1. In order to easily understand current concepts in the biological basis of human function and health, a cellular approach is necessary; which means that we need a… (Brief) Introduction To Metabolic Biochemistry And Cell Biology

2. The basic assumption of most of these “lectures” is that everyone already has had an introductory course in Biology and Chemistry and knows what a cell is, an organ is, and the difference between a brain cell, a muscle, and a liver (only just a little facetious there, eh?)… The other major concept of these and other sciences is that actually, all that is necessary to really understand health is what most would call high-school level chemistry and biology (with a little physics thrown in here and there). What makes the study of health so difficult is integrating everything so that it all makes sense. Everything that happens inside cells, of course, is chemistry! Many different chemicals can react with each other to produce other chemicals. These reactions are commonly referred to as chemical reactions. Some chemical reactions produce other chemicals that in turn, are useful for cells. Although “chemistry” per se, will not really be discussed, many many chemical reactions do happen within a cell and they are necessary to maintain cellular function, structure, & integrity. These chemical reactions are usually known as biochemical reactions; and as such various biochemistry processes (and not biochemistry) is a major component of many of these “lectures”.

3. Most biochemical reactions are simply chemical reactions that are catalyzed by a biological agent, usually an enzyme. Enzymes are necessary because the chemical reactions catalyzed by them simply cannot happen fast enough to be useful for a cell without them. Enzymes are proteins that catalyze a specific chemical reaction. In the cartoon to the left, the enzyme chymotrypsin catalyzes the hydrolysis of a peptide bond, i.e.: the bond joining the amino acid tyrosine to the amino acid alanine is broken apart with the addition of water. Without the enzyme, the chemical reaction might happen once every 10 years… in the presence of the enzyme, this chemical reaction can happen about 40x each second. (this is probably the most common text-book example of an enzyme reaction…) Completely different enzymes, (cartoons not shown) are necessary to create the peptide bond(s) in the first place (see protein synthesis stuff). For a great tutorial on enzyme mechanisms; go to: http://www.steve.gb.com/science/enzyme_mechanisms.html

4. There are many different mechanisms through which activity of an enzyme can be regulated, from modifying the structure of the enzyme, to changing the amount of the chemicals they “work with”, to changing the number of enzymes in the first place by modifying rates of synthesis of the enzyme (protein synthesis). Regulating enzyme content through regulating rates of protein synthesis is an extremely important area of study commonly called cell biology. All of the different mechanisms responsible for regulation of the protein synthesis processes, and the mechanisms responsible for controlling all of the biochemical reactions of the various metabolism processes are highly integrated and constitute what is known as “integrated cell biology” (my own definition of the integration of metabolism and cell biology, so it may not be the greatest…). The metabolism part, is the total of all biochemical reactions in a cell that are necessary to supply every molecule that the cell needs in order to maintain its structure and function… while cell biology usually refers to all those processes that are involved in regulating the rates of protein synthesis for all the different proteins necessary for the cell to perform its functions… In the case of the overall concept of metabolism, non-enzyme mediated chemical reactions also are considered to be an important part of metabolism, especially those relating to reactive oxygen species (ROS, aka oxygen radicals) and hydrogen-bonding reactions between electrolytes and proteins.

5. The chemical: ATP, provides the molecular form of energy that cells need in order to build and repair cell structures, membranes, and other components. We consume oxygen when the molecules in the food are metabolized by a variety of different enzymes to CO 2 and H 2 O and some of the energy from this process is conserved by building (or more accurately, regenerating from ADP and Pi) the ATP molecule; which cells use as chemical energy… Details of these processes will follow... Some (Oversimplified) Metabolism Stuff

6. And now a very brief overview of some cell functions that are very important for overall (cellular- & whole body-) health will be reviewed…

7. Membranes are molecular structures that surround cells and the organelles inside of cells. These membranes are predominantly made up of phospholipids and proteins and these structures are very sensitive to physical trauma and chemical attack as described later (several important organelles are pictured)

8. Graphic from: Biochemistry by Mary K. Campbell Phospholipids automatically arrange themselves into a bi-layer as pictured. The phospholipids are actually complex fats with a water-soluble end (blue circles) and a fat-soluble end (pink squiggly lines). They form a water-tight barrier and a large variety of specialized proteins can be embedded into the membrane to regulate what gets in and out of the cell as well as to help regulate cell functions (discussed later), and to provide the special physical properties that a cell membrane needs in order to protect the cell.

9. As already mentioned… phospholipids (PL) are extremely important molecules that make up the lipid (barrier) bi-layer. A phosphorus molecule gets attached to the “third” carbon of a glycerol molecule while 2 hydrocarbon chains (fatty acids) get attached to the first and second carbon of the glycerol. Different fatty acids can get joined to the glycerol molecule and this will change the physical properties of the PL (make it more or less fluid depending on the degree of unsaturation of the fatty acids) or provide for special functions such as being a precursor for prostaglandin synthesis… if arachidonic acid is joined to the second carbon (as pictured here)

10. Because water soluble molecules, and especially the charged ones such as the electrolytes, have a very hard time crossing the lipid bi-layer, membranes allow the environment outside of a cell to be very different from the inside and many different specific trans-membrane transport proteins need to be synthesized in order to regulate the transport of these charged molecules. In the cell membrane pictured here a chloride transport protein in the membrane pumps chlorine out of the cell while a sodium/potassium ATP’ase transport protein pumps sodium out of the cell and potassium into the cell, creating a trans-membrane gradient of these important electrolytes; gradients that provide useful functions for the cell. Calcium ATP’ase transport proteins pump calcium from the cytosol of the cell into the endoplasmic reticulum producing a store of calcium that can be used by the cell for a variety of very important signaling functions (and other calcium pumps pump it out of the cell-ensuring very low amounts of calcium in the cytosol). Both of these membrane-bound proteins use adenosine triphosphate (ATP) as the source of chemical energy to move the specific ions to where they belong; hence the common name ATP’ase. Specialized cells will have different (additional) proteins in their membranes to produce the special functions. For example, nerves and muscle cells are capable of propagating action-potentials. In order to do this, they not only need the sodium-potassium ATP’ase transporter proteins (as all cells have); they also need, in addition, voltage-gated sodium channels and voltage-gated potassium channels (which all cells do not have). These are specialized proteins that only allow the respective ion to move through the membrane under very specific conditions.

11. A few of the cell-structures & organelles that will be discussed in a little more detail are pictured here:

12. Although the nucleus is sometimes called the “control center” of the cell… this is not a very accurate description. The process of transcription (converting the DNA code into a mRNA code) and translation (converting the mRNA code into a sequence of amino acids (joined together by peptide bonds) is actually controlled by a variety of proteins that are activated by molecules produced as a result of metabolism… meaning metabolism controls protein synthesis, not the nucleus! The nucleus contains all of the chromosomes. Chromosomes are made up of extremely long strands of DNA molecules joined together and coiled up very tightly. The long strands of DNA molecules are organized in very specific sequences with the order of DNA molecules for many segments of the long strand corresponding to the amino acid sequence of specific proteins (ok… actually to the sequence of mRNA molecules that code for the amino-acid sequence). The order of DNA molecules in a segment that provides the code for a sequence of amino acids in a protein is known as a gene. There are many genes on each chromosome and all of the chromosomes contained in each nucleus (23 pairs) contain all of the codes necessary for the cell to build all of the functional proteins necessary for all of the cell functions. During the process of synthesizing one protein, the DNA code for the one gene is transcribed into an RNA sequence (messenger RNA, mRNA) inside the nucleus. This means that the cell synthesizes a strand of RNA molecules in a sequence that corresponds to the sequence of DNA molecules in the gene. The mRNA strand that contains the code for the amino acid sequence for the protein is then transported out into the cytosol. The mRNA strand is then used as the “blueprint” from which the proper order of amino acids are joined together in a process called translation. The actual synthesis of the protein occurs on ribosomes which are attached to the rough endoplasmic reticulum.

13. A sequence of DNA molecules codes for a sequence of amino acids of a protein. Different sequences of DNA molecules (genes) code for different proteins (gene products). Transcription of DNA sequence into mRNA sequence is tightly controlled by a variety of transcription factors (proteins) than can initiate, enhance, or repress transcription; transcription factors that are in turn controlled by a variety of metabolic, hormonal, or other signaling processes. Just imagine what might happen if you screw up any of the processes of metabolism, or hormone function, or signal transduction, or get toxic damage to the structure of any DNA base!

14. Endoplasmic reticulum is an extensive membranous network of tubules and flattened sheets that “coil” through the entire cell. Granular (or rough) ER has ribosomes attached, which are the sites of protein synthesis and the rER also provides a convenient “highway” for the transport of newly synthesized proteins to their proper destination (cytosol, one of the organelles…). The smooth ER stores and releases calcium (among other functions) and this calcium storage/release is extremely important for cell function: because calcium pumps in the cell membrane pump calcium out of the cell and calcium pumps in the sER pump calcium into the sER tubules, the calcium concentration inside of cells is extremely low. Calcium then becomes a convenient signaling molecule because of this. Anything that activates a calcium channel, or anything that damages a membrane, will allow calcium to move into the cytosol (along its concentration gradient) and the calcium will bind to various signaling proteins and activate them. Which signaling proteins get activated depends on how much calcium gets into the cytoplasm and how long it stays there… remember… calcium pumps are always active and always pumping calcium out of the cytosol.) As mentioned before, there are many different proteins embedded into the cell membrane. Some of these cell-membrane proteins indirectly regulate the entry of calcium into the cell cytoplasm. Many signalling molecules coming from other cells can bind to these cell-membrane proteins (many are called G-proteins) and the G-protein starts a cascade of events that eventually opens up calcium channels in the ER, thus letting in calcium. Different G-proteins let in different amounts of calcium for different amounts of time and therefore stimulating different G-proteins can produce different changes in cell function.

15. The mitochondria are highly specialized organelles that are responsible for an amazing amount of metabolic reactions – including the oxidative production of ATP (metabolism overview is a little later). Mitochondria also have calcium pumps in their membranes so they also help to control calcium levels in the cell. In most cells, the mitochondria is actually a network of coiled tubes, similar to the ER; and not a discrete organelle as pictured in this cartoon... The mitochondria has 2 sets of membranes... kind of unique... The outer membrane and the inner membrane have quite different sets of proteins embedded in them to provide for different functions; in fact, the ability of the mitochondrion to regenerate lots of the ATP molecules depends on the ability to produce very different environments between the matrix part (in red) and the space between the two membranes (in green). The mitochondria are responsible for generating the vast majority of the chemical called ATP which is used as our “universal” source of chemical energy. In essence, mitochondria utilize the chemical energy from the food we eat to convert it into the chemical energy form of ATP. This ATP provides us with the chemical energy necessary for all of the cells’ life processes; such as protein synthesis, lipid synthesis, membrane repair, DNA synthesis, maintenance of membrane potentials, etc., etc., etc Unfortunately, some of the electrons removed from the molecules end up “falling off” of some of the enzymes and they are picked up directly by molecular oxygen (not joined to the oxygen by enzymes), producing oxygen radicals. These oxygen radicals (made through normal metabolism) can end up being very damaging to cell structures if there are too many of them.

16. Cells spend a tremendous amount of energy moving various compounds across the cell membrane (and across any of the organelles’ membranes) through membrane-bound Active Transport proteins. Active transport means the transport protein uses the chemical energy ATP to provide the necessary energy to move the compound across the membrane. Many of these active transport proteins are responsible for maintaining differences in ion concentrations across the cell membranes. Some of these concentration gradients can then be used to drive biological processes while some are simply a result of excretion or uptake processes. Some of these biological processes are discussed in other “lectures”… The ATP energy for these processes comes mainly from the mitochondria. If mitochondrial function declines, then total cell function declines because the many different ion gradients can’t be maintained at the proper level. If mitochondrial function declines enough, the cell can die. If a sufficient number of cells dies (within one organ) then organ failure can happen. This obviously might be fatal if the organ in question is your heart, or liver… or brain…

17. Active transport proteins are responsible for more than 20% of all resting metabolism in humans…. That is LOTS of energy… and an awful lot of ATP regeneration… The chloride, sodium/potassium, & calcium transporters are just a few of them…

18. Some of the metabolic pathways highlighted here are important for regenerating ATP from ADP+Pi: Glycolysis: produces pyruvic acid for acetyl CoA production in mitochondria, produces NADH (electrons) for ETC in mitochondria, anaerobic production of ATP, and more….. MK & CPK: anaerobic production of ATP TCA: accepts acetyl-CoA for citrate synthesis, production of NADH (electrons) for ETC, “anaerobic” production of GTP β-oxidation: produces acetyl CoA for TCA Transamination: produces pyruvic acid, or acetyl CoA, or TCA intermediates ETC: electrons from TCA cycle & glycolysis are “joined” to oxygen to make water & the production of ATP In order to supply lots of ATP regeneration to keep all of the transporters functional, lots of metabolic (chemical) reactions are necessary. They are organized into metabolic pathways: a metabolic pathway is a series of chemical reactions catalyzed by enzymes; the conversion of a 6-carbon molecule called glucose into 2 x 3-carbon molecules called pyruvic acid requires a series of enzyme-catalyzed chemical reactions in the metabolic pathway called glycolysis… there are many different metabolic pathways and each one consists of an enzyme, or rather, a series of enzymes responsible for picking up a particular molecule and ultimately converting it into another molecule that is useful for one (or more) cell function or structure. (The biochemistry-metabolism presentations cover these pathways in detail…)

19. From the preceding slide, several different metabolic pathways are represented, and they are responsible for an amazing amount of metabolic biochemistry. Products produced from the metabolic pathway can be used for triglyceride synthesis, fatty acid synthesis, anaerobic and aerobic ATP production, DNA and RNA synthesis, and many more… obviously, if these metabolic pathways were compromised in any way, there would be serious repercussions to cellular function and even to cellular survival… A by-product of all the metabolism that goes on in cells not often thought of is: HEAT. No chemical reaction is perfectly “symmetrical”, (ok, some are, but very rare)… there is almost always a release of heat energy at the same time chemical energy is being converted from one form of chemical energy to another during the various chemical reactions. This heat needs to be carried away from the cells or they will become too hot and their lipids in their membranes will “melt”, they will soften up and become leaky – a very bad thing. That is one major reason why we have a cardiovascular system… the blood flowing through our tissues removes the heat resulting from the chemical reactions and the heat is then transferred to the atmosphere through our skin (get hot and sweaty, skin gets red from higher blood flow and the sweat helps to get rid of the heat). Too much heat and those membrane gradients can’t be maintained and cells lose function and you pass out from heat-stroke and die very quickly (unless someone dumps you into a barrel of ice real quick!!!)

20. This next set of slides is here to introduce concepts of toxicity that are mainly responsible for many of the chronic diseases; diseases that are, in part, responsive to specific dietary factors… or exacerbated by environmental toxins…

21. The membranes which surround cells and organelles are very sensitive to physical trauma, or to heat, or to chemical damage caused by oxygen radicals, or by chemical radicals… and any damage would result in leaking membranes… ruining those gradients that cost so much energy to maintain… and causing problems… Membranes & Necrosis

22. You already know that an intact cell membrane acts as a barrier for (among other things) charged particles such as calcium… a good thing since calcium is such an important signaling molecule…

23. A damaged cell membrane will allow (among other things) charged particles to enter the cell or organelle. Agents that damage cell membranes include heat, physical trauma, oxygen radicals, and chemical radicals (aka toxins)… and even continual physical contact can cause leaks by stretching the phospholipids apart just a little bit… just rub the back of your hand for 10 minutes with your finger and see what happens!!!

24. Any damage to cell membranes or to endoplasmic reticulum membranes allows calcium (and other ions) to leak into cell cytoplasm – increasing osmotic pressure and causing swelling… which, of course, stretches the membrane and makes the leaks worse… you all remember from your high-school chemistry that osmotic pressure is the pressure that forces water to move from an area where not many things (the number of molecules dissolved in a volume is important, not the size of the molecules) are dissolved in the water to an area where lots of things are dissolved… entry of many small molecules into a cell increases osmotic pressure to force entry of water into the cell too… Membranes & Necrosis

25. As the membranes stretches and the spaces between the phospholipids get bigger, the increased membrane permeability leads to swelling of the endoplasmic reticulum and even more calcium (among lots of other things) leaks. Calcium can bind to calcium-activated enzymes in the lysosomes which digest lipids leading to even more membrane leaks… and with enough lysosomal activation, the cell can even digest away so many lipids that the cell will die… unless, of course, the leaks are stopped and all the damage repaired… Lysosomes are organelles within cells that contain a variety of digestive enzymes (including those for lipids). These enzymes can be activated when cellular content of calcium is elevated for too long. This can happen if ER or cell membranes get damaged and they leak too much calcium. The lysosomes act, essentially, as the garbage-disposal system and will digest any damaged and no-longer-functional molecules. If lysosomes are activated for too long, sustained digestion of membrane lipids can occur, leading to increased membrane damage. This can become highly problematic for cell function.

26. Mitochondria will take up calcium too, but this greatly reduces ATP synthesis – leading to poor repair of membranes and other damaged structures. Mitochondria also can swell up because of the leak-induced changes in osmotic properties within the cell while cellular membranes fragment and lysosomal enzymes continue to digest cellular components… things can really get bad… this is actually a kind of a “safety” mechanism; when there is just a little too much damage to be easily repaired “in time”, mitochondrial function is compromised to ensure insufficient energy for repair... Leading to a guarantee of cell death through this necrosis process: it is simply not in the best interests of any organism to have severely damaged but still-living cells around. The swelling of mitochondrial membranes makes the inner membrane and outer membrane leak and then without much difference between the matrix (look again at the previous mitochondrial slide) not much works… Membranes & Necrosis

27. When the cell membrane is damaged, or “dying”, the cell synthesizes and releases a variety of proteins called cytokines which then initiate an inflammatory process… calcium IS a signaling molecule and it binds to a variety of signal-transduction enzymes and stimulates them to become active… and these activated enzymes produce a variety of signaling molecules that turn on a lot of different cellular functions, both inside the cell and outside the cell… one of the activated cell functions leads to the production of signaling molecules called prostaglandins, leukotrienes, and thromboxanes that are made from arachidonic acid (remember that AA, comes from some of the phospholipids) and they initiate a variety of actions that lead to inflammation. (details in PPT-Innate Immune Function-Human Disease). Another of the cell functions activated by the activated signal transduction pathways is protein synthesis; of a variety of cellular proteins including those proinflammatory signaling proteins called interleukins. Membranes & Necrosis

28. A local inflammatory response is actually necessary to get the cellular repair processes started… it also initiates a series of events that may not be so helpful… depending on what caused the damage in the first place… (These events here are explained in detail in the PPt presentation “V” & “U” from the human disease series) In summary, the damaged endothelial cells (in in this case) release the AA-derived signaling molecules into the blood where they stimulate vasodilation to cause swelling, make platelets sticky to cause blood clots (even if you don’t want them), make blood vessels more leaky so things get out of the blood more easily (causing edema), and attract inflammatory cells into the damaged tissue; both monocytes and neutrophils enter the damaged region to kill off any infection… even of you don’t need it… and the stimulated inflammatory cells also release their own inflammatory signals...

29. Various newly synthesized proteins called interleukins are also released from the damaged & inflammatory cells and they bind to membrane proteins and initiate synthesis of a variety of other signaling proteins - also all part of a local inflammatory response… leading to attraction and stimulation of inflammatory cells, stimulation of growth/repair enzymes by growth factors, and scar formation to close the wound… even if you don’t need it… (details in PPt- “V” & “U” -Human Disease)

30. Some of the recruited inflammatory cells, especially neutrophils and macrophages, get stimulated by these same signaling molecules to produce reactive oxygen species to kill infectious organisms… even if you don’t need it… which, of course, causes damage to other cells in the region…

31. Some of the interleukins and growth factors produced as part of the inflammatory response can lead to increased rates of protein synthesis and cell division by activating some of the signal transduction pathways (RAS in the illustration) that then activates another, which then activates another… to produce a cascade of protein-protein activations that eventually lead to transcription activation of proteins needed for protein synthesis to enhance rates of damage-repair and to turn on cell division in order replace any dead cells… even if you don’t need it… (that G-protein cascade mentioned earlier!!)

32. As a result of all of the inflammatory signaling molecules being produced, a variety of cellular responses occur in the affected tissues: 1. Increased blood flow to increase “in-flow” of inflammatory and immune cells, 2. Infiltration of the tissue with inflammatory cells to kill off infectious agents, 3. Blood clots to stop the bleeding, 4. Scar tissue formation to close up wounds, 5. Increased protein synthesis to help repair the damage, 6. Increased cell division (signals) to replace any dead cells, 7. Initiation of an immune response resulting in the production of antibodies (and other things)... So... Exactly why do I want to know all this? Well… if you study human disease, this in excruciatingly important, if you study any other health-related area, it is JUST as important!!!

33. So… Why is all this cell-function stuff important? Well, we are made up of billions of cells and the proper function of our organs (each made up of a different array of specialized cells) depends on the proper functioning of the individual cells. Normal processes of metabolism in cells are the processes that ensure proper cell function. These processes, however, produce small amounts of potentially damaging molecules; reactive oxygen species (ROS) for example. Normal functions of protein synthesis and lipid synthesis enzymes and repair enzymes ensure that the damage is repaired and cell function continues. In fact, it is the same ROS that can cause damage that also activate the signal transduction pathways to increase rates of repair – so ROS are actually important regulators of cell function in terms of activating protective functions. [Well, as long as you don’t get TOO many ROS; some are essential for proper cell function; too many cause damage and disease.] When there is more extensive damage from, say, a cut to the skin, liver damage from drinking a little turpentine, or lung damage from breathing in smoke from burning plastic, or damage to respiratory-tract tissues from a bacterial or viral infection, then an inflammatory response will occur to accelerate the repair/replace processes to ensure that the damage is repaired, dead cells are replaced, and tissue (or organ) function is returned to normal.

34. If the repair process just isn’t fast enough, cells die. If a whole bunch of cells in a single organ die at once, we just might be in trouble; if a bunch of heart or brain cells die, we may go along with them. Using liver as an example… If a whole bunch of liver cells get damaged and die, causing a massive inflammatory response (hepatitis) we may suffer some form of liver failure… if the damage is “mild” the inflammatory response may save our liver. The rest of our cells in our other organs, however, suffer until the liver is completely repaired because they depend on the liver to produce many different molecules that are necessary for their proper function and to handle the wastes produced by all of our cells. If the liver damage is too severe to repair quickly, then other organs can fail because they don’t get what they need, and again, we will die along with them. In essence, cells in each of the different organs are all inter- dependent on the function of all the other cells in other organs. Obviously without the inflammation response to start the repair & replace process you would die at the first scratch… on the other hand, anything that inappropriately enhances this response, or sustains it for long periods of time; such as overexposure to toxic chemicals (cigarette smoke, toluene, carbon tetrachloride, chlorine…) or a lack of specific nutrients and beneficial phytochemicals… inevitably leads to continual local production of growth signals and ROS from the inflammatory cells: resulting in continual damage, fibrosis and eventually a slow & painful death from organ failure, cardiovascular disease, cancer, or some other degenerative disease… Ultimately, our health depends on optimal function of our cells! And this function can be impaired by aberrant inflammation.