of confusion about the meaning of the term "lipid hypothesis" in pop science books, blogs, and other places.

Often times, the lipid hypothesis is confused with the diet-heart hypothesis.  The two are very different.  The lipid hypothesis concerns the role of lipids in the blood.  The diet-heart hypothesis concerns the role of lipids in the diet.

The term "diet-heart," as best as I can tell, was coined in the 1960s to describe the National Diet-Heart Study, which was designed to test the effect of reductions of dietary saturated fat and increases in dietary polyunsaturated fat on blood cholesterol and heart disease risk.  In 1969, a panel of the American Medical Association headed by Edward Ahrens Jr. called this "the diet-heart question."  

In 1976, Ahrens first popularized* the term "lipid hypothesis" to refer specifically to the hypothesis that increased levels of cholesterol in the blood increase the risk for heart disease.  He published a paper in the Annals of Internal Medicine entitled, "The Management of Hyperlipidemia: Whether, Rather than How."  Here's what he wrote:
What is The Lipid Hypothesis?

The Lipid Hypothesis is the postulate, based on Framingham (8) and similarly derived data, that reducing the level of plasma cholesterol in an individual or in a population group will lead to a reduction in the risk of suffering a new event of coronary heart disease.  It is a premise based on the undisputed fact that people with higher plasma cholesterol levels have more and earlier coronary heart disease than do others with lower cholesterol levels; but the premise has not yet been proved true to the satisfaction of epidemiologists and biostatisticians or of the medical community at large.  The Lipid Hypothesis, then, is simply an inference derived from accepted facts (Figure 1); though the hypothesis has been put to the test repeatedly in the past two decades, completely satisfactory evidence has not yet been advanced either pro or con.
His figure is meant to demonstrate the difference between association and causation:

On the left, we see the facts as they had been gathered at the time in Framingham, that blood cholesterol is correlated with the risk of heart disease.  On the right we see a depiction of the hypothesis used to explain those facts.  The arrow within the curved line represents a treatment used to lower the level of blood cholesterol.  The arrow on the left side of the picture represents a hypothetical decrease in the risk of heart disease that results from the treatment.  This hypothesis is the lipid hypothesis.

I have been writing about the need to distinguish between the lipid hypothesis and the diet-heart hypothesis for three years, since first reviewing Daniel Steinberg's The Cholesterol Wars (you can read my review here).  

I credited Steinberg with making that proper distinction (indeed, I was not even aware of it until reading Steinberg's book), and criticized Uffe Ravnskov for conflating the two hypotheses in The Cholesterol Myths (you can read that review here).

I also emphasized this distinction in my last Wise Traditions lecture, "Heart Disease and Molecular Degeneration: The New Paradigm."

Whether we view the lipid hypothesis as true, partly true, or false, I think it is important to make this distinction clear.  The alternative is to jumble up various hypotheses and make it difficult to find the truth.

Read more about the author, Chris Masterjohn, PhD, here.

*I say "popularized" because Ahrens was not the first to use the term, even though this paper quickly became referenced as the first to define the lipid hypothesis.  For example, see Epstein's 1977 paper, "Preventative trials and the diet-heart question: wait for results or act now."  Indeed, Ahrens' paper is the first indexed for pubmed that is searchable by the term "lipid hypothesis," and appears to be the first paper ever written specifically devoted to defining the lipid hypothesis.  However, Daniel Steinberg used the term in 1974 as a presentation to the Drugs Affecting Lipid Metabolism conference in Milan.  Steinberg was the Chair of the planning committee for the Corornary Primary Prevention Trial, and described the committee as being charged to "to design a feasible study to test the 'lipid hypothesis,' i.e., the hypothesis that intervention to reduce serum cholesterol levels does reduce risk of clinically manifest coronary artery disease."  Reference (provided by Dr. Steinberg): Steinberg, D. Planning the Type II Coronary Prevention Trial of the Lipid Research Clinics (U.S.A.) in Advances in Experimental Medicine and Biology 63: 417-426, 1975. Also cited as a book: (in) Lipids, Lipoproteins and Drugs (eds. D. Kritchevsky, R. Paoletti,and W.L. Holmes) Plenum Press, New York, 1975.  Note that Steinberg's definition is the same as Ahrens'.  It is possible that earlier print references exist; however, none appear to have popularized the definition until Ahrens' paper.  The actual hypothesis, however, dates to Anitschkov's cholesterol-fed rabbit in 1913.

Wednesday, February 16, 2011

The New Genetics -- Part IV: Who's In the Driver's Seat? How Cells Regulate the Expression of Their Genes

by Chris Masterjohn

Who is in the driver's seat?  The gene, the cell, or the organism?

It's a complex question, and in a future post in this series, I'll attempt to identify ideal metaphors we might use to understand this question.  For now, I'd like to focus on how the cell utilizes its genes and controls their expression.  

This story should probably begin with Barbara McClintock, who won the Nobel Prize in Physiology or Medicine in 1983 for her discovery of mobile genetic elements (you can read her Nobel lecture here).  McClintock discovered in the 1940s that certain pieces of the corn plant's genome could switch positions on the chromosome and change the expression of nearby genes, controlling pigmentation and many other traits.  

The idea that gene expression could be regulated was revolutionary.  Genetics was in its infancy: the structure of DNA had not even been determined, and genes were viewed simply as static determinants of heredity.  McClintock perceived the reaction of the scientific community as "hostile" to her work, and in 1951 ceased openly publishing the results of her research for twenty years.  Nowadays, the regulation of gene expression is textbook material.

If we simply look at the myriad cell types within an organism or how an organism interacts with its environment, we will see why gene expression has to be regulated.

Take, for example, different cell types like a neuron (nerve or brain cell) or an adipocyte (fat cell).  They look and function in completely different ways.



Even though these cells look radically different from one another and perform radically different functions, they have the same DNA.  If you take the nucleus of an adult skin cell from a frog and inject it into a frog's egg that's been robbed of its own nucleus, you get a tadpole.

If the DNA were the sole determinant of the cell's structure and function, the DNA of the skin cell would have to be unique and when you injected it into an egg you'd get a skin cell instead of a tadpole.

Cells must also respond to their environment, and different cell types often respond in different or even opposite ways.  For example, when we haven't eaten for some time, our adrenal glands release hormones called glucocorticoids.  When these hormones reach the liver cell, the liver cell turns on the gene for tyrosine aminotransferase, an enzyme that helps the cell make glucose from protein and send it out into the blood.  In fat cells, however, the same hormones have the opposite effect, and in other cell types they have no effect at all.

The typical human cell only expresses between one and two thirds of its genes during its whole life cycle.  The particular cell type determines not only which genes are expressed but how much they are expressed as well. 

How do cells regulate the expression of their genes?  

To answer that question from a conventional perspective, let us turn for now to chapters 4, 6, and 7 of Molecular Biology of the Cell, the definitive guide to mainstream molecular biology.  The first author, Bruce Alberts, was President of the U.S. National Academy of the Sciences for twelve years from 1993-2005.  The information in this blog post will come from this source except where otherwise stated.

The Genome Is Three-Dimensional

The first thing we must realize in order to understand how our cells regulate the expression of our genes is that the genome is not simply a two-dimensional, linear sequence of its basic building blocks, called nucleotides, but is a complex, dynamic, three-dimensional structure.

In fact, it just has to be three-dimensional.  The typical human cell contains two meters of DNA that it must pack into a nucleus measuring about six micrometers in diameter.  A micrometer is one millionth of a meter, so this is like packing 24 miles of thread into a tennis ball!

But that misses the point.  The three-dimensional structure of DNA is dynamic and highly regulated, and the cell uses it to regulate gene expression in both transient and heritable ways.  Thus it is important both in minute-to-minute, day-to-day function, as well as in forms of epigenetic inheritance.

Here is a diagram showing the different levels of organization imposed upon DNA:

The DNA begins as a two-stranded spiraled thread called a double helix, likely a familiar term to most of us who passed high school biology recently enough to have remembered anything from it.  But then it is wound around proteins called histones and thereby arranged into structures called nucleosomes that are arranged along the DNA like beads on a string.  This beads-on-a-string structure, coated in a number of other non-histone proteins, is called chromatin.

The chromatin is then organized into a number of different layers of folding.  The familiar X-shaped chromosome is compacted 10,000-fold, but only occurs during mitosis, the process by which a parent cell divides into two daughter cells.  In a non-dividing cell, the chromatin is compacted on average 500-fold.

The importance of histones eluded scientists for decades.  The fact that DNA is the vehicle of inheritance was determined in bacteria, which lack histones.  Thirty years ago, scientists figured histones were, in the words of the authors of Molecular Biology of the Cell, "relatively uninteresting proteins," simply aiding in the packing of chromatin but acting as "uninvolved bystanders" in gene expression.

Nothing could be further from the truth.

The mass of histone proteins within chromatin is equal to the mass of DNA.  The precise amino acid sequence of histone proteins is so critical to function that organisms as disparate as the cow and the pea — yes, those little green suckers we eat! — differ in sequence less than two percent.  That's roughly the sequence difference between chimpanzees and humans for the average gene.

In yeast, nearly every mutation in a histone protein tested has proved lethal.  The few mutations that are not lethal cause changes in gene expression and other abnormalities.  This is rather remarkable because, according to a 2008 paper, when yeasts are faced with nutritional abundance, 80 percent of their genes can be deleted with no obvious effect at all.  Clearly, histones are not "uninvolved bystanders."

The cell possesses a vast array of enzymes used to modify histones.  These include enzymes that chemically modify histones with methyl, acetyl, or phosphate groups, to strengthen or loosen their bonds with the DNA.  They also include dozens of different chromatin-remodeling complexes that slide the nucleosome along the DNA and, in conjunction with histone chaperone proteins, can even remove part or all of the nucleosome.  

These are all essential to the expression of genes, because the chromatin must be fully unwrapped in order for a gene to be expressed.

The chemical modifications of histones can exist in thousands of different combinations, and are thought to constitute a "histone code."  Cells also insert and remove special "histone variants" into the chromatin as part of this code.  In the fruit fly, over 50 proteins have been identified that act as code-reader/code-writer complexes.  The code can signal the beginning of DNA replication, the need for DNA repair, or the expression level of a gene.
Some histone modifications are often reversed almost immediately — for example, the sliding or removal of histones during the active expression of a gene — while others may persist for generations, and thus constitute a form of epigenetic inheritance. 

While most of the chromatin within the nucleus is packaged as euchromatin, corresponding roughly to the third level of organization shown in the picture above, over ten percent is packaged as heterochromatin, which is dependent on histones, non-histone proteins, and small RNA molecules called interfering RNAs.  This is a highly condensed form that silences genes not only within the heterochromatin, but even nearby it.  

There are likely to be over ten unique types of heterochromatin that have different magnitudes and types of reversibility.  Different heterochromatin patterns are found in different tissues.  Thus, heterochromatin likely plays a role in day-to-day function as well as epigenetic inheritance.

Heterochromatin is particularly concentrated in the telomeres located at the ends of chromosomes, and the centromeres that constitute the middle of the "X" shape.  The centromere contains a special histone variant and a number of other unique proteins.  In humans, there is no DNA sequence that dictates the centromere, so this is another form of epigenetic inheritance.  Since a gene's distance from the centromere will influence its level of expression, this is an important form of epigenetic inheritance.

Of course, unraveling the three-dimensional structure is just the first thing that the cell must do to express a gene.  Much more must happen at the site of the two-dimensional, linear sequence.

Gene Regulatory Sequences

Genes are associated with regulatory sequences that lie just upstream from them in the DNA sequence, as well as far, far away from them.  These interact with regulatory proteins that communicate the needs of the cell and the organism to the cellular machinery that will express the gene.  

Some genes require just a handful of regulatory proteins to be expressed and others require hundreds.  Humans have about 2,000 different regulatory proteins that are believed to constitute about eight percent of the human genome.  

In addition, there are certain proteins that always need to be there.  These include RNA polymerase, which actually synthesizes the mRNA transcript, five "general transcription factors" that are actually complexes of 27 total proteins, and a giant complex called Mediator that is composed of 24 individual protein subunits. 

Here is a picture of a "gene control region" being activated:

In this picture, the light blue protein and most of its conjoined smaller subunits is the RNA polymerase complex, which contains about 100 protein subunits.  The giant purple guy is Mediator.  In the first panel, two of the general transcription factors are shown on the left.  Up at the top, we see a protein marked "activator," which is one of the gene regulator proteins.

This model is simplistic, as there are often hundreds of activators, and they often act at many different sites instead of one.  The corresponding picture from the current edition of Molecular Biology of the Cell, which is not available online, shows four different sites of activation instead of one.

This picture also is not depicting the many later events involved in expressing the gene, discussed in the last post in this series, such as the shedding of these proteins and the tethering of several hundred other proteins and other components to its long tail to form an "RNA factory."

Nevertheless, even from this picture we can see that the activator is actually binding to a location quite far away from the gene, as indicated by the dotted lines in the DNA sequence on the left side.  This distant part of the DNA sequence becomes close to the gene by folding of the chromatin.  Thus, even in this relatively unraveled state, expressing a gene is a three-dimensional operation.

In fact, the average gene size is 27,000 nucleotides  — and as we will see below, only an average of 1,300 of these actually code for the protein — but this elaborate control region can often span 100,000 nucleotides.

The job of the activator proteins is to attract RNA polymerase and the associated transcription factors and Mediator complex, appropriately position them, and chemically modify them so they can get going along the DNA strand.  They also must recruit histone modification enzymes, chromatin remodeling complexes, and histone chaperone proteins, so that those "beads on a string" can be plowed through.

There are other gene regulator proteins called "repressors," and in fact some proteins can act as "activators" in one context and "repressors" in another.  As an example, the nuclear receptors for the fat-soluble vitamins A and D can act as repressors in the absence of these vitamins, but as activators in their presence.

In addition to protein activators and repressors, humans also express 400 different micro RNAs (miRNAs) that can form RNA-induced silencing complexes that regulate at least one third of all human genes.  These complexes as well as direct addition of methyl groups to cytosine nucleotides within the DNA provide additional mechanisms of epigenetic inheritance.

The authors of Molecular Biology of the Cell therefore state that "each eukaryotic gene is therefore regulated by a 'committee' of proteins, all of which must be present to express the gene at its proper level," and that given the thousands of proteins involved, "there would seem to be almost limitless possibilities for the elaboration of control devices to regulate eucaryotic gene transcription."

Here is quite a profound statement the authors offer about the ability of the cell to regulate its gene expression in response to its needs and the needs of the organism according to changes in the environment:
This large number of genes reflects the exceedingly complex network of controls governing expression of mammalian genes.  Each gene is regulated by a set of gene regulatory proteins; each of those proteins is the product of a gene that is in turn regulated by a whole set of other proteins, and so on.  Moreover, the regulatory protein molecules are themselves influenced by signals from outside the cell, which can make them active or inactive in a whole variety of ways.  Thus, we can view the pattern of gene expression in a cell as the result of a complicated molecular computation that the intracellular gene control network performs in response to information from the cell's surroundings.
They conclude the section on transcriptional regulation by noting that scientists who try to reduce this regulation to its component parts and design their own predictable systems as a test of whether they understand the regulatory systems inevitably make mostly predictions that fail.  This indicates that scientists still have yet to grasp the level of intelligence that exists within a cell.

But gene regulation doesn't stop there!  So far all we've done is regulate the production of an mRNA transcript.  The cell can still regulate the processing of the mRNA transcript, its stability, its rate of translation into protein, and then can act on the protein to alter its activity in various ways.

The Cell Edits mRNA, Blurring the Definition of "Gene"

As noted above, the average gene is 27,000 nucleotides long, but on average only 1,300 of them code for protein.  What's up with the rest?

Here's a picture of two different genes, a small one on the left, and a larger one on the right:

The red stripes are the parts of the gene that code for proteins.  These are called exons because they are expressed.  The intervening orange sequences are called introns.
Before the mRNA exits the nucleus, the introns must be spliced out.  Why, then, do they exist?  One explanation given by the authors of Molecular Biology of the Cell is that it would allow exon shuffling during the course of evolution, so that distinct functional domains could be mix-matched in a copy-and-paste manner to generate new proteins.

However, it turns out that not all introns are thrown away.  Indeed, the 150 guide RNAs that are involved in the production of the ribosome, the factory where proteins are synthesized, are often encoded by introns.  Similar RNA molecules have just recently been discovered that are only produced in the brain, where they are believed to organize the direct chemical modification of mRNA transcripts.

But as we'll see, the most important reason is that splicing allows another level of regulation.

Many genes exhibit alternative splicing patterns, meaning that the cell can make more than one protein from the same mRNA transcript.  mRNA transcripts from a full 75% of human genes undergo alternative splicing, often generating dozens of different proteins.

Like the process of transcribing the mRNA in the first place, the cell has activators and repressors with which it can regulate the alternative pathways of mRNA splicing. 

In addition, mRNA transcripts from about 1,000 human genes are believed to undergo RNA editing, which involves the chemical modification of some of the nucleotides.  For example, we possess enzymes that convert the nucleotide cytosine to uracil, and others that convert adenine to inosine.

This makes it difficult to define what a gene is, and to count the number in the human genome.  At one time, it was thought there was a gene for each protein, but this is clearly false.  Some have suggested a gene be defined as each unique mRNA, in which case there could be hundreds of thousands.  The authors of Molecular Biology of the Cell suggest that a gene be defined as a closely associated cluster of exons that code for a closely related family of proteins, as alternative splicing usually leads to proteins of related function in humans.  Very few examples have been identified where radically different proteins are produced from the same sequence, and in these cases this can be considered two distinct genes that overlap in the DNA sequence.

Regulation of mRNA Stability and Translation

The final mRNA transcript doesn't last forever.  The cell can regulate how long it lasts, as well as the rate at which it is translated into protein.  This begins in the nucleus when the poly-A tail and the poly-A-binding proteins are added, but it continues in the cytosol.

For a nutritional example, the cell has an elegant way of regulating its concentration of iron.  There is a sequence of untranslated RNA called an iron response element (IRE) in the mRNA both for ferritin, an iron storage protein, and transferrin, an iron transporter.  Ferritin binds iron and thus makes less available for use, whereas transferrin helps the cell take up more iron from the blood.

In ferritin, a single IRE exists in the 5'-untranslated region, near the cap.  In transferrin, multiple IRE's exist in the 3'-untranslated region, near the poly-A tail.

When the cellular concentration of free iron gets low, a protein called iron regulatory protein (IRP)  loses one of its iron atoms.  This causes it to bind to the IREs in the ferritin and transferrin mRNA transcripts.

Because of the different locations of the IREs, IRP-binding causes opposite effects in ferritin and transferrin.  It prevents the translation of ferritin protein from its mRNA transcript.  By contrast, it prevents the degradation of the transferrin mRNA transcript, causing an increase in the level of mRNA and thus more of the protein to be produced.

Thus, when the iron concentration of the cell gets low, the cell makes more transferrin and less ferritin, both of which increase the cellular concentration of usable iron.

Another way of regulating mRNA degradation is to induce it with little molecules of RNA called interfering RNAs, the same molecules that are sometimes involved in the formation of heterochromatin. There are also a great number of proteins involved in the process of translating the protein, and the cell has a variety of enzymatic networks for controlling these proteins.

Once the protein is produced, there are a great number of ways to regulate its activity, which will be considered in a future post in this series.

Other Forms of Regulation in Viruses and Bacteria

Regulation of gene expression in bacteria is a lot different.  Their genomes are much simpler, lacking histones, introns, and true chromosomes, usually regulating lots of genes at once as part of a complex called an operon, and using an expansive list of enzymes to carry it all out, but nevertheless a much smaller list than we use.

Nevertheless, bacteria and even viruses have some sophisticated ways of regulating gene expression that, to date, have not been considered very important in humans and other higher organisms.

Although I learned in genetics class about five years ago that the genetic code has, since the days of Watson and Crick, been considered "non-overlapping," meaning that each set of three nucleotides constitutes a distinct codon, this is untrue in certain viruses.   

Retroviruses, for example, use translational frameshifting to produce their protein shell, called a capsid, and the enzymes reverse transcriptase and integrase that they use to insert their genetic information into their host, all from the same mRNA transcript.  They do this by changing the "reading frame," so that that the codons begin with different nucleotides.

There are cases in bacteria where the bacteria literally restructure their own genome to regulate their genes.  For example, Salmonella has a specific 1000-nucleotide DNA sequence that it inverts in order to express one of two different types of flagella proteins.  It uses this process to exhibit phase variation.

The bacterium that causes gonorrhea uses a different process called gene conversion to transfer DNA sequences from an unexpressed "library of silent 'gene cassettes'" to a site in the genome where the genes are expressed, as if playing the "gene cassette" in a "genetic tape player."  This allows it to induce a heritable change in its surface properties and thereby evade an immune attack.

We have already considered organized genome restructuring in humans in the case of antibodies and T cell receptors in the first post in this series.  Whether such organized restructuring of the genome has contributed in a greater capacity to who we are on evolutionary and other levels will be considered in a future post.

What Are the Roles of Epigenetics?

So far we've considered histones and associated proteins, RNA-induced silencing, and DNA methylation in epigenetic inheritance.  In the last post, I considered protein-folding, which I would consider non-genetic but some would consider epigenetic.  What roles do they play?

Certainly, they contribute to cell type.  Epigenetic switches and gene expression feedback loops are important in maintaining the structural and functional differences between different types of cells.

They likely also transmit information from the mother's womb and her environment during fetal development, some of which may persist through life.

Do they also contribute to inheritance from one generation to another?  That will be the subject of a future post.

Read more about the author, Chris Masterjohn, PhD, here.

Tuesday, February 15, 2011

The New Genetics -- Part III: Genes Don't Express Themselves

by Chris Masterjohn

Part III of The New Genetics

If we are to understand the role of genes within a living organism, the first thing we must understand is that genes, by themselves, are inert.  Put a gene in a petri dish with all the nutrients needed to make a protein and it will do nothing.  Nada.  Genes do not express themselves.  Cells express genes.

A eukaryotic cell  — a cell from an organism of higher complexity than bacteria — must use hundreds of proteins to make a single functional protein from a gene.  Here is a schematic showing the different steps a cell takes to make a functional protein:

The cell can regulate each step in response to its own needs and the needs of the organism of which it is part, constantly adjusting the expression of its genes to fit its environment.

What follows is a brief summary of chapter six of Molecular Biology of the Cell, the definitive guide to mainstream molecular biology.

The first thing the cell must do is make an RNA transcript called messenger RNA or mRNA that will carry the information from the DNA within the nucleus to the cellular fluid that lies outside, called the cytosol.  This process is called transcription because the code is staying within the "language" of nucleic acid, but changing form somewhat, like converting a handwritten message into a typed message.  

Although RNA polymerase is the enzyme that assembles the mRNA, this process generally requires well over a hundred proteins to get going.

Once the polymerase begins transcribing the gene, it sheds this first assembly of proteins, called transcription factors, and begins associating with a number of proteins called elongation factors required for it to keep moving along.

Before the nucleus exports the mRNA, the mRNA must be edited.  The splicing machinery required for this process involves five RNA-based enzymes and as many as 200 additional proteins.  

The mRNA must also have a cap made from a modified form of guanine, one of the nucleotides that makes up the basic structure of DNA.  It takes three enzymes to make the cap.  Several other sets of proteins are required to give the other end of the mRNA transcript a poly-A tail made of a string of adenine nucleotides.  Adenine is another basic building block of DNA like guanine.  The cap and tail of the mRNA help identify it as a transcript destined for protein synthesis and provide the cell with several additional mechanisms to control the amount of protein it makes from the transcript, as discussed in more detail in the next post in this series.

The RNA processing enzymes are generally seen to be stockpiled in granules poised nearby transcription sites, ready for use.  But when transcription begins, they become tethered to a long tail protruding from RNA polymerase, so Molecular Biology of the Cell calls the polymerase an "RNA factory."

Much of the spliced material is discarded, and the nucleus uses specific receptors and nuclear pore complexes to transport the usable mRNAs into the cytosol.  In order to make proteins out of it, however, the cell must have protein-making machines made primarily of RNA called ribosomes.  A growing mammalian cell contains ten million ribosomes.  

The ribosome is a ribozyme, a word used to describe enzymes that are made from RNA instead of protein.   While four different RNA molecules make up two thirds of the ribosome, each ribosome also has fifty different proteins that lock into the holes and crevices in its surface, where they appear to stabilize its shape and facilitate its movement. 

In order to make enough RNA to manufacture millions of ribosomes, multiple copies of the four unique RNA-encoding genes are required.  Humans therefore have 200 genes located on five chromosomes that code for ribosomal RNA.

In order to actually assemble each ribosome, 150 different "guide RNAs" are required to make approximately 200 specific chemical modifications to the ribosome. This occurs in  a structure within the nucleus called the nucleolus, which Molecular Biology of the Cell calls a "ribosome factory."

While the cell's ten million ribosomes are primarily involved in using mRNA transcripts to make proteins, the first role of the ribosome is to enforce quality control (QC) on the mRNA transcripts as they exit the nucleus.  During the mRNA splicing we discussed above, each splice site is tagged with special proteins.  As the mRNA exits the nucleus, a special ribosome examines it to make sure the "stop" message occurs after the last of these labels.  If not, the QC managing ribosome will identify it as an mRNA that was either incorrectly spliced, or produced from a defective gene, and will target the mRNA for destruction.  If so, the QC manager removes the labels and allows the transcript into the cytosol where it can be used to make proteins.

The process of making a protein from the mRNA transcript is called translation, because it is translating the message from the language of nucleic acid to the language of protein.

Several sets of proteins are involved in carrying the amino acids to the ribosome in a way that ensures accurate reading of the genetic code.  There are 48 unique transfer RNAs (tRNAs) encoded in multiple copies by 500 genes.  These tRNAs are shaped like a cross or a three-leaf clover and attach an amino acid on one end, and read the genetic code of the mRNA transcript on the other end.

There are 20 aminoacyl-tRNA synthetases, which are enzymes that attach the amino acids to the proper tRNA molecules.  These enzymes also contain an "editing" capacity that fixes mistakes where the wrong amino acid gets joined to the tRNA molecule.

Finally, several elongation factors are also required to increase the speed and accuracy of protein production at the site of the ribosome.

The protein exits the ribosome through a tunnel in a mostly unfolded, linear state.  Its final functional properties, however, depend on its three-dimensional structure after it has folded.  The information for the three-dimensional structure is partly contained in the linear sequence of amino acids, but not always entirely.  

As the protein exits through the ribosomal tunnel, chaperone proteins often help it fold properly.  Once it has exited, other chaperone proteins sometimes form a barrel-shaped "isolation chamber" around the protein to help it finish folding, or to help misfolded proteins refold.  There are several major families of chaperone proteins, and they are often specific to certain proteins and even to certain organelles within the cell.

Alternative ways of folding a protein are most commonly known in disease states, such as in the prion theory of mad cow disease.  However, alternative protein folding states have been shown to contribute to the formation of different cell types in certain fungi, and it is possible that protein folding could represent a form of non-genetic inheritance, as discussed in more general terms in the second post in this series.  

Some researchers have made the controversial suggestion that alternative forms of protein folding could contribute to memory in complex organisms such as humans.

Once the final protein is folded, the cell still has many ways of modifying the protein to make it active, inactive, or to change how it functions or just how active it is.

This has been, of course, a simple summary of what is contained in Molecular Biology of the Cell.  Despite all of the complexity that has been discovered, the authors still state that "an enormous amount of ignorance remains; many fascinating challenges therefore await the next generation of cell biologists."

What we do know, however, should make it clear that genes do not express themselves.  It should, by now, be abundantly clear that cells express genes.

In the next post, we will take a look at the fascinating way in which cells control the expression of their genes in response to their environment, in ways that fulfill their own needs and the needs of the organism of which they are part.  Now that we've covered some of the basics, things will really get interesting.  Stay tuned!

Read more about the author, Chris Masterjohn, PhD, here.

Sunday, February 6, 2011

The New Genetics -- Part II: Some Biological Heredity Is Neither Genetic Nor Epigenetic

I feel bad for the gene.  Why?  Well first, just take a look at his face.
Why is Gene so sad?  I think it's because we're always talking about him behind his back, saying things like "it's genetic" and, "so-and-so has the gene for _____."  Because we decided who he was decades before we even met him.  And the more we learn about him, the more we continue to talk about him as if we've learned nothing.  The sadness upon his face seems to say, "How come you never listen?"

The word "gene" was introduced in 1909, but the structure of DNA was not determined until the 1950s.  The word "gene" was introduced to mean, basically, a unit of biological heredity responsible for a specific heritable trait.  

The idea that each trait had a specific unit of heredity behind it was of great service to the eugenics movement, as I discussed in my introduction to this series.  Soon after, it became very useful to the neo-Darwinian Modern Synthesis that unified Mendel's laws of inheritance with Darwin's theory of natural selection as the primary creative force responsible for the diversification of all life from a common ancestor.

Nevertheless, the discovery of molecular genetics, from its inception, began to add much more to the story.  

No doubt genes carry heritable information.  And no doubt there are variants of each gene called alleles, which contribute to variation in the traits of an organism that are subject to natural selection. 

But is that all a gene does?  Quite certainly not.  With the discovery of DNA, molecular biology began showing that genes are designed to bestow flexibility upon an organism, not simply the ability to inherit characteristics, but the ability to change characteristics in response to the environment and in response to the organism's own needs.

The remarkable ability of the cell to access and utilize its genome, much like we use our computer programs, will be the topic of the next post in this series.

In this post, I'd like to focus on another reason that Gene might be sad.  Perhaps when we assume that a heritable disease is "genetic" Gene feels unjustly blamed, and perhaps when we assume that the marvelous and beautiful functional and aesthetic traits of an organism are "genetic" Gene feels flattered but somewhat uneasy of the high expectations laid upon him.  Perhaps we are being somewhat demanding in assuming that Gene should carry the burden of biological heredity all by himself.

Please forgive my anthropomorphizing for a moment, as I promise this post actually contains some science, following shortly below.

There is a type of biological inheritance that is not encoded in the DNA within the nucleus of our cells called cytoplasmic inheritance.  The cytoplasm consists of the fluid that surrounds the nucleus called the cytosol and all the organelles within it.  Organelles are the "little organs" of the cell. 

While we inherit half of our nuclear DNA from our mother and half from our father, we inherit mostly our mother's cytoplasm.  This includes our mother's mitochondria, which contains its own genome, but it also includes all the other organelles of our mother's egg cell, and these all carry heritable information that is not encoded in any DNA at all, as will be discussed further below.

Cytoplasmic inheritance is commonly discussed in genetics and molecular biology textbooks.  One modern way of demonstrating cytoplasmic inheritance is to take a nucleus from one species and transfer it to a denucleated cell of another species.  This technique usually fails, demonstrating the need for compatability between the nucleus and the rest of the cytoplasm, but the few success stories are quite fascinating.

Six years ago, for example, Chinese researchers successfully transplanted nuclei from a common carp genetically engineered to express human growth hormone into the egg cells of a goldfish.  Remarkably, the cloned hybrid shared certain characteristics that were intermediate between the two types of fish, rather than simply exhibiting the characteristics of the carp.  Here's a picture of the hybrid and its parental species:

Sun YH, Chen SP, Wang YP, Hu W, Zu ZY.  Cytoplasmic Impact on Cross-Genus Cloned Fish Derived from Transgenic Common Carp (Cyprinus carpio) Nuclei and Goldfish (Carassius auratus) Enucleated Eggs.  Biology of Reproduction. 2005;72:510-515.

The cloned hybrid is on the left.  The hybrid's nucleus came from the carp, shown in the middle, and its cytoplasm came from the goldfish, shown on the right.

The authors say there was little difference in the appearance of the hybrid from the carp, and indeed, the hybrid looks a lot closer to the carp than to the goldfish.  However, other authors have pointed out that the body shape of the hybrid is more rounded than the carp and thus somewhat intermediate between the carp and goldfish.  Personally, the tail looks to me like a cross between the two types of fish.

In any case, the authors provided a remarkable finding about the number of vertebrae.  The goldfish had 26 vertebrae and the carp had 33-36 vertebrae.  Even though the nuclear DNA came from the carp, most of the cloned hybrids had 27 or 28 vertebrae while another had 26 and another had 31.  This shows that the cloned hybrid had a number of vertebrae closer to the goldfish that provided the cytoplasm than to the carp that provided the nucleus.

Moreover, all of the cloned hybrids were sterile.  Sterility is common among true hybrids of different species where half of the nuclear DNA comes from one species and half from another.  Here, the same results are achieved when the nuclear DNA comes from one species and the cytoplasm comes from another, suggesting a certain type of equivalence between the importance of information in nuclear DNA and information in the cytoplasm.

In general, cytoplasmic inheritance is tacitly assumed to be a function of mitochondrial DNA.  This is based more on faith than on evidence, and there are a two good reasons to think it is probably not entirely true.

First, although the mitochondria contains its own DNA, most of its proteins are actually encoded by nuclear DNA, and mitochondria do not appear to export any of the proteins that they make themselves.  Thus, the mitochondrial DNA might make a meaningful but perhaps relatively minor contribution to the characteristics of an organism.

Second, there is a massive amount of information contained in the cytoplasm itself that is not encoded by any genes, whether nuclear or mitochondrial, but is nevertheless inherited from generation to generation.

To understand how this is possible, let's take a look at what the inside of a cell looks like.
Image hosted by PubMed's searchable but not browsable version of the 2002 edition of Molecular Biology of the Cell

The cell is organized into a number of different compartments by a continuous system of membranes.  This system of compartmentalization is absolutely essential for cellular function and for producing the biological characteristics of an organism in conjunction with the information coded in DNA.

The image is, of course,  simplistic.  Even within one organelle's membrane, there are further regional divisons in structure and function, and there are many fascinating localized structures that are not shown in the picture.

What is important here, though, is another point.  The information needed to produce these membranous compartments is only partly coded for in DNA.  Part of it resides in the membrane itself, is heritable, and is not coded for anywhere in the DNA.

Molecular Biology of the Cell, the definitive guide to mainstream molecular biology, decribes why this is so in its twelfth chapter, "Intracellular Compartments and Protein Sorting."

Each membranous organelle is to some degree distinguished by the types of lipids it contains, but these are in turn determined by its unique set of proteins.  The proteins themselves are major determinants of the oragenelle's function.  These proteins have signal sequences associated with them that direct them to specific organelles.

But here's the catch!  What facilitates the matching of the signal sequence to the membranous organelle for which the protein is destined?  The proteins in the membrane.  That's right, if there are no protein transporters in, for example, the endoplasmic reticulum, then those signal sequences that destine a protein to the endoplasmic reticulum can't be recognized by anything and have no meaning at all.

In the case of the endoplasmic reticulum, the phenomenon is even more striking, since the endoplasmic reticulum actually has to make those proteins before they can get transported anywhere anyway.

Thus, it is not just the nuclear DNA that defines the membrane, but the membrane itself.  Here's the conclusion of the authors of Molecular Biology of the Cell (p. 704) in their own words:

Thus, it seems that the information required to construct an oragenlle does not reside exclusively in the DNA that specifies the organelle's proteins.  Information in the form of at least one distinct protein that preexists in the organelle membrane is also required, and this information is passed from parent cell to progeny cell in the form of the organelle itself.  Presumably, such information is essential for the propagation of the cell's compartmental organization, just as the information in DNA is essential for the propagation of the cell's nucleotide and amino acid sequences.
Thus, there exists absolutely critical information that is biologically heritable that is not coded for in the DNA.

To what extent do variations in this information contribute to variation between and within species? 

This is a mystery that will only begin to be unraveled when more scientists drop the myopic and completely false view that organisms are largely vehicles meant as containers for self-propagating genes — a view that was always preposterous but is now more than ever completely proven false — and join the ranks of computational systems biologists who look at physiology as an integrated whole and attempt to actually ask and answer such questions.

That's not to say that most scientists actually hold such a myopic view, but scientists who study heredity essentially universally study variations in DNA sequences.  The fact that we know of many examples where variations in DNA contribute to variations in heritable characteristics and do not have a similar body of knowledge about non-genetic heritable information may simply be a result of the many people asking the first type of question and almost no one asking the second type of question rather than being a result of a greater relative frequency of that type of biological inheritance.

On the other hand, though we still have a great deal to learn about gene expression, we do have a very impressive body of knowledge about how cells access, utilize, control, and even in some cases restructure their genomes in response to their own needs and the needs of the organisms of which they are part.  That will be the subject of the next post in this series. 

Stay tuned!

Read more about the author, Chris Masterjohn, PhD, here.

Friday, February 4, 2011

Some Things I Like About "The New Evolution Diet"

by Chris Masterjohn

My overall review of Art De Vany's The New Evolution Diet probably came across pretty negatively, but there's a lot of stuff I like about the book that's worth summarizing. 

Unfortunately, many of the poorer ideas in the book shine forth with a brighter light simply because these good ideas have been discussed in many other places, and that tends to steal credit away from Dr. De Vany, who has apparently been discussing these ideas on his blog for years.  Some of them are also much more difficult than throwing away egg yolks, making it difficult to tell whether the person of average motivation would really benefit from the book. 

Nevertheless, here they are.

Intermittent Fasting.  My interest in intermittent fasting began just under eight years ago when many of us on the Yahoogroup Native-Nutrition, a spinoff of the Weston A Price Foundation that eventually took on its own life, began discussing Ori Hofmekler's book, The Warrior Diet: Switch on Your Biological Powerhouse for High Energy, Explosive Strength, and a Leaner, Harder BodyI never actually got around to reading The Warrior Diet, but I did read Hofmekler's later and more technical book, Maximum Muscle, Minimum Fat: The Secret Science Behind Physical Transformation, which discusses intermittent fasting as well as cold exposure and other ideas currently popular in the Paleo movement.   Hofmekler recommended eating most of your food in a four-hour window in the evening in those books, whereas De Vany recommends the gentler approach of skipping a single meal or a whole day's worth of food once a week

I don't think practicing intermittent fasting is necessary to be healthy, but I do think it has the potential to lengthen life, prevent cancer and other forms of degeneration, expand dietary flexibility in a practical way, and offer the opportunity for spiritual growth for those interested.

Against Snacking.  De Vany suggests three meals a day and comes out clearly against snacking.  He also recommends exercising in the fasting state.  In my experience, these recommendations are extremely helpful.  I first read of these principles in Byron Richards' Mastering Lepting: Your Guide to Permanent Weight Loss and Optimum Health, another book that was discussed on Native-Nutrition as an outgrowth of our discussion of The Warrior Diet.  Richards recommends eating two or three meals a day with no snacks, fasting at least three hours before beginning exercise, and at least three hours before going to bed.  While this book has some horrible grammar to trudge through and is often somewhat frustrating to read, these recommendations were extremely helpful to me in overcoming some of my sleeping problems.

I have encountered people with putative adrenal problems who could not handle the fasting involved in The Warrior Diet or even Mastering Leptin, so I would not recommend either of these approaches for everyone, but I do think the idea of three meals a day with no snacks and exercising on an empty stomach are very accessible and would benefit most people.

High-Intensity Interval and Strength Training.  De Vany recommends exercising in short bursts rather than for long durations, and getting cardio by doing intense and quickly repeated bursts of strength training.  I've been into kettlebells for years, which achieve precisely that.  These bad boys also came up in discussions on Native-Nutrition almost eight years ago.  I also generally do weight lifting routines with low bouts of rest between sets, about one minute in length.  I've only recently discovered what havoc four minutes of Tabata squats with only ten seconds rest between sets can wreak on my back and thighs through a session at CrossFit NYC

High-intensity interval training (HIT) is quite in fashion nowadays, and popular magazines you'd find at the gym like Men's Health often recommend it.  I've even seen discussions of intermittent fasting in these magazines.  And you can even buy kettlebells at Wal-Mart now.  I think one could probably make an argument for other types of exercise depending on your goals, but I do think there is some good research favoring HIT.

Against Calorie Counting.  De Vany writes that "a smart diet reduces the amount of energy (meaning food) you feel like consuming at the same time that it increases the amount of energy you feel like spending.  And this occurs spontaneously, without any thoughts of cutting calories, or exercising more, or anything else.  It just happens."  While I think many people would find themselves consuming too few calories on the low-fat, low-carb plan that De Vany provides, I agree with the general principle that a diet should be satisfying and make you energetic.  Of course, a diet that promotes leptin sensitivity and proper thyroid functioning, among other important physiological benefits, will improve even the energy you expend at rest.  I suppose Gary Taubes' Good Calories, Bad Calories, and Why We Get Fat will prove to be the quintessential deconstructions of the calorie-cutting myth, and I'll be reading and reviewing those books on this site soon.

"Posture Is a Full-Time Event."  I definitely agree with this statement, and believe that posture is incredibly important to health.  One of the coaches for the ballroom dance team I'm part of, Mark Sheldon, often drills home the point that a few hours practicing dance a week isn't going to compensate for spending 6-8 hours hunched over a laptop every day.  I have little doubt that disruptions of posture have significant metabolic effects.  I'm looking forward to reading Esther Gokhale's 8 Steps to a Pain-Free BackHere's a lecture of hers:

Nassim Nicholas Taleb on Randomness.  Taleb, author of The Black Swan: The Impact of the Highly Improbable and Fooled by Randomness: The Hidden Role of Chance in Life and In the Marketsbooks I should probably put on my to-read list, wrote an excellent afterword to De Vany's book.  He makes some great points about the limitations of science, the importance of tradition, being blinded against empirical evidence by pre-conceived notions of "what makes sense," and the importance of variety, change, and occasional intensity in one's diet and lifestyle.  If you're the type to cough up $14 for a ten-page essay, this is the best part of the book.

Some Good Food Recommendations.  Despite trashing egg yolks, fat, and red meat, the diet does get rid of junk food and eliminates non-Paleo foods, which might represent common food sensitivities.  Of course, opposition to junk food can be found almost everywhere in the health world, and there are a lot of Paleo books already out now.  And I agree with Chris Kresser that Paleo restrictions should be seen as a starting point, and that many neolithic foods should often be included in a person's diet according to their individual needs and tolerances.  I also think a comprehensive diet book should discuss food preparation methods to render grains and legumes non-toxic, even if these are deemed impractical or imperfect, and a diet purportedly based on "evolution" should cover variation in human responses to these foods, as well as how these responses might change over time with genetic, epigenetic, non-genetic (e.g. intestinal flora), and technological adaptations. 

Unfortunately, many of these ideas are overshadowed by the inconsistent and incoherent presentation of physiology, the difficult and likely often hypocaloric recommendations of low-fat, low-carb eating, and some of the bizarre ideas about metabolism, genetics, and evolution presented in the book that I covered in my primary review

Had this book come out eight years ago, much of it would seem revolutionary to me and I would be inclined to overlook some of these negative attributes.  As it stands, it seems to have little to offer to the "inititated" who are already familiar with these concepts, and has potential dangers for the newcomer who might be more enamored with negative recommendations like trashing egg yolks while intimidated by some of the better, but harder suggestions.  Certainly, this book has something for many a curious person that lies somewhere in between, so I won't make a blanket recommendation against the book.  Instead I'd like to give kudos to Dr. De Vany for all the good he's done over the years, but just provide the warning: reader beware.

Read more about the author, Chris Masterjohn, PhD, here.