Mast Cell Heparin

Mast cells are sentinels in tissues. They respond to invading pathogens by releasing their stored histamine, enzymes and heparin. The heparin modifies the activity of enzymes and cytokines.

What are mast cells and why are they loaded with heparin (left)? Mast cells start in the bone marrow, like many other components of the immune system. They then move into the blood stream and offload in most of the tissues that typically encounter pathogens and parasites. Thus, the typical commercial source of the mast cell-produced heparin is pig intestines or cow lungs, i.e. since heparin is made and stored in mast cells and mast cells are abundant in lungs and intestines, those are the sources of crude heparin. Proteins bound to the crude heparin are removed as the heparin is cleaned up to be used as an anti-clotting drug.

Mast cells are sentinels near the surface of mucus membranes that line the airways of the lungs and the digestive tract. Diseases of the lungs and intestines, e.g. asthma and inflammatory bowel disease, that have an inflammatory and/or autoimmune component yield high levels of mast cells in the affected tissues. Pathogens or parasites coming in contact with mast cells trigger the sudden release of vesicles full of histamine, enzymes and heparin.

Heparin stored in vesicles in mast cells can also be readily visualized by staining the mast cells in microscope sections using the fluorescent dye berberine (left). Berberine binds quite specifically to heparin and is also used in herbal medicine as a treatment for many inflammatory diseases, such as arthritis. It would be very interesting to know whether berberine has any effect on asthma.

Mast cells display a variety of receptor proteins on their surfaces. Protein receptors work by binding target molecules, ligands, changing their shapes and transmitting a signal through the cytoplasm. A key aspect of the signal transmission is the requirement for the ligand binding to bring together receptors in pairs. The pairing of receptors during ligand binding is facilitated by the binding of heparin to both ligands and receptors. Two ligands, e.g. cytokine peptides, such as TNF, can bind to adjacent sites on a heparin molecule and this pair can then bind to two receptors brought together on the surface of a cell. The receptors bind to the ligand and to the heparin. Some ligands will bind to their receptors without heparin, but the presence of heparin greatly accelerates and intensifies the reactions.

Heparin is synthesized in the vesicles of mast cells and binds to enzymes, e.g. tryptase, also present in the vesicles. The tryptase enzyme proteins form tetramers with heparin wrapped around the edge (left, edge view showing one pair of tryptase proteins with heparin bound diagonally to blue heparin-binding domains; other pair of tryptase proteins is hidden).

Interestingly the active site for each tryptase in the tetramer faces a hole where the four proteins come together. Thus the tetramer can degrade small peptides, but large proteins cannot get access to the blocked active sites. Monomers change shape and are no longer active.

Activated mast cells release their vesicle contents with some enzymes active and their bound heparin is replaced by the heparan sulfate attached to adjacent cells. Other enzymes are initially inactive bound to heparin and are activated by dissociation of the heparin once they are released from the vesicles. In both cases some of the heparin is released from the mast cells into the surrounding tissue. The free heparin can bind to cytokines released from other cells and the combined pairs of cytokines bound to heparin can in turn bind to appropriate receptors on other cells. The abundance of heparan sulfate bound to other cells will determine whether additional heparin is required for receptor responses from particular cytokines. Cells with abundant heparan sulfates will sweep heparin binding ligands toward receptors aggregated in lipid rafts, as the heparan sulfate proteoglycans are internalized for recycling.

Mast cells can be activated by allergens, because of IgE receptors. IgEs are antibodies that trigger allergic responses. The IgEs produced by antibody producing B lymphocytes circulate in the blood serum and bind to mast cell receptor proteins. Allergen molecules bind to the IgE-receptor complexes, trigger the activation of the mast cells and release histamine. The histamine binds to receptors on other cells and produces the symptoms of allergy or asthma.
Heparin can be sprayed into the lungs of asthma sufferers and reduce symptoms. This suggests that the ratio of heparin to cytokines is important and that cytokine signaling required for asthma episodes of airway constriction can bind individually to different heparin molecules and minimize mast cell triggering and histamine release.

Asthma also responds to a general decrease in chronic systemic inflammation. Thus, an anti-inflammatory diet and lifestyle, can reduce episodes and potentially reverse symptoms. Omega-3 oils and glucosamine, for example are both effective.

Tryptase model: Sommerhoff CP, Bode W, Pereira PJ, Stubbs MT, Stürzebecher J, Piechottka GP, Matschiner G, Bergner A. 1999. The structure of the human betaII-tryptase tetramer: fo(u)r better or worse. Proc Natl Acad Sci U S A 96(20):10984-91.


Berberine staining of mast cell heparin: Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, Takahashi HK, Morgan ES, Dvorak AM, Fehling HJ, Rodewald HR. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 25:6199-210.

Asthma

Asthma is chronic constriction and inflammation of the airways of the lungs. Aggrevation of these symptoms can result from allergens, exercise, stress or infection. Once lungs become asthmatic the episodes of symptoms can be controlled by drugs that usually include broncodilators. But what is the cause of asthma?

Risk factors suggest the source of the disease. Exposure to smoke or inhaled pollutants, antibiotic use, formula use, obesity (metabolic syndrome), use of chlorinated swimming pools all increase the likelihood of developing asthma. The common factor in all of the risks is chronic systemic inflammation combined with some irritation of the lungs. Systemic inflammation of asthmatics is frequently the result of altered gut flora. Use of formula or antibiotics, for example, lead to a disruption of normal newborn gut bacteria, replacement with hospital strains, e.g. Clostridia spp., and inflammation. Obesity can also lead to inflammatory gut flora. Systemic inflammation combined with lung irritants, e.g. allergens, chlorinated organic compounds, infections, can lead to allergic responses and in the lung this can mean asthma.

An asthmatic attack starts with spasmodic contraction of the airway muscles, followed by inflammation. Initially, so called cholinergic receptors located in the membranes of the smooth muscles that surround the airways of the lungs, are stimulated by the binding of neurotransmitters. The result is that these receptors trigger the cleavage of phosphoinositol diglycerides to release inositol with three phosphates (IP3) and a glycerol with two lipid chains (diacylglycerol, DAG). The IP3 (which mimics heparin, by the way) binds to receptors controlling the internal stores of calcium (in the endoplasmic reticulum), causing a surge of intracellular calcium. The rise in intracelllular calcium is what causes the muscle cell actin and myosin to contract and the rings of muscle on the airways to constrict. This is ridiculously simplified to show the basic components of stimulating receptors, IP3 increase, calcium increase and muscle contraction.

Early attempts to treat asthma used plant extracts that blocked the action of the cholinergic (as in acetylcholine) receptors. These are familiar plants such as tobacco, coffee, tea and cacao. The cholinergic blockers are nicotine (pictured and in computational model below), caffeine, theophylline and theobromine. Datura stramonium, Jimson weed, also produces atropine, that is another cholinergic inhibitor. There are modern forms of the cholinergic antagonists, which are used to treat episodic constriction.

I have illustrated in the model above, how nicotine binds to the cholinergic receptor, between a tryptophan (bottom, yellow) and two tyrosines (above, orange).

The IP3-calcium constriction system can also be stimulated by allergen-based mast cell release of histamine. The histamine binds to another set of receptors and triggers IP3 release. In this context, anti-histamines are moderately effective, but the initial response is so rapid, that the histamine phase is quickly passed.

The rapid constriction phase is followed six to eight hours later by inflammatory release of leukotrienes that also cause constriction via IP3. Leukotrienes share the same precursor, omega-6 arachidonic acid, as the inflammatory prostaglandins made by the cyclooxygenase, COX 1&2, but in this case an alternative enzyme, lipoxygenase, is used. The leukotriene receptors on muscle cells act similarly to the catechol receptors and stimulate constriction.

Another approach to preventing constriction or actively relaxing airway smooth muscles is by stimulation of adrenergic receptors that have the opposite effect of cholinergic receptors. The adrenergic receptors trigger cyclic AMP production, that compromises the IP3-calcium signaling. Cholinergic receptors are activated to minimize the damage to inhaled toxins or irritants. Adrenergic receptors open the airways to increase airway capacity for flight or fight in response to adrenalin. Some of the drugs to treat asthma stimulate adrenergic receptors and open airways.

Treatment of asthma is centered on constriction episodes and not on reversal or prevention of the disease. Thus, treatment or prevention of an asthmatic episode of airway constriction can utilize drugs that block steps along the cholinergic cascade (receptor, IP3 increase, calcium increase) or by stimulation of adrenergic receptors.

These treatments do not address the cause or reversal of asthma. The inflammatory basis of most asthma risk factors suggests that lowering chronic inflammation, particularly in young children, should be a high priority in preventing asthma. The association of asthma with the use of formula and antibiotics, implicates inflammation, as well as inflammatory gut flora, as the foundations of asthma and emphasizes the essential role of diet and lifestyle in the development of asthma.

Tryptophan, sugars, inflammation

Tryptophan binds basic amino acids, sugars and alkaloids. Examples of tryptophan binding include glycosidase, importins, arginine transporters and cytokine receptors. Molecules that mimic and interfere with these interactions are important drugs.

I have always been interested in nuts, bolts and mechanisms. That has progressed from Erector sets and Lincoln Logs, to chemistry sets and microscopes, and ultimately to computational models. So when I think about inflammation and disease, I think in terms of how molecules interact to produce the symptoms of disease. I seek the molecular basis of inflammation, and how proteins and small molecules interact to make people sick. In most of the interactions that I study, that means how proteins bind to carbohydrates, e.g. heparin, and how alkaloids, e.g. curcumin from turmeric, disrupt those interactions.

Enzymes bind to sugars and catalyze particular biochemical changes in the sugars through amino acids on the surface of the enzyme. Examination of the surface of the enzyme would show pits or clefts with exposed tryptophan residues. The tryptophan does not hydrogen bond with water, i.e. it is hydrophobic. Similarly the top and bottom faces of sugar, such as glucose, are hydrophobic. If a sugar crashes into a tryptophan, as a result of thermal motion, the sugar will stick, because exposing the hydrophobic of both surfaces to water is a higher energy state; the sugar is bonded to the tryptophan.

I have taken a mathematical model of a sugar-binding protein and highlighted the surface of the protein, the helical twists of the protein backbone, the yellow tryptophan and the red and white sugar. You can see that the faces of the sugar and the tryptophan are touching.

I have explored hundreds of protein structures derived by X-ray crystallography and NMR. Sugar-binding enzymes, such as glycosidases, glycanases, lectins, etc., usually have tryptophans to bind the sugars. Examination of enzymes that bind long chains of sugars have a series of tryptophan stepping stones that are spaced and oriented to bind the faces of the sugars.

Some of the structures that molecules display are just amazing. Glucose chains are very long in the polysaccharides that we are familiar with as starch. Small lengths up to about a dozen sugars would be call dextrins. If these dextrins are connected end to end, they become cyclodextrins. The cyclodextrins can be of different sizes, but instead of being bracelets for different sized wrists, the cyclodextrins bind to molecules of different sizes. Since the inner surface of the cyclodextrins is hydrophobic, other small hydrophobic molecules that can slip into the rings, will get stuck. These bound molecules, which would otherwise interact with your senses to produce smells, are odorless. Thus, cyclodextrins can eliminate odors and that is just what they do in the product called Febreze.

Cyclodextrins and short dextrins can also be visualized as they bind to an enzyme, amylase, that would normally hydrolyze starch to dextrins. I have highlighted the structures of an amylase mathematical model derived by X-ray crystalography, to show how the cyclodextrin on the upper left surrounds an aromatic amino acid (in this case tyrosine) and the dextrin binds to a series of aromatic amino acids in the enzymes active site with several orange tyrosines to the right.

Sugars and tryptophans also bind to the hydrophobic arms of the basic amino acids, arginine and lysine. I have illustrated this in the case of a disaccharide bound between tryptophan (yellow) and arginine (blue) in the binding site of a lectin, a protein that binds to specific sugars.

Polysaccharide, such as heparin, that have bulky hydrogen-bonding sulfates bound to one face can still bind a tryptophan to unobstructed faces, and a basic amino acid to the other face. The basic amino acids bind in a two step process. The positively charged nitrogen at the end of the amino acid first binds to the negatively charged oxygens of the sulfates and then subsequently the nitrogen forms hydrogen bonds with the sulfates and hydrophobic interactions as the arm of the amino acid lies across the surface of the sugar.

Alkaloids, aromatic molecules with positively charged nitrogens and other negatively charged plant molecules interact with the aromatic amino acids and polysaccharides and alter human physiology. Berberine, from Barberries, has been used to treat arthritis, but it also binds and makes heparin fluorescent. Thus, berberine can be used as a fluorescent dye to visualize mast cells, which are the source of heparin. I have used berberine to stain heparan sulfate proteoglycans on cartilage producing chondrocytes in cell culture. Quinine is also similar in structure to berberine and college students should know that quinine in tonic water fluoresces in black light (UV). Putting the two together, I successfully stained my chondrocytes with tonic water!

The significance of these investigations is the potential to explain how traditional herbal remedies work and to develop new approaches for the prevention and treatment of inflammation based diseases.

Rheumatology Patients Vitamin D Deficient

Vitamin D deficiency is rampant in patients seeking care for pain and inflammatory diseases.

A recent study (reference below) of patients in England who were being treated by rheumatologists for inflammatory arthritis and chronic pain found that most were diagnosed as deficient in vitamin D at the start of treatment, were prescribed a daily dose of vitamin D that is now known to be inadequate and remained deficient in vitamin D during treatment for their primary complaint.

These results suggest a very odd situation in medical treatment for inflammation-based diseases, which include cancers, degenerative diseases and autoimmune diseases.

1. The symptoms of inflammatory diseases are the same as those of vitamin D deficiencies.
2. Patients with symptoms of inflammation are frequently not screened for vitamin D deficiency or are screened incorrectly (20% of physicians order the wrong test).
3. Prescriptions for vitamin D supplements are inadequate to correct deficiencies.
4. Treatment is not reassessed to confirm that the deficiencies have been remedied.
5. Vitamin D deficiencies are routinely left untreated.

A Modest Proposal: This might mean that a huge fraction of the inflammatory epidemic could be remedied by having people roll up their sleeves and pant legs and spend 20 minutes in the midday sun. After two weeks of this treatment, the U.S. health care budget could be reduced by a detectable percentage (10% reduction in symptoms?)

Mouyis M, Ostor AJ, Crisp AJ, Ginawi A, Halsall DJ, Shenker N, Poole KE. 2008. Hypovitaminosis D among rheumatology outpatients in clinical practice. Rheumatology (Oxford) 47(9):1348-51.

Vitamin D

Vitamin D is in the news. It decreases cancer and most degenerative diseases, at least in part, because it is anti-inflammatory. As with most vitamins, the minimum daily requirement to prevent its associated deficiency disease, rickets, is about one tenth of what is required for optimal health.

The Vitamin D Council makes the following statement:
Current research has implicated vitamin D deficiency as a major factor in the pathology of at least 17 varieties of cancer as well as heart disease, stroke, hypertension, autoimmune diseases, diabetes, depression, chronic pain, osteoarthritis, osteoporosis, muscle weakness, muscle wasting, birth defects, periodontal disease, and more.

To read the newspapers and blogs, one would think that vitamin D is the cure-all, the panacea or the snake oil of today (Note that the original snake oil was very effective and high in omega-3 oils.) As with much of today’s medical advice from the media, the problem with our vitamin D metabolism is yesterday’s medical advice. Not only are the previous minimum daily requirements too low by an order of magnitude, but the dietary advice is in conflict with life style suggestions to minimize sun exposure. And all of the advice is prejudiced by commercial considerations. (Note that the way that most sun blocks are used to permit longer time in the sun, actually increases the rate of skin cancer.)

The previous recommendation was to have 200 IU of vitamin D per day in your diet. How can this number make any sense, when 20 minutes of sun exposure can produce 20,000 IU of vitamin D? And what about the storage of vitamin D in fat and the obvious problem that obese people can easily store more vitamin D than they consume in their diet, i.e. they are always deficient even on what appears to be an adequate diet?

It is also confusing to measure vitamin D in the blood. The active form of vitamin D is calcitriol and it is made in the kidney from the short-term stock of vitamin D that is released from the liver. This suggests to me that there are at least three types of protein carriers for vitamin D: 1) point of origin carriers that take vitamin D from fat, skin and intestines to the liver, 2) liver-to-kidney carriers that transport to the kidney and 3) calcitriol carriers that take the active form to organs where it is used for calcium transport, etc. Which form of vitamin D should be measured? The answer is that the major circulating form, not the active, calcitriol, should be measured, because even people with severe deficiencies can have normal levels of calcitriol.

Active vitamin D, calcitriol regulates calcium distribution between diet, serum and bone. The serum calcium level must remain constant or fundamental cellular processes of signaling and secretion will be disrupted. The result of lowered calcium would be loss of neural and muscular function.

Calcium present in the serum goes through a two step process in the kidney; calcium and urea pass out of the blood vessels and then 95% of the calcium is reabsorbed and passed back into the serum. This loss and any additional requirements for bone growth must be compensated by uptake from the diet. Calcium uptake from the second stage of the kidney and the intestine is from the action of calcium transporters. Synthesis of the transporters as well as the expression of a hundred other genes is controlled by calcitriol.

Calcitriol is a prohormone. Calcitriol binds to a cytoplasmic receptor and the calcitrol/receptor complex is transported to and into the nucleus. The hormone complex then acts as a transcription factor by binding to specific control elements of genes and controlling gene expression. Thus, the amount of calcitrol/receptor determines the presence of calcium transporter proteins on the surface of kidney or intestinal epithelial cells and the amount of calcium loaded into the serum.

Since calcitrol is synthesized in the kidney, calcium reuptake by the kidney is directly related to calcitrol production. If there is inadequate calcium in the serum, the calcium level will also lower in the calcitrol synthesizing cells of the kidney and this will increase the activity of the calcitrol/receptor complexes. This should be adequate to produce enough calcitrol in the serum to enhance calcium uptake in the intestines. If the level of calcium is still inadequate, then the parathyroid glands secrete PTH that stimulates both more calcitrol production and release of calcium from bone. Chronic release of bone calcium result in osteoporosis.

Obesity is a problem, because of the solubility of vitamin D in fat. Vitamin D is transported to fat cells, just as it is transported to the liver. Presumably the same carrier protein is used to transport to and from the fat cells. Thus, there is an equilibrium between the vitamin D stored in fat droplets of the fat cells and the vitamin D stored in the liver. Unfortunately, the capacity of the fat cells is much greater than the liver. As a result, a huge quantity of vitamin D is required to saturate the fat storage of an obese person and there is a constant deficiency punctuated by the dietary contributions of vitamin D after each meal. This is one of the reasons for the increased rates of most degenerative and autoimmune diseases for obese individuals.

There is one last point that I want to make about nonsense in the news. It is stated that exclusively breastfed babies are at risk for vitamin D deficiency, because mother’s milk is low in vitamin D by comparison to the higher value in formula. This is ridiculous in the same way that formula should be better, because it has more iron. The reality is that formula (even a single bottle at night in the hospital nursery) destroys the normal newborn gut flora and leads to chronic inflammation. In fact, the added iron in formula is particularly disruptive and enhances colonization by inflammatory bacterial species. The point is that babies should not be continuously swaddled and kept away from the sun. Babies need to be exposed for brief intervals (never long enough to cause reddening) to the sun. In fact, because of the compromised intestines, newborns who are fed formula are probably in greater need of sun exposure for vitamin D production.

The bottom line is that you need to have your arms and face, or arms and legs, exposed to the sun for 15-20 minutes several times a week. This is very important if you carry a few extra pounds and/or show symptoms of chronic inflammation. It may also make sense to supplement your diet with 1-2,000 IU of vitamin D. Don’t forget to follow the rest of the anti-inflammatory diet and lifestyle suggestions.

New Topics

Here are some topics that I am working on for articles in the near future:

Cyclodextrins: These interesting bracelets of sugar molecules show how sugars bind to flat aromatic rings structures, such as aromatic amino acids and alkaloids. This molecule illustrates why I am interested in the interplay of heparin, inflammation and disease.

Vitamin D: This vitamin is a prohormone that binds to a cytoplasmic protein and then acts like a transcription factor to control gene expression. It is also synthesized by a reaction that required ultraviolet light in the skin and it is stored in fat. Vitamin D deficiency is linked to cancer and many of the same chronic diseases associated with inflammation. Interestingly the ends of some collagen molecules have structures that are similar to the serum transport proteins for steroids.

COPD: Chronic obstructive pulmonary disease is an inflammatory disease that is unfortunately very common.

Asthma: This allergic condition causes major restructuring of airways in response to inflammation. Atomized heparin has been used as a treatment to minimize inflammation.

Suggestions: I am always looking for suggestions of topic that are of special interest to my readers, so if you would like a subject discussed, just leave a comment.

Transglutaminase and Polyglutamine

Transglutaminase 2 can cross-link proteins and is implicated in many diseases, including cancer, celiac and Alzheimer’s. Polyglutamine stretches of amino acids in wheat gluten and human proteins can have lethal interactions with this ubiquitous enzyme.

I spend a lot of my time trying to fill in blanks or connect the dots -- I seek a grand conceptualization of the biological world. I want the molecular world to make sense to me. The problem is all of the loose ends and the task is to bring all of the loose ends together to make one beautiful whole. All of the components of the living world make sense, but they make sense only if the activities and interactions of most of the components are understood. Unfortunately, in many areas enough of the pieces are not understood, so the the whole can’t be envisioned. Let’s use an enzyme, transglutaminase 2, as an example.

Transglutaminase 2 (TG2) can replace the amino (-NH2) group at the end of a glutamine amino acid residue of a protein and attach its own sulfhydryl (-S-) in its place. Thus, the enzyme becomes covalently connected to another protein. Then, depending on what molecules crash into this conjugate, the protein can be transferred to the amino group of a lysine on second protein, to cross-link the two proteins, or a water molecule could interact to free the protein, but leaving a glutamic acid instead of the original glutamine. This explains the essence of the transglutaminase activity of the enzyme.

You can expect that I would have checked out the structure and amino acid sequence of TG2 and peeked at the literature on the enzyme. The enzyme has strong heparin-binding domains and those domains explain the ability to purify TG2 on heparin-sepharose, sticking of TG2 to the extracellular matrix (heparan sulfate proteoglycans), internalization and translocation into the nucleus.

TG2 can also cross-link proteins to toughen up the sloughed off layers of the skin and gut. It also can cross-link proteins in the cytoplasm as part of programmed cell death, with the result being protein aggregates that are easily endocytosed by phagocytic cells. TG2 in the nucleus can cross-link and stabilize histones.

TG2 can also be involved in pathology and where ever you see clumps of proteins in cells or tissues, TG2 should be suspected. TG2 is produced in close association with inflammation and the TG2 gene has an NFkB promoter, so TG2 is expressed along with other inflammatory genes.

Proteins cross-linked by TG2 are not readily degraded in cells. TG2 can of course reverse its cross-linking and separate proteins.

There are nine different diseases, including Huntington’s disease in which the protein products have long stretches of polyglutamines. Polyglutamines are excellent substrates for TG2 and proteins with long polyglutamine stretches are readily aggregated by TG2. Neurons suffering from this type of disease die stuffed with undigestable protein aggregates.

So what happens when you eat proteins with polyglutamines? The gluten proteins in wheat and other grains are these kinds of proteins. Most people digest grain proteins just fine, but an unlucky minority suffer when their intestinal TG2 attacks and gets cross-linked to the gluten proteins. The end result is production of antibodies to both the gluten proteins and to TG2, killing and scarring of the intestinal epithelium, and the symptoms of celiac (gluten intolerance). This may be an example of how plants have avoided being eaten. Insects and other herbivores must ingest polyglutamine-rich proteins in order to eat grains. These proteins destroy the gut of insects that are not adapted, because their TG enzymes are inactivated and aggregated. Most grain eating herbivores need a system to bypass the polyamines, but I don’t know what that is.

TG2 is also implicated in many cancers and I have invoked TG2 as the mechanism whereby glucosamine is anti-inflammatory. I have not touched on the role of TG2 as a signal molecule controlled by binding nucleotides, i.e. as a G protein, nor have a elaborated on all of its neurophysiological activities. Clearly TG2 is an enzyme with many activities and interactions, and it will be puzzling me for many years.

Metastasis, Osteopontin, Heparin

Metastasized cancer stem cells respond to osteopontin produced by primary tumors, NFkB is activated and bone stem cells are recruited. The cancer-bone cell aggregates in this inflammatory environment proliferate to produce new tumors.

Cancers are thought to start in one location and spread to other sites by a process called metastasis. This spread of cancer to multiple sites is what typically leads to death. The primary tumor is the nursery for the production of cells that are able to leave the tumor, migrate to blood vessels and infiltrate other tissues. Thus lung cancers typically kill smokers by terminal brain tumors.

The primary tumors appear to have a controlling influence over subordinate satellite tumors and can keep the subordinates quiescent or turn them into aggressively growing secondary tumors. Production of osteopontin, a cytokine-like hormone associated with bone production, by primary tumors can stimulate quiescent metastasized cells into proliferation.

Recent reports show that a primary human breast tumor established in a mouse can stimulate proliferation of quiescent colon cancer cells. Osteopontin produced by the primary tumor stimulates invasiveness and growth of the secondary cancer cells and also recruits bone marrow stem cells that further enhance proliferation.

Other studies have shown that inflammation is required for cancer and osteopontin activates NFkB, the inflammation transcription factor. It is interesting that the presence of the heparan sulfate proteoglycan, syndecan 4, blocks this process.

Human osteopontin has very strong heparin-binding domains and internalization signals (blue R and K) that suggest that this protein would rapidly bind to surface heparan sulfate proteoglycans and be internalized. The adjacent pairs of basic amino acids also suggest that osteopontin my be transported to and into nuclei, and have transcription modifying capability.

MRIAVICFCLLGITCAIPVKQADSGSSEEKQ

LYNKYPDAVATWLNPDPSQKQNLLAPQNA

VSSEETNDFKQETLPSKSNESHDHMDDMD

DEDDDDHVDSQDSIDSNDSDDVDDTDDSH

QSDESHHSDESDELVTDFPTDLPATEVFTP

VVPTVDTYDGRGDSVVYGLRSKSKKFRRPD

IQYPDATDEDITSHMESEELNGAYKAIPVAQ

DLNAPSDWDSRGKDSYETSQLDDQSAETHS

HKQSRLYKRKANDESNEHSDVIDSQELSKVS

QELSKVSREFHSHEFHSHEDMLVVDPKSKE

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Osteopontin is activated by the serum protease thrombin, also known for activating fibrinogen into fibrin to form clots. The cleavage site (green) for thrombin on osteopontin is just to the left of a strong heparin-binding domain to which the heparin of syndecan 4 binds. Thus heparin binding to osteopontin blocks access to thrombin and leaves osteopontin in an inactive form. Also notice that the upper half, N terminal, of osteopontin is dominated by acidic, negatively charged amino acids, D and E, whereas all of the positively charged, heparin-binding domains are on the other half that activates inflammation.

These studies highlight the roles played by inflammation and heparin in cancer. Anti-inflammatory diets and lifestyles enhance heparin production and reduce the potential for cancer. This also suggests that heparin therapies should be explored for use in cancer prevention and treatment.

Humming Nitric Oxide

Access to sinus-infecting bacteria is minimized by poor blood circulation in congested sinus spaces. Dilation of the blood vessels in the sinuses is dependent on nitric oxide present in exhaled air. Humming can break up the static air spaces, replenish nitric oxide and enhance the immune defenses in the sinuses.

Antibiotics must come in contact with bacteria to be effective and that intimate contact is difficult in sinus infections. Congestion of the nasal sinuses yields a thick mucus layer that can separate infecting bacteria from antibiotics or defensive molecules and cells present in adjacent blood vessels. Congestion also increases the distance from the respired gases of the air spaces of the sinuses to the blood vessels of the surrounding tissues. Nitric oxide present in the respired air from the lungs usually provides signals to keep the sinus blood vessels dilated. Blocking the source of nitric oxide causes constriction and reduced blood flow. The result is that congestion isolates the sinuses from the circulation and immune system. This is why antibiotics are relatively ineffective for sinus infections.

Humming vibrates air and can even shake up the sinuses. Persistent humming can provide enough nitric oxide to the sinuses to dilate the blood vessels and reestablish effective circulation. Humming alone is usually as effective as antibiotics in treating sinus infections and will also augment the use of antibiotics.

It is not surprising that traditional practices to augment health include breathing exercises that will enhance circulation in the upper and lower respiratory tract. These practices also include the prominent use of humming, aummm.

Estrogen and Brain Damage

Estrogens provide protection from some forms of inflammation and reduce symptoms of acute trauma as well as chronic inflammation.

Researchers were trying to figure out why some people died of brain injuries that others sustained with minimal lasting damage. Rats showed the same differences, that were readily sorted into a group of females with minimal damage, females with moderate to severe damage and dead males. Supplementing the females with estrogens protected them all. Supplementing the males with estrogens protected them too.

Head trauma and various kinds of strokes show essentially the same protective quality to estrogens. Estrogens appear to diminish brain inflammation following trauma and lowering inflammation apparently minimizes lasting damage. It is as if inflammatory swelling and not the initial injury causes damage in brain trauma.

Estrogens work like all steroid hormones by binding to cytoplasmic receptors. The activated, hormone-bound receptors are then transported to and into the nucleus, where they act as transcription factors and regulate gene expression. The activated receptors may also act directly on the inflammatory transcription factor, NFkB and interfere with inflammation signaling. The net result is that estrogen-treated cells are less responsive to inflammatory signals, which also include pyrogenic bacterial wall fragments, such as LPS.

The periodic rise and fall of estrogens in the menstrual cycle of women results in a corresponding general cycle of susceptibility to inflammation. Women can become pregnant at the low ebb of inflammability, but when estrogens are low and inflammability is high, women display symptoms of any suppressed underlying inflammation. Chronic inflammation can be displayed in inflammatory symptoms such as acne, arthritis pain, depression, etc. Chronic inflammation can be so high that inflammatory disease symptoms appear throughout the menstrual cycle and even lead to infertility. Some women may experience relief from their inflammatory disease symptoms by taking birth control pills that suppress inflammation with higher levels of estrogen.

Menopause will also reveal underlying chronic inflammation and women going into menopause would be advised to be particularly vigilant and proactive to reduce sources of inflammation. The best advice that they could get would be to aggressively pursue an anti-inflammatory lifestyle and diet.

Hot Springs

My wife and I took a quick break at Gold Fork Hot Springs, south of McCall, Idaho. The hot springs are laced with lithium salts, but I began to wonder if the more profound healing effects of hot mineral waters are due to the warming of inflamed, or perhaps chilled and hibernating, bodies.

The heat sensors in your skin respond to vanillin-like molecules, e.g. hot pepper capsaicin, or elevated temperature to cause a sensation of warmth. Thus the vanillin receptors are proteins embedded in the membranes of nerves and they bind the molecules of the appropriate shape, the receptors change shape and start an electrical signal that moves to the brain. We experience that as warm or hot, depending on the intensity of the signal.

The nervous system also responds by a returning nerve signal that releases anti-inflammatory hormones in the tissue from which the original hot signal arose. This is the reason that heat applied to an inflamed wound will reduce swelling, redness, etc. Capsaicin or castor oil applied to the same general area will also reduce inflammation, because capsaicin and castor oil (ricinoleate) bind to the same vanillin receptors.

I think that hot springs are anti-inflammatory, because they stimulate vanillin receptors over a large portion of skin and stimulate body-wide suppression of inflammation. This would suggest that many inflammation-based diseases would benefit from hot springs, saunas, sweat lodges and other types of heat treatments. Because cold/menthol receptors are involved in a parallel anti-inflammatory response, the sauna to snow alternation may make a lot of sense for reducing chronic inflammation.

Fever and chills may also be related to these effects. Sudden shifts in the temperature of tissues may disrupt the equilibrium between the tissue and a quiescent pathogen. Heat treatments may similarly disrupt the chronic, bacterially-induced inflammation that some postulate to be the basis for many degenerative diseases.

Toxins, BPA, Estrogens

Toxins and estrogen mimics are potential threats to our health, but diet-induced inflammation is a major contributor to disease and makes us susceptible to environmental toxins.

The world is full of toxins. More industrial toxins are produced each year and many escape. The drugs and bioactive molecules that people consume seeking health end up down the sewer and into waterways via inadequate sewage treatment. Estrogens from birth control pills end up in our waterways in sufficiently high concentrations to alter the fertility of some fish species and impact ecosystems. The big question is, “Are these molecules toxic to us at very low environmental concentrations?”

Can we argue that if some people are sensitive enough to suffer from a millionth of a peanut, that vanishingly low concentrations of a molecule, e.g. bisphenol A that interferes with the binding of estrogen to its receptor, can cause severe development problems and disease? This may be a suitable comparison, because in both the case of peanut allergy and an acute sensitivity to a particular chemical, the sensitivity may be the key issue. Most people may suffer no problems, because a healthy body processes toxic materials and keeps functioning. Those people with defective immune systems, in the case of allergies or unusual sensitivities may be the canaries in the mine -- warning the rest of us of accumulating toxins. Alternatively, the unusual sensitivity may be similar to the allergies and provide a dangerously aggressive response to an otherwise innocuous environmental material, such as a peanut.

How do we know if the toxins in our midsts are real threats? Lets treat the easy cases first. Cigarettes are not too dangerous until they are burned, but then the smoke and the filters or unused butts are quite dangerous. I don’t think that most biochemistry research laboratories could get permission to bring the hazardous materials from a burned cigarette into their laboratories. Special handling, including air purification would be needed. Clearly this type of toxic material that can cause damage at very low concentrations must not leach into our environment. Cigarette butts should be handled as hazardous waste and should be never left unattended. It should certainly be illegal to discard this material where children could come in contact. Radioactive materials similarly should also be handled with great care.

Our bodies can handle relatively large amounts of some toxic materials -- detoxifying enzymes are present in the walls of our intestines and in the liver to convert toxic dietary molecules into other molecular forms that are less toxic and more readily excreted in our urine. Thus, we can eat some plants that would kill our pets, because dogs and cats lack the ability to handle common plant toxins. Some manmade toxins may be similarly handled by functional detoxifying systems in healthy people. A classic argument in this context is whether organic vegetables are healthier than those produced by chemical farming. Organic vegetables have higher levels of naturally occurring, but nonetheless potentially hazardous toxins produced by opportunistic fungi, i.e. mycotoxins, and the plants themselves, e.g. phytoalexins. Chemical farming produces veggies higher in manmade toxins. Which is safer? I would choose the organically grown, but that is probably just my personal prejudice, because the research is fairly clear that organically grown veggies don’t actually make people healthier. It is healthier to eat more veggies from any source. Unfortunately, just adding vitamin supplements also doesn’t improve health. That is why I recommend an anti-inflammatory diet supported by an anti-inflammatory lifestyle.

But what about the bisphenol A that is in the news. As the name implies, this molecule has two ring structures, phenols, that are connected and this combined structure vaguely compares to the structure of steroid hormones. The bisphenols were originally synthesized as potential birth control drugs. They aren’t very effective in competing for binding to estrogen receptors or to the enzymes that are involved in the synthesis of steroid hormones. I don’t know how quickly the bisphenols would be excreted from the body, but I doubt if they accumulate. So BPA should be excluded as an added ingredient in shampoo, for example, but I personally would not worry about the small amounts that leach from polycarbonate bottles.

There is one worrisome observation that I heard in a news report, and that is that BPA may disrupt the release of adiponectin from fat tissues. Adiponectin is anti-inflammatory, so this got my attention. I guess I will keep drinking my water from a glass.

I will leave with a final anecdote. My wife encouraged me to contribute to a listserve that was discussing the dangers of soy baby formula, based on the use of genetically modified soybeans and glyphosate herbicide. Since I had told my wife that glyphosate, which readily breaks down into an amino acid and phosphate in soil, is one of the few safe and environmentally friendly herbicides, I agreed to present my views that genetically modified soybeans were probably a better source for the baby formula than traditionally grown soybeans. (This doesn’t suggest that I think that any formula is safe for babies -- I am only considering the relative safety here.) So after I went out on the limb here and supported glyphosate, I read that this herbicide modifies the metabolism of the soybean plants and increases the level of estrogenic compounds! I had recommended a treatment that potentially exposed this group of soy formula-fed babies to higher levels of phytoestrogens. So, beware of unknown consequences and stick to mother’s milk.

In summary, my personal feeling is that the toxic molecules in our environment are a minor impact on our health. Environmental carcinogens, for example, are a rare source of mutations in our cells compared to the natural mutation frequency due to replications errors of our cellular machinery. Unexposed to any environmental insults our cells would produce at least one hundred times as many potential cancer cells as result from common environmental carcinogens (smoking accepted). I think that we should focus our attention on reducing the big problem of diet-induced chronic inflammation, thereby develop a higher general level of health that would reduce degenerative and autoimmune diseases by at least 90% and then consider the smaller problem of environmental toxins. Most healthy people would resist the minor impact of toxins, as well as their personal genetic predispositions, to disease.

Rattlesnake Venom

Plants attack pathogens, pests and herbivores with toxic chemicals, whereas snakes attack other animals with heparin-binding protein toxins. The rattlesnake toxin, crotamine is an example of a small peptide cross-linked by disulfide bonds, which attach to cells via heparin-binding domains.

I had a sabbatical in Singapore, at the National University. I was seated on the patio of the university canteen eating one of my typical lunches: curried mutton with hot lentils, rice and a durian milkshake, served on a banana leaf. I struck up a conversation with a biochemist who studied the structure and function of snake venom proteins. He was systematically analyzing the proteins in various venoms looking for proteins that interact with essential features of cells. This was a potential initial step for the design of new drugs. I remember asking him how he knew which part of the venom proteins was important and which parts just served as a rigid platform to display the active parts. He said that it was simple, the water-binding, hydrophilic amino acids that formed amorphous loops bordered by sulfhydryl-bonding cysteines were his targets. Ten years layer, I observed that these loops also have the basic amino acids (K, lysine and R, arginine) that form heparin-binding domains.

His conversation came back to me a couple of days ago when I ran across the structure and function of the crotamine toxin from the venom of the South American Rattlesnake, Crotalus durissus terrificus.

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG

The heparin-binding domains were evident in both the amino acid sequence as well as the protein structure. As is true of many small proteins, or peptides, they are held in their functional shape by -SS-, disulfide bonds, between cysteines (C). The presence of two well-defined heparin-binding domains (blue) also predicts that the toxin would be anti-bacterial and that it would bind to phospholipids, i.e. membranes. The observed toxic quality of the toxin is its ability to disrupt ion transport through membranes and it has a shape similar to the mammalian anti-bacterial peptides, defensins. Most venom toxins bind to the heparan sulfate proteoglycans (HSPGs) of their victim's cells and then as the HSPGs are brought close to the cell surface during recycling, the toxins attack the membrane proteins and kill the cells.

I am tangentially interested in snake venom, because the proteins that mediate its toxic effects are related to the hormones that mediate inflammation.

It is interesting that a simple discussion over curried mutton ten years ago would be so consistent with a major shift in my research interests to study inflammation.

Simple Remedies

Simple is usually best. Traditional herbal cures are tried and true. I learn a lot about biology by studying what works in herbal medicine. Some simple plant products, such as Vicks Vaporub, are very potent cures for what typically ails you. Castor oil is an excellent topical pain killer.

Herbs and spices excite our senses and dominate cuisines. Cultures are identified by their food, but the use of particular plant materials to food is not a random act of history, nor is it limited to the regions where the herbs and spices first appeared. Research by Paul Sherman at Cornell, and others has shown that herbs and spices that are used in a culture are also the most effective at inhibiting pathogens and parasites where that food is traditionally served.

I must talk about some related experiences that touch on the same subject, but are simply fun explanations of cultural practices. Milk is used in some interesting cultlural practices, because it has very potent anti-viral, anti-bacterial and anti-fungal components -- milk keep newborns well nurished, but also safe from nasty germs, etc. while the immune system the baby matures. Two astoundingly disparate applications of milk come to mind: pruning fruit trees and walking hot coals. I have observed both. I previously worked with plant pathologists and I watched pruners sanitize their shears in milk between trees. The milk stopped the spread of viruses and bacterial pathogens. I also observed firewalking in the Sri Mariamman temple in Singapore. The firewalkers stepped from the coals into a pool of milk to stop infections of their singed feet. In both examples, milk provided an abundance of anti-microbial molecules that were retasked from protecting babies to protecting trees or adult feet.

Herbs and spices are plant products that are toxic to plant pathogens or herbivores, which are retasked to protect people. Some of these, such as the curcumin in turmeric, are potentially more effective that commercial drugs. I want to point out some of the common plant materials that are very useful in our diets and to remedy common infections, aches and pains.

My current champion cure-all is Vicks Vaporub. This thick ointment has the pleasant scent of its ingredients, menthol, eukalyptol, camphor and terpentine. I associate the smell with childhood treatment for congestion. I also remember that my father used to rub it on his arthritic hands to loosen them up before a day’s work. There is a solid physiological basis for the action of Vicks. Many of the ingredients are powerful antibiotics effective against a variety of bacteria and fungi. Vicks is one of the most effective topical treatments for athlete’s foot and ringworm fungal infections. The menthol is cooling, because it binds to the cold sensing receptors and it is an effective analgesic and anti-inflammatory, because it triggers acupuncture like responses through the vagus nerve. I would also try Vicks on autoimmune conditions of the skin, because of both the anti-inflammatory and anti-bacterial properties. I have even seen Vicks recommended for the same reasons for the treatment of acne. TMJ pain and inflammation apparently responds to the menthol.

Castor oil binds to heat detecting receptors of the skin and works similarly to hot pepper capsaicin. Castor oil can be used to stop many aches and pains in arms and legs by topical applications. Since most of these plant products act through the pain sensors in the skin, they don’t actually penetrate to the joints involved, but rather they trigger release of neurotransmitters from nerves that do penetrate to the sites of interest. I also think that the use of castor oil packs applied to the skin of the abdomen, may have systemwide anti-inflammatory impact.

Garlic is the most anti-bacterial of the herbs, but most of the common herbs added to food probably affect the gut flora and shift it to a more anti-inflammatory composition. Many herbs and spices are used as topical cures for acne, because of their combined antimicrobial and anti-inflammatory qualities. Plant materials are very potent. They contain many compounds that are highly effective at low concentrations in neutralizing plant pathogens and herbivores, and so they are also very potent in their impact on the bacteria of our gut and potential on our own systems. Plants are powerful, but just because they are natural does not mean that they are safe. Plants are also rich sources of poisons. Domesticated plants are safer, because we have selected for variants that have lower levels of the compounds that the plants need to otherwise protect themselves. This also means that the compromised varieties need to be sprayed with antibiotics, e.g. apple trees sprayed with streptomycin, fungicides and herbicides. We have traded one group of plant toxins for manmade toxins. All this aside, plants are necessary for our health, but it is better to browse over many different plants than eat a lot of just one. Grains are a relatively recent addition as large components of the human diet, and should also be limited because of their high starch and inflammatory omega-6 oil content.

Turmeric, red pepper and black pepper are commonly ground together and used to enhance many dishes in a variety of different cuisines. It turns out that the curcumin in turmeric and the capsaicin in red pepper are very potent anti-inflammatory agents, but they are enzymatically modified as they are absorbed through the intestines. The black pepper piperine inactivates the enzymes of the intestines and enhances the effectiveness of the other two chemicals. Thus, there has been a lot of trial and error optimization in the use of spices. It makes a lot of sense to eat the way that locals eat when traveling.

Chronic Disease and Bacteria

There is increasing evidence that many, if not all, chronic degenerative and autoimmune diseases have a bacterial component and that antibiotics may be much more broadly useful.

Antibiotics are fed to children with ear infections, fed to cattle to fatten them, sprayed on apple orchards to stop fireblight and used to treat AIDS patients with nuisance to nasty infections. Bacteria are in many ways better adapted for exploiting nutrients than plants or animals, but antibiotics level the playing field.

Fungi have the same problems with bacteria stealing their food and they have adapted their biochemistry to kill off competing bacteria with their antibiotics, while they digest the world around them with enzymes. Thus, the classic antibiotic, penicillin, is made by a green mold and this antibiotic kills bacteria by mimicking one of the ingredients used to make bacterial walls -- the unsuspecting bacterium incorporates the penicillin into its wall and the compromised wall stretches apart. Bacteria grow themselves to death in the presence of penicillin.

Antibiotics discriminate between bacteria and us, because the bacteria use different molecular machinery, e.g. ribosomes, cell walls, than we do. So antibiotics kill bacteria and our cells are spared. Some antibiotics are somewhat specific and can be used to kill just certain types of bacteria, while other antibiotics are broad spectrum and rather indiscriminate.

Use of antibiotics is problematical, because most of our bacteria are necessary for our health. Antibiotics kill a substantial fraction of the beneficial bacteria that live in and on us, and leave us exposed to opportunistic pathogens and parasites. Everyone is familiar with yeast infections following a course of antibiotics and the rapid acquisition of hospital strains of antibiotic resistant bacteria.

Another problem with antibiotics is that they fail to reach every nook and cranny of the body. Sinuses, for example, provide vast distances that separate bacteria from the blood stream. Similarly, pieces of metal that pierce cartilage can produce bacterial infections that can’t be reached by antibiotics, because cartilage inhibits capillary production, so their is no blood circulation to carry the antibiotics to the bacteria. In the same way, the depletion of capillaries in the extremities of diabetics with poorly controlled blood sugar, makes infections difficult to treat with antibiotics.

Even under the best of conditions it is difficult for antibiotics to reach lethal concentrations for resident bacteria and biofilms make survival of bacteria certain. Bacteria have adapted to the hostile environment of human tissue by making structured colonies. The outer layers of bacteria are sacrificed to spare the inner, proliferating bacteria. Bacterial biofilms are highly resistant to antibiotics and to attack by the cellular defenses of the immune system. Unfortunately the bacteria in biofilms, and in other forms adapted to human tissue and have a different metabolism and stain differently from the same bacteria grown on laboratory agar plates. Thus, it is quite possible for pathogenic bacteria to be undetectable and fail to be associated with a particular disease.

Bacteria may avoid scrutiny by pathologists, but they cannot fail to release cell wall fragments and debris, which triggers inflammation and other responses from the surrounding tissue. Numerous researchers have implicated mycobacteria, chlamydia and many other bacteria as causal agents of degenerative and autoimmune diseases -- there is a distinct correspondence between inflammatory diseases and these bacterially-associated diseases. There are also recent patents supported by provocative experimental data, that claim that aggressive antibiotic treatments (usually supported by anti-inflammatory lifestyles and diets) can cure these diseases.

I expect that antibiotic cures for many degenerative and chronic diseases will be demonstrated with a huge accompanying upheaval of traditional medicine. There will be, and there has been for the last century, substantial resistance to antibacterial approaches. Even though Helicobacter pylori was implicated as the causal agent of ulcers, antacids and blockers of acid production persist as the predominant treatment. The role of bacteria in chronic disease will be substantiated, but for me the lingering question will be, “where are the bacteria that are being treated?” I still think that the best candidate is the gut. Afterall, the gut is the body’s major interface with the outside world and there is a compelling research literature on the impact of gut flora on health and disease.

Migraine, Heparin, Glucosamine

Is it possible to repair the damage that leads to migraine headaches? If chronic inflammation is a risk factor, then depletion of brain heparan sulfate proteoglycans could be repaired temporarily with heparin or gradually with glucosamine.

Mast cells, the secreters of histamine in allergic reactions, also secrete heparin at the same time. In fact, cattle and swine intestines are the sources of commercial heparin. The crude source of heparin became dangerously apparent in recent scandals over the adulteration of Chinese heparin raw materials with hypersulfated chondroitin sulfate. But why is heparin secreted along with histamine in mast cells and what does this have to do with migraine headaches and glucosamine?

A basic observation is that migraineurs who receive heparin or glucosamine treatments for other symptoms, see relief from their headaches. Heparin treatments can produce dramatic effects that may be shortlived. Glucosamine may require substantial amounts taken orally for four to six weeks before migraines abate. Heparin and glucosamine are both effective, but how do they work?

Heparin and glucosamine therapies are both awkward. Heparin is rapidly internalized and degraded by cells. Glucosamine is also rapidly taken up and processed. In both cases, ingested heparin or glucosamine do not enter the blood stream. I think it is highly unlikely that either taken orally will have their effects directly in the brain or joints, simply because they are commonly metabolized molecules. Glucosamine may be a building block for heparin and other glycosaminoglycans, but it just passes directly into glucose metabolism, so that logical connection is unimportant in joint pain or migraines. I covered glucosamine in more detail as an anti-inflammatory molecule in another article, so here I will only emphasize that glucosamine is very effective for prophylaxis of migraine headaches.

Heparin can have an impact orally for some bowel diseases, but for headaches it must be administered IV or by inhaling. The major point here is that heparan sulfate proteoglycans (proteins with long heparan polysaccharides) mediate most of the hormonal signaling by mediating the binding of hormones to their receptors. Heparan fragments, called heparin, are a mixture of molecules that may interfere with or augment signaling, dependent on the quality of the heparinoids and the signal pathways under consideration. In most cases, there are so many different hormones and receptors involved, that it is more straightforward to try heparin to see if it works, rather than attempt to sort out all of the side reactions. If heparin does work, it should also be noticed that the amount of heparin required for an anticoagulant effect is on the high side, reflecting the depletion of circulating heparin.

Mast cells and histamine release have been implicated in migraine, but we have to return to the question of why heparin is normally released at the same time. From my own experiments with chondrocytes, the cartilage secreting, developmentally related sisters of the arterial endothelial cells, I would expect that inflammation inhibits heparin synthesis. Prevailing inflammation may reduce the production of heparin by mast cells and neuronal cells. As a result, triggering mast cells may release histamine into heparin-depleted brain tissue. If heparin normally serves to control the spread of the inflammatory signal from mast cells, then the absence of adequate heparin may lead to a spreading inflammation, a kind of neurological shock and awe. Addition of circulating heparin may temporarily repair the blood brain barrier, just as it does the lining of the bladder with interstitial cystitis or the kidneys in diabetes or the intestines in protein lossing enteropathy.

It appears that migraines are based on chronic inflammation of the brain and an associated compromise of the blood brain barrier. The source of the chronic inflammation may be shared with other degenerative and autoimmune diseases that lead to migraine attacks. Underlying infections may be hard to identify. The solution is to reestablish the blood brain barrier by eliminating chronic inflammation. Separate reports indicate that glucosamine, omega-3 fatty acids and anti-oxidants are all effective in reducing migraines. It seems obvious that the first step would be to begin an anti-inflammatory lifestyle (including exercise, dental hygiene, etc.) supported by an appropriate diet.

NO Migraine

Migraine attacks are based on inflammation and nitric oxide production in the capillaries and nervous tissues of the brain. Drugs that impact NO have a major impact on migraine attacks.

What do angina/nitroglycerine, impotency/Viagra, and migraine/L-NMMA have in common? The answer is nitric oxide, NO, a potent vasodilator and messenger molecule derived enzymatically from the basic amino acid arginine. NO is ubiquitous, short-lived and associated with inflammation. One of the NO synthetases (iNOS) that make NO is inducible as part of the suite of inflammation genes under the control of NFkB. Other NOS enzymes are controlled by cellular calcium levels and since we will be talking about neurons, it is important to mention that neurons ultimately release neurotransmitters when an action potential reaches the synapse and causes a sharp increase in calcium.

NO acts on a cell by activating cellular GMP cyclase to produce cyclic GMP. cGMP in turn can activate MAP kinase or NFkB. Viagra, by the way, enhances the vasodilation effects of NO by inhibiting the breakdown of cGMP and thereby causing more dilation with a limited amount of NO. Nitroglycerine/glyceryl trinitrate (GTN) increase the amount of NO available to endothelial cells and relax coronary arteries.

People who suffer from migraines, migraineurs, are sensitive to NO donors such as GTN and have a migraine attack after a delay of 4-6 hours. The headaches are not usually preceded by the typical visual auras. Non-migraineurs have no response. These results suggest the critical role of NO in the early stages of the migraine attack. Inhibition of NOS by L-NMMA reduces symptoms in most migraineurs after a spontaneous attack starts. Metabolites of NO also increase during migraine attacks.

Viagra, which blocks breakdown of cGMP, also induces migraine attacks in migraineurs and it does not cause dilation of the middle cerebral artery, suggesting that vasodilation may be only associated with migraine attacks, but not integral. Histamine can also cause attacks, but requires the NO pathway.

NO appears to act by increasing the release of the neuropeptide called CGRP, because NO initiated attacks can be stopped by drugs that block the CGRP receptor.

Inflammation is both a precondition for migraine attacks and a response to administration of NO donors such as GTN. The inflammatory transcription factor NFkB is activated by GTN and elevated levels of inflammatory cytokines, IL-1, IL-6, are released. Pretreatment with parthenolide, the active ingredient in feverfew, which is used as a migraine prophylaxis, reduced activation of NFkB by GTN. This observation reinforces the view that migraine pain results from NO induced inflammation.

The most effective and selective treatment for migraine headaches are drugs that block the action of the 5-hydroxytryptamine (5-HT) receptors, e.g. triptans, such as pizotifen and methysergide. The 5-HT receptor stimulation can result in NO production and blocking these receptors, blocks subsequent NO production, inflammation and pain. Prophylactic administration of NOS inhibitors, such as L-NMMA or NO scavengers, such as hydroxocobalamin, can reduce attacks.

There is substantial evidence that anti-inflammatory lifestyle and diets have a significant impact on the frequency and intensity of migraine attacks, because they minimize the participation of inflammation and its product, NO.

Neeb L, Reuter U. 2007. Nitric oxide in migraine. CNS Neurol Disord Drug Targets. 6(4):258-64.

Toulouse-Lautrec

Cathepsin K and Heparin

The power of simple sequence pattern analysis in predicting protein behavior is illustrated in the case of Cathepsin K, a papain-like cysteine protease, involved in many degenerative diseases, bone development and Pycnodysostosis (Toulouse-Lautrec syndrome.) Triplets of basic amino acids are typical of heparin binding proteins that are internalized.

I admit that I am obsessed with inflammation and heparin. My daughters automatically yell out “Give him heparin!” when a patient on ER has a severe migraine attack. They think it is a good joke until the savvy doc reveals the latest approach and actually injects heparin with satisfying results. Heparin and inflammation are intimately involved and I predict that blood tests that determine the quality and quantity of protein-bound heparin, will ultimately be used as measures of chronic inflammation, as well as revealing a variety of diseases.

I have a habit of examining the molecular basis of diseases that I encounter on TV, in newspapers or in books. Wikipedia is my first source, followed by the National Center for Biotechnology Information (NCBI). As soon as I find the genes/proteins involved, I check to see if the structures has been determined by X-ray crystallography or NMR, and then I look at the amino acid sequence. I check for pairs or triplets of basic amino acids. Invariably the pairs are matched with a neighboring basic amino acid, and that is a putative heparin-binding domain. Triplets almost always indicate secreted proteins that are brought back into cells dependent on strong affinity for recycled heparan sulfate proteoglycans. Within minutes of hearing about a new disease, I usually know something about the molecules involved and particularly whether or not inflammation is going to be a major factor.

I was just reading Outlander by Diana Gabaldon and one of her characters has the short stature and disablity of Toulouse-Lautrec syndrome. I literally ran to my computer, because I am particularly interested in diseases of cartilage and bone. Since TLS is a genetic disease, I checked the NCBI Online Mendelian Inheritance in Man (OMIM) site and found that the genetic defect is in the cathepsin K gene. Cathepsin K is a protease similar to papain, which is intimately involved in many different facets of development, as well as cartilage and bone production. The cells that degrade cartilage to remodel bone, osteoclasts, use cathepsin K to degrade collagen.

I found a structure for cathepsin K bound to chondroitin sulfate. The structure looked all wrong, based on my prejudices -- the sugars of the polysaccharide should have been bound to the basic amino acids or to surface aromatic amino acids. The accompanying amino acid sequence told the whole story:

---DYRKKGYVTPVKNQGQCGSCWAFSSVGALEGQLKKKT---

There were two triplets of basic amino acids (R, arginine or K, lysine), indicative of internalization and strong heparin binding. I performed a quick literature search for heparin binding and internalization and found a reference that confirmed my hunches (note the title):

Nascimento FD, Rizzi CC, Nantes IL, Stefe I, Turk B, Carmona AK, Nader HB, Juliano L, Tersariol IL. Cathepsin K binds to cell surface heparan sulfate proteoglycans. Arch Biochem Biophys. 2005 Apr 15;436(2):323-32.

The article demonstrated that cathepsin K bound only to the surface of cells that produced heparin sulfate and was internalized only by heparin-producing cells. Moreover, cathepsin K changed shape as it bound to heparin, but not to chondroitin sulfate.

This story underscores the predictive power of simple generalizations derived from the dominating interactions between heparin and proteins. Heparin-binding domains, because of their positive charges, stay on the surface of the protein, don’t tend to fold well into helices or other secondary structures and are readily recognized in amino acid sequences of proteins. Stronger heparin-binding domains involved in internalization or transport into nuclei are even more stereotyped as triplets or quadruplets, respectively, of basic amino acids.

There are some complicating special cases involving basic amino acids, since these amino acids are also involved in glycosylation, nucleic acid binding, inositol phosphate interactions, phospholipid interactions, protein folding/chaperone binding, and protease action, but the generalizations outlined here provide a starting point for exploring the exciting area of heparin binding.

Knowing just a few typical patterns, you can just look at a protein sequence and amaze people by telling them that they should be able to purify their protein on heparin Sepharose! You can also point and gasp at the fact that in the early 1990’s in China the bird flu hemagglutinin picked up a new sequence with a quartet of four basic amino acids. When I saw that I called the CDC and explained the new cell receptor! It still has me scared -- am I the first one to notice the potential for a pandemic much more severe than the 1918 Spanish flu?

Inflammation Protects

Inflammation in response to infection mobilizes the immune system and serves a vital purpose in defense against disease and parasites. Too much or too little inflammation is unhealthy.

A recent report (ref. below) provides a note of caution in the use of anti-inflammatory dietary supplements. Modern diets are inflammatory and provide a major predisposition to degenerative and autoimmune diseases, but the inflammation has some qualified advantages in providing protection to infectious diseases. Densely packed humans survive against the constant threat of epidemics, in part because of heightened dietary inflammation.

The paper listed below shows that habitual use of the potent anti-inflammatory compound in turmeric, curcumin, can effectively lower chronic inflammation. Inhibition of inflammation is not a good thing, however, if the source of inflammation is a chronic parasitic infection of Leishmania. While this article indicates that suppression of inflammation may not be uniformly advised, it does demonstrate the effectiveness of natural products, such as curcumin.

A different study of tuberculosis comparing  land-locked versus genetically related, fish-fed populations, found that the fish eating group was more susceptible to the disease. The interpretation of the results was that the high omega-3 fatty acids of the fish diet reduced chronic inflammation in the coastal community and thereby increased the susceptiblity to TB.

The bottom line for me is that a healthy, anti-inflammatory diet is much safer than the typical modern inflammatory diet of high carbs, high vegetable oils (omega-6), low fruits and vegetables (few antioxidants, low in vitamins C, D and A) and harsh for supportive gut flora. It does leave open the possibility that during an unusual infection a return to fast food may shift your body into a state of high alert.

Adapala N, Chan MM. Long-term use of an anti-inflammatory, curcumin, suppressed type 1 immunity and exacerbated visceral leishmaniasis in a chronic experimental model. Lab Invest. 2008 Sep 15. [Epub ahead of print]

Steroid Hormones

Numerous cholesterol-derived hormones (testosterone, estrogen, corticosterone, vitamin D, vitamin A, retinoic acid) influence gene expression by directly binding to cytoplasmic transcription factors. These hormones control inflammation, as well as development and numerous examples of cellular differentiation.

Hormones are a form of molecular communication between cells in different locations in the body connected by the blood stream. Cells, for example in the pituitary of the brain, receive a nerve or chemical signal and respond by the secretion of a steroid hormone that changes the behavior of cells in other parts of the body. Steroid hormones are produced enzymatically in mitochondria by the enzymes (P450) related to those used to detoxify dietary molecules.

Steroid hormones are useful for transmitting messages, because when they stick to the surface of a protein, they can make it change shape. The shape change is the result of the hybrid structure of steroids -- they are large flat molecules that don’t bind to water, i.e. hydrophobic, on their faces, but form bonds with water, i.e. hydrophilic, on the edges. This dual, amphipathic, nature means that the steroids will bind to hydrophobic pits in proteins and as the steroid squeezes into the pits, it will change the shape and ultimately the activity of the protein. Sugars, on the other hand, have small rings of carbon atoms that are hydrophobic, but their small size means that they bind to small aromatic amino acid residues in shallow surface pits that don’t usually change the shape of the proteins. Hence, there aren’t sugar-responsive transcription factors, but large, highly charged polysaccharides, such as heparan sulfate proteoglycans, can dominate the structures taken by proteins, e.g. most protein hormones and their receptors.

Steroid hormone receptors are cytoplasmic, whereas receptors for proteins hormones, such as insulin, are found on the surface of cytoplasmic membranes. It is commonly assumed that steroid hormones, because of their amphipathic characteristics, can diffuse through cytoplasmic membrane and move directly from blood to the cytoplasm of cells. I doubt the evidence and think that diffusion is too slow to account for the action of steroid hormones. It is more likely that steroid hormones bind to carrier proteins in the blood, and general transporters transfer the steroids either directly from their carriers to intracellular receptors (testosterone, estrogen, corticosteroids) or the carriers are transported all the way to the nucleus for transfer of the steroids to nuclear receptors (vitamin D, vitamin A, retinoic acid). Binding of the steroids to cytoplasmic receptors displaces chaperones (heat shock proteins) and expose nuclear localization receptors (groups of basic amino acids that would function as heparin-binding domains, if the proteins ever found themselves in the extracellular environment) that result in transport into the nucleus.

Receptors with bound steroids act as transcription factors in the nucleus -- they bind to unique DNA sequences and control gene expression. Thus, anti-inflammatory steroids can block the signaling via NFkB from a bacterial infection (LPS triggering the TLR) by directly interfering with transcription (binding to inhibitory control elements) of inflammatory genes. The presence of estrogen receptors in a wide variety of tissues provides an overall decrease in inflammation in the presence of estrogen. Thus, women going through menopause and reduction in estrogen, may experience a sudden increase in inflammation-based symptoms that were previously suppressed by anti-inflammatory estrogen.