Spider Inflammation

Happy Halloween! It's time for creepy, crawly things that bite... and cause inflammation.

The brown recluse has the bite that keeps on giving. Its venom contains a phospholipase that cleaves membrane phospholipids to release arachidonic acid (omega-6 fatty acid precursor of inflammatory eicosanoids) and lysophosphotidylcholine that binds to multiple receptors to signal NFkB-based inflammation.

It’s Halloween and a time to talk about things that bite and sting. After all, the symbol of Halloween is the Jack-O-Lantern, the flame within, i.e. inflammation. Venom is Nature’s answer to the need for instant inflammation.

The brown recluse is adapted for hunting, killing and eating. The killing bite is the subject tonight. The recluse injects a venom designed to get the biggest bark for the bite. It uses the least amount of protein to immobilize its prey/meal. Of course, since spiders are suckers, it helps if the venom also liquifies its meal.

The venom also has a secondary use as a defense. In this case, if something too big to eat encounters a brown recluse, the goal is to encourage the big beast, you or me, to leave. No pain, no gain.

Venom provides a convergence of enhanced meal making and avoidance of being made into a meal. The answer is, of course, enzymatic inflammation. Venom contains enzymes that convert common cellular components into potent pyrogens, inflammation inducers. The wicked witch of the world of inflammation is phospholipase A2 (PLA2).

Brown recluse venom PLA2 clips off the arachidonic acid from membrane phospholipids and leaves an amphipathic detergent, lysophospholipid (LPL). The LPL can destabilize membranes and rupture cells. It is also a potent activator of several cell receptors that signal inflammation via NFkB. The COX-2 induced by inflammation has a readymade substrate present, arachadonic acid, and produces inflammatory prostaglandins, that continue the signaling frenzy. The result is inflammation, pain and necrosis.

Recluse bites are hard to treat. I think that some people recommend topical application of gunpowder. I guess that would make sense, because one of the problems of recluse bites is actually constriction of blood flow by other venom components, and nitroglycerin patches apparently provide some relief. Both the patches and nitrates of the gunpowder cause vasodilation, and would remedy some of the circulation problems of the venom. Glucocorticoids are also partially effective, since they are anti-inflammatory. Then there is heparin. I would always try heparin, at least as a last resort, because heparan sulfate proteoglycans are an essential component of most extracellular signaling systems, e.g. cytokines. Heparin should disrupt or activate all of these systems. It is unpredictable. It turns out that heparin is helpful against the brown recluse. I would also like to try a topical potion made of Vick’s Vaporub (menthol, eucalyptol, camphor, turpentine), castor oil and heparin. That should fix about anything. It even smells good, sooths painful joints and makes your skin soft.

Happy Halloween. Enjoy trick or treating, but watch where you put your hands.

Cartilage as Rejuvenation

I have studied cartilage secreting (chondrocytes) for the last few years. Chondrocytes are normally derived by differentiation of mesenchymal stem cells (MSCs) that grow in the bone marrow. MSCs can differentiate to produce bone secreting cells (osteoblasts), muscle cells (myoctes), fat cells (adipocytes) and insulin-secreting cells (beta-pancreatic islet cells.) I chose to study a rat chondrosarcoma (RCS) cell line, because this is a type of cell that shares the properties of many other important cells and it will continue to grow in cell culture. Thus, I can dilute some RCS cells in a solution with all of the nutrients required for growth and the cells will stick to the surface of the plastic dishes that I use, grow and differentiate. If you stain the cultures for cartilage, you get the following micrograph.

At first the cells stretch out and move about the surface of the dish. Then they become progressively less adhesive to the surface and more spherical as they start to produce and secrete the polysaccharides (glycosaminoglycans, such as chondroitin sulfate and heparan sulfate) and proteins (collagen) of cartilage. Finally they produce thick layers of cells that are separated and embedded in cartilage. After a little more than a week in culture, the cells are moving through the cartilage matrix by enzymatically degrading the cartilage ahead of them and secreting new cartilage in their wake. The cells that eat their way to the dish surface separate the cartilage layer from the dish and the colonies of cells begin to slough off from the dish surface. Normal chondrocytes would eventually stop dividing under culture conditions, but the cancer line that I use continues to grow quite happily and can be diluted and plated continuously. Chondrocytes in cartilage live in cavities within the cartilage and are surrounded by heparan sulfate attached to proteins of their cell membranes, i.e. heparan sulfate proteoglycans.

Cut cartilage will grow back together as the chondrocytes mine, secrete and gradually knit the two surfaces together with strands of new cartilage. Movement through and renewal of cartilage, e.g. in the connective tissue of skin, is restricted if the collagen fibers that are assembled outside of the secreting cells are cross-linked. This is why sunbathing ages skin. High fructose corn syrup also accelerates cross-linking. This cross-linking is also what makes meat less tender. The cross-linking and toughness can be measured by inserting fluorescence-measuring probes into meat, because the protein cross-links fluoresce in UV light.

A consequence of the development of chondrocytes on the ends of bones, is that the nutrients for the cells change. Initially the chondrocytes enjoy the abundant glucose and oxygen of the blood stream and gradually they are remove further from blood vessels. (Note that cartilage actively inhibits vascularization, so there are no blood vessels in cartilage. This lack of blood vessels and associated enhanced risk of disfiguring infection, is a reason to discourage piercings that involve cartilage.) Chondrocytes snug in their little cartilage cavities no longer eat sugar or breath oxygen, they dine on cartilage and ferment.

What happens if you expose mature chondrocytes to a new source of rich nutrients? I think that the answer is rejuvenation. Quite literally, the chondrocytes regress and return to the lifestyle of their youth. Instead of producing mature, weight-bearing, dense cartilage, these rejuvenated cells start to produce the weaker matrix of their youth. This weak cartilage is readily damaged by abrasion and is not suitable for joint surfaces. This is one of the consequences of arthritis. Inflammation of cartilage brings rejuvenating, damaging nutrients to chondrocytes. The mechanical damage leads to destructive cycles of further inflammation.

Chondrocytes in cartilage also respond to mechanical stress and this stress maintains their maturity. Persistent weight bearing at the same orientation leads to bone production. Thus, after joint injury it is important to use the correct regimen of rehabilitation to maintain mobility of the joint and mineralization of the bone.

Palm Oil

One of my favorite chocolates is Truffettes de France. I tell myself that just one serving (1.5 oz) or five pieces will provide an excellent supplement to may daily plant anti-oxidants. I am almost tempted to consider it one of the five recommended servings of fruits and vegetables. Alas, examination of the ingredients lists palm oil first, sugar second and low fat cocoa third.

Palm oil is a story in itself. This oil is about half palmitic acid (C16), one third oleic (C18) and one tenth linoleic (C18). The soap made from palm oil was developed in the US by the Palmolive company. Notice the name, palm-olive, from the two prominent fatty acids, which are also named after their original sources, palm and olives.

Another rather shocking use of palm oil is with naphthalene to produce a sticky flammable mixture called napalm.

Oil palms were brought to Malaysia from West Africa in the beginning of the 20th century. When I stayed in Singapore in 1997 for a research sabbatical, the monsoon season was delayed so that the rainforests of Malaysia and Indonesia were drier than usual. Entrepreneurial plantation owners took advantage of the opportunity to send out thousands of workers to start fires. Fire is the tool used to clear forests and subsistence farmers from lands that cannot be used for profit, and to turn those cleared lands into plantations that use the landless as employees. The smoke was so thick throughout the whole region that street traffic in some areas of Malaysia and Indonesia had to be restricted for safety. I could see smoke even in the lecture halls at the National University of Singapore and my daughters had to check the smoke level outdoors before traveling to the university swimming pool. There were reports that birds fleeing the burning forests were lost at sea and took refuge on ships. Orangutans in Borneo Sumatran Tigers and the Asian rhinoceros are endangered by encroaching plantations.

My big worry about eating the delicious truffles is the linoleic acid. This shouldn’t be too much of a worry, even though it is an omega-6 fatty acid, because it is too short. Most vegetable fatty acids are shorter than the C20 needed to make inflammatory prostaglandins. The trouble is that short fatty acids can be elongated from 18 to 20 to 22 by cellular enzymes and omega-6 fatty acids block the elongation of omega-3 fatty acids. Omega-6 fatty acids that are heavily marketed by the US agribusiness industry, are essential in small amounts and a health risk when present in the diet in a higher ratio to omega-3 oils of greater than approximately three or four to one. The US diet is typically more than 10:1, highly inflammatory.

The single serving of five truffles contains about a gram, 1000 milligrams, of linoleic acid, so it will take about a single fish oil supplement capsule to balance that much omega-6 fatty acid. Maybe I will try to cut back to just a couple of truffles and eat more kale.

Osteoporosis Treatment

Osteoporosis is an imbalance between bone production and loss. Most loss is due to dietary use of omega-6 vegetable oils. Treatment should focus on elimination of demineralizing oils and minimizing inflammation.

It is hard for me to discuss osteoporosis without the image of Sally Field advocating the use of Boniva (the bisphosphonate Ibandronate, see figure) popping into my head. Advertising is very powerful. Bisphosphonates stop bone loss by killing osteoclasts, the cells that demineralize bone during bone remodeling.

Normally demineralization of bone by osteoclasts is followed by secretion of osteoid containing osteocalcin by osteoblasts. Osteocalcin initiates mineralization. Thus, a large fraction of bone is being rejuvenated by balanced osteoclast/osteoblast action at any given time. Cessation of this remodeling process in bone as in cartilage and other connective tissue, e.g. skin, gives the symptoms of aging.

Osteoporosis, loss of bone density, means that there is a net conversion of bone hydroxyapatite (calcium phosphate crystals) into blood calcium, followed by calcium loss in urine. This means that the continuing development and activity of osteoblasts and osteoclasts is out of control. Osteoblasts develop from stem cells that are also the origin of fat adipocytes. The transcription factor that controls the alternative destiny of these stem cells is PPARgamma. Omega-6 fatty acids are converted into molecules that stimulate PPARgamma and result in adipocyte production in bone marrow instead of osteoblasts. In general terms, vegetable oil (except olive oil) makes fat cells instead of bone cells.  This is particularly true in postmenopausal women.  It is no wonder that the emphasis on the use of vegetable oils to avoid saturated fats has resulted in a pandemic of osteoporosis.

Omega-3 fatty acids (fish oil) are anti-inflammatory and they do not stimulate the production of PPARgamma. As a consequence omega-3 fatty acids, DHA and EPA, enhance bone production and density, and are very important to maintain normal osteoblast production.

Osteoporosis treatments that block osteoclast development or activity can be expected to have long term side effects, because normal renewal of bone is being disrupted. Inhibiting bone demineralization can lead to a slowing of osteoporosis, but it does not get to the cause of the osteoporosis.

Because of the prevalence of diet-based chronic inflammation, one might expect that diet and lifestyle are also the foundation of most osteoporosis.  With both inflammation and osteoporosis vegetable oils appear to be the major culprit.  Aging is also associated with osteoporosis.  Most of the symptoms of aging can be attributed to mismanagement of chronic inflammation.  Now it turns out that osteoporosis is a dietary problem (vegetable oil) compounded by physical problems of inflammation that limit activity.  Loss of muscle mass, i.e. sarcopenia, and replacement with fat around organs and in bone marrow can be explained by diet-based chronic inflammation and inadequate weight-bearing exercise.

A major risk factor for osteoporosis is omega-6-rich vegetable oils, e.g. corn, soy, etc. In simple terms, the first step that I would recommend for anyone concerned about osteoporosis is to shift to an anti-inflammatory diet. Eliminate all vegetable oils, except olive oil, from the diet and supplement with omega-3 fish oils (short-chain omega-3 oils in most vegetable sources are much less effective.)

The biomedical literature is very clear. Osteoporosis is not normal, is not a part of aging and can be avoided. There are some genetic predispositions to osteoporosis, but most can be overcome by meaningful diet and lifestyle changes. An anti-inflammatory diet and lifestyle (sunlight for vitamin D and exercise) is the cheapest, safest and most effective way to prevent and treat osteoporosis.

Osteoporosis

Bone density is based on the balance between bone production and demineralization. Inflammatory cell signaling is required for release of calcium. Muscle building exercise favors increased bone density.

Newborns do not have fully formed bones in their limbs. The reason that milk has so much calcium, is that babies mineralize their cartilage bone scaffolds after they are born. Cartilage is made by chondrocytes (sisters of blood vessel endothelial cells and fat adipocytes, with the same stem cell parents) and the chondrocytes will continue to burrow through existing cartilage and make new cartilage, if mineralization does not take place. The cells that synthesize bone are called osteoblasts. They adhere to a framework of cartilage and begin to secrete collagen I, the major protein of bone and osteocalcin, the calcium binding protein that initiates the deposition of hydroxyapatite [Ca5(PO4)3(OH)]. As the bone forms, the osteoblasts become trapped in lacunae within the bone and stop secreting osteocalcin and begin to secrete hormones in response to the mechanical stress on the bone.

Bone is degraded by osteoclasts that colonize the completed bone after migrating from bone marrow. The total bone mass and density is determined by the dynamic balance between the deposition of bone by osteoblasts and disassembly of bone by osteoclasts. Approximately 10% of bone is being remodeled at any time and the porus trabecular bone in the pelvis, hips, wrist and spine is most actively remodeled. If there is an imbalance that leads to a bone deficit, it usually shows in weak trabecular bone.

Problems with low bone density, i.e. osteoporosis, can result from decreased estrogen (menopause), inadequate vitamin D/sunlight/dietary calcium, or medication, e.g. heparin or warfarin.

The ability of heparin to cause osteoporosis with prolonged use caught my attention. Heparin is anti-inflammatory and inflammation reduces heparin production. Thus, the inflammation caused by high blood glucose levels in diabetics results in loss of heparin production in kidneys and loss of protein from the urine. If heparin causes loss of bone mass, then it might be decreasing inflammation that is needed for bone accumulation.

Osteoclasts are activated by the RANK (receptor activator of nuclear factor κB) system. As the name states, RANK is a receptor that activates the inflammatory transcription factor NFkB. The cytokine that binds to RANK is the corresponding ligand, RANK-L, which is related in structure (and function) to TNF. RANK-L is secreted by osteoblasts, binds to RANK on osteoclasts, activates NFkB and stimulates bone demineralization. A protein called osteoprotegerin, is a soluble receptor of RANK-L that binds the bone and immobilizes the RANK-L and keeps it from activating osteoclasts.

Heparin could interact with many of these components. For example, the binding of RANK and RANK-L is mediated by heparan sulfate proteoglycans. The heparin deficiency that usually accompanies inflammation, and in this case excitation of osteoclasts, could be decreased by administration of heparin. Thus, demineralization would result in osteoporosis.

Warfarin-based osteoporosis could be based on upsetting vitamin K metabolism in osteoblasts. Vitamin K recycling is inhibited by warfarin and vitamin K is needed for a special modification of glutamic acids in particular proteins, such as osteocalcin. The action of osteocalcin in binding calcium is based on three glutamic acids that have been carboxylated using vitamin K. This is shown in the figure as three green calcium atoms bound to red dicarboxylic glutamic acids. You can also notice that the osteocalcin also has a substantial heparin binding domain (blue) at the top. Thus warfarin could cause osteoporosis by disrupting mineralization.

When I was trying to figure out the warfarin/osteoporosis relationship, I tried to find protein structures in the NCBI data base, which had warfarin bound. All I found was warfarin bound to human serum albumin, the protein that carries warfarin and many alkaloids through the blood. I was always suspicious of the use of heparin and warfarin somewhat interchangeably in many different settings in which the mode of action was assumed to be anticoaggulation of blood. I was not surprised when I found that the aromatic rings of warfarin (oxygens in red) were bound to arginines (blue) in a ligand-binding pit on the serum albumin.

A practical note on osteoporosis is that this disease is an exception to many of the degenerative and autoimmune diseases that are based on an inflammatory diet. Osteoporosis is more similar to the problem of gut injury by aspirin. Aspirin blocks COX-2 the enzyme that produces inflammatory and anti-inflammatory prostaglandins from omega-6 and omega-3 fatty acids, resp. Taking aspirin can block inflammation, but the integrity of the lining of the stomach and intestines requires inflammatory prostaglandins, so aspirin can also lead to a bleeding gut. Osteoclasts require NFkB signaling and other aspects of bone production may also require an inflammatory environment. This may explain why corticosteroids also lead to osteoporosis.

Deposition of bone is stimulated by weight bearing exercise that is consistent with the anti-inflammatory lifestyle.

Farmer and Chief

Michael Pollan has articulated the relationship between U.S. agricultural policy, diet, health and lifestyle. In an open letter to the new President-Elect in the October 9, 2008, issue of The New York Times, he reiterates his logic in a plea for reasonable action. Farmer in Chief is at once an indictment of the greed of our food processors, a shrug at the myopia of our medical system, and a hopeful guidance for a future path that provides a sensible alternative.

Asthma Nitric Oxide

Arginine is the source of nitric oxide using the enzyme NOS. Inducible NOS is expressed in response to triggers of inflammation mediated by NFkB. NO in turn activates guanylate cyclase to increase cyclic GMP and cGMP relaxes muscle actin and myosin.

Studies are being made to assess the status of asthma by measuring nitric oxide (NO) in respired air. Since NO is produced as a product of the action of induced nitric oxide synthase (iNOS), and iNOS is synthesized when the inflammatory transcription factor NFkB is activated, then the inflammation that is a characteristic of asthma should be signaled by the production of NO.

If NO is involved in asthma, then one would expect relaxation of muscle to occur. That is the opposite of what is observed; contraction of the muscle cells surrounding airways is responsible for airway constriction is asthmatic episodes. Thus, during asthmatic episodes NO must be reduced.

Since NO is made from the amino acid arginine, then the ability to produce NO can be decreased by reducing available arginine. Arginine can be reduced by the enzyme arginase. The product is ornitine that can in turn be converted into purtrescine, the simplest of the polyamines that are used as counterions in the secretion of heparan and chondroitin sulfates.

Arginase is produced in high levels in asthmatics, but not in normal lungs. Also elevated putrescine is found in asthmatic lungs, indicating that arginine is being converted by arginase into ornithine and on to purtrescine. Ornithine is the precursor for proline that is a key amino acid in collagen production and the increased extracellular matrix that thickens the tissue of asthmatic lungs is rich in collagen.

A major point of this discussion is that NO can only be effectively lowered if there is a limited supply of arginine. Thus arginine supplementation should be helpful to asthmatics. Nitroglycerine patches may also be a means of enhancing sources of NO. Another possible approach would be the use of NO donors conjugated to NSAIDS. These conjugates delivered in an atomized form could provide the lungs with both NO and anti-inflammatory agents. Asthmatics also respond well to rigorous application of the anti-inflamatory diet and lifestyle.

Palmitoleate: omega-7 lipokine

Palmitoleic acid is responsible for keeping people healthy (lipokine) and for the smell of old people (nonenal). Overproduction of this lipid by blocking uptake of fats results in resistance to type II diabetes and atherosclerosis.

If you listen to the commercials on television, you know that there are two sources of fats/cholesterol; you either make it in your cells or take from your diet. Recent research shows that mice that have been genetically modified to lack cytoplasmic lipid carriers, can’t store dietary fat, so they make more of their own lipids. Specifically, they make more palmitoleic acid (C16:1n7). By U.S. standards, those defective mice are very healthy. It turns out that palmitoleic acid acts as a lipid hormone that communicates between fat tissue and other organs, and maintains a healthy metabolic balance.

Lipids are hydrophobic and require protein carriers to be moved from their source, such as the intestines, through the blood and to be offloaded into tissues. Most of the lipids enter the diet as triglycerides, i.e. a three carbon glycerol with three fatty acids attached. Those fats are extracted from food with bile, which is a mixture of modified cholesterol salts that acts as a detergent to dissolve fats. The dissolved fats, in the form of chylomicrons (big fat droplets coated with a lipid layer and proteins) are produced by intestinal cells and released into the blood stream. During transit, a lipase removes the fatty acids from the triglycerides. As in all of the lipid transport systems, the protein carriers determine how the lipid contents are distributed.

If a fatty acid or triglyceride is added to a cell membrane, the lipid would get stuck in the membrane's double layer of phospholipids. Fat droplets in cells are nothing more than fats that are loaded into the membrane of a cellular vesicle until a droplet covered by a half membrane forms. The alternatives for lipid transport are the HDL and LDL (protein coated lipids of the blood), and the intracellular fatty acid-binding proteins. The proteins bound to the surface of LDLs and HDLs bind to receptors on cell surfaces and control transfer of lipids to and from cells. One example is apolipoprotein E4. This protein is intimately involved in determining risk for athersclerosis and Alzheimer’s. ApoE4 binds to its cell surface receptor via heparin. Note the blue basic amino acids that form a massive heparin-binding domain down one side of the protein.

Fatty acid-binding proteins (FABPs) are just globular proteins, with hydrophobic amino acids arranged in the center and hydrophilic, water-bonding, amino acids on the surface. I have drawn the structure of a FABP using a graphics program called Chimera to visualize X-ray crystallographic data in the National Center for Biomedical Information (NCBI) database. The continuous chain of amino acids is shown as a white ribbon and the surface of the protein is shown as a transparent overlay. The protein chain makes a cage with the hydrophobic parts of the protein pointing toward the center to make a hydrophobic-lined container for the trapped fatty acid (pink). The two ends of the protein vessel are held closed by interdigitation of tryptophans (yellow) and basic amino acids (arginine and lysine, dark and light blue). The FABP also has a nuclear translocation signal, a group of four basic amino acids and other concentrations of basic amino acids displayed linearly across the surface of the cage (not shown), that probably are involved in transport of the trapped fatty acid from the cell surface to the surface of the nuclear envelope, which is involved in phospholipid assembly.

Mice engineered to have the human ApoE4 gene develop atherosclerosis and type II diabetes. If the FABPs of the fat cells of these mutant mice have also been removed, then the mice are essentially normal. Removal of the FABPs blocks the uptake of dietary fatty acids and stimulates the production of endogenous lipids, including the omega-7 fatty acid palmitoleic acid. This fatty acid is a lipid hormone, lipokine that stimulates normal metabolism and provides protection against several inflammation-based diseases. Interestingly, palmitoleic acid accumulates abundantly in the skin of old people and is converted to nonenal that has the smell of old books.

Cao H, Gerhold K, Mayers JR, Wiest MM, Watkins SM, Hotamisligil GS. 2008. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 134(6):933-44.

Heparin Binding

This little video clip that I put together shows how a growth factor binds to its receptor. Heparin mediates the process. The growth factors bind to receptors in pairs with the heparin threaded through all four proteins and holding the complex together. The receptors would be embedded in the membrane at the bottom. This clip starts with a single receptor to which the heparin binds first, followed by the first growth factor.  The basic amino acids, arginine and lysine are shown in blue.  In the first image, the amino acid backbone is shown as a ribbon with blue bars.  In the second, the atoms of the amino acid side chains are filled in and the blue bars become blue atomic balls that reveal the surface of the protein.  The blue areas are positively charged and groups on the surface are heparin-binding domains.  The heparin is shown as a polysaccharide with atoms colored by element, O-red, S-orange, N-blue.  The proteins bind to the heparin like beads on a string.  Pairing of the receptors in a particular orientation determined by the heparin and growth factors, is responsible for triggering the cytoplasmic signal that the growth factor is present.

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

EDKHLKFRISHELDSASSEVN

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.